Polishing of Anaerobic Secondary Effluent and Tuoyuan...
104
Polishing of Anaerobic Secondary Effluent and Symbiotic Bioremediation of Raw Municipal Wastewater by Chlorella Vulgaris Thesis by Tuoyuan Cheng In Partial Fulfillment of the Requirements For the Degree of Master of Science King Abdullah University of Science and Technology, Thuwal Kingdom of Saudi Arabia © April 2016 Tuoyuan Cheng All rights reserved
Transcript of Polishing of Anaerobic Secondary Effluent and Tuoyuan...
Polishing of Anaerobic Secondary Effluent and
Symbiotic Bioremediation of Raw Municipal Wastewater by Chlorella Vulgaris
Thesis by
Tuoyuan Cheng
In Partial Fulfillment of the Requirements
For the Degree of
Master of Science
King Abdullah University of Science and Technology, Thuwal
Kingdom of Saudi Arabia
© April 2016
Tuoyuan Cheng
All rights reserved
2
EXAMINATION COMMITTEE APPROVALS FORM
The thesis of Tuoyuan Cheng is approved by the examination committee.
Committee Chairperson: Dr. TorOve Leiknes Committee Members: Dr. Noreddine Ghaffour, Dr. Chunhai Wei
3
ABSTRACT
Polishing of Anaerobic Secondary Effluent and
Symbiotic Bioremediation of Raw Municipal Wastewater by Chlorella Vulgaris
Tuoyuan Cheng
To assess polishing of anaerobic secondary effluent and symbiotic bioremediation of primary
effluent by microalgae, bench scale bubbling column reactors were operated in batch modes
to test nutrients removal capacity and associated factors. Chemical oxygen demand (COD)
together with oil and grease in terms of hexane extractable material (HEM) in the reactors
were measured after batch cultivation tests of Chlorella Vulgaris, indicating the releasing algal
metabolites were oleaginous (dissolved HEM up to 8.470 mg/L) and might hazard effluent
quality. Ultrafiltration adopted as solid-liquid separation step was studied via critical flux and
liquid chromatography-organic carbon detection (LC-OCD) analysis. Although nutrients
removal was dominated by algal assimilation, nitrogen removal (99.6% maximum) was
affected by generation time (2.49 days minimum) instead of specific nitrogen removal rate
(sN, 20.72% maximum), while phosphorus removal (49.83% maximum) was related to both
generation time and specific phosphorus removal rate (sP, 1.50% maximum). COD increase
was affected by cell concentration (370.90 mg/L maximum), specific COD change rate
(sCOD, 0.87 maximum) and shading effect. sCOD results implied algal metabolic pathway
shift under nutrients stress, generally from lipid accumulation to starch accumulation when
phosphorus lower than 5 mg/L, while HEM for batches with initial nitrogen of 10 mg/L
implied this threshold around 8 mg/L. HEM and COD results implied algal metabolic
pathway shift under nutrients stress. Anaerobic membrane bioreactor effluent polishing
showed similar results to synthetic anaerobic secondary effluent with slight inhibition while
4
symbiotic bioremediation of raw municipal wastewater with microalgae and activated sludge
showed competition for ammonium together with precipitation or microalgal luxury uptake
of phosphorus. Critical flux was governed by algal cell concentration for ultrafiltration
membrane with pore size of 30 nm, while ultrafiltration membrane rejected most
biopolymers (mainly polysaccharides). Further research would focus on balancing cell
growth, specific nutrients removal, and specific COD change by utilizing rotating biological
contactor.
5
ACKNOWLEDGMENTS
This thesis fulfilled by Tuoyuan Cheng is kindly supervised by Prof. TorOve Leiknes,
patiently supported by Dr. Chunhai, and generously allowed by the committee to revise. He
is also grateful to the assistance offered by Yasmeen Najm and Ryan Lefters, as well as
cooperative arrangements contributed by lab staff.
6
TABLE OF CONTENTS EXAMINATION COMMITTEE APPROVALS FORM ........................................................... 2
ABSTRACT .......................................................................................................................................... 3
ACKNOWLEDGMENTS ................................................................................................................ 5
TABLE OF CONTENTS .................................................................................................................. 6
LIST OF ABBREVIATIONS ........................................................................................................... 9
LIST OF FIGURES .......................................................................................................................... 10
LIST OF TABLES ............................................................................................................................. 12
1. Introduction ............................................................................................................................... 13
1.1 Microalgae in Wastewater Phycoremediation ................................................................ 14
1.2 Microalgae in Biofuel Production ................................................................................... 17
1.2.1 Harvesting .................................................................................................................. 20
1.2.1.1 Flocculation ....................................................................................................... 20
1.2.1.2 Flotation ............................................................................................................. 21
1.2.1.3 Gravitational and Centrifugal Sedimentation ............................................... 22
1.2.1.4 Filtration ............................................................................................................. 22
1.2.1.5 Immobilization .................................................................................................. 23
1.2.2 Drying ......................................................................................................................... 24
1.2.3 Extraction ................................................................................................................... 24
1.2.4 Downstream Processing ........................................................................................... 25
1.3 Major Factors in Algal Cultivation .................................................................................. 26
1.3.1 Microalgae Species .................................................................................................... 26
1.3.2 Carbon Source and Energy Source ......................................................................... 27
1.3.3 Nutrient Source ......................................................................................................... 28
1.3.4 Light Intensity ............................................................................................................ 29
1.3.5 Cell Density ................................................................................................................ 30
1.3.6 pH ................................................................................................................................ 30
1.3.7 Temperature ............................................................................................................... 31
1.3.8 Dissolved Oxygen (DO) .......................................................................................... 31
1.3.9 Biotic Factors ............................................................................................................. 32
1.3.10 Reactor Configuration .............................................................................................. 32
1.3.11 Mixing ......................................................................................................................... 33
1.3.12 Hydraulic Retention Time (HRT) ........................................................................... 34
1.3.13 Operation Mode ........................................................................................................ 35
7
1.4 Scope of this Research ...................................................................................................... 35
2. Materials and Methods ............................................................................................................. 37
2.1 Inoculum Preparation ....................................................................................................... 37
2.2 Reactor Design and HRT ................................................................................................. 37
2.3 Carbon Source and pH ..................................................................................................... 38
2.4 Energy Source and Light Intensity .................................................................................. 39
2.5 Feed Water Composition and Seeding ........................................................................... 40
2.6 Water Quality Characterization ....................................................................................... 41
2.7 Filtration Process and Critical Flux ................................................................................. 44
2.8 Data Processing ................................................................................................................. 45
3. Results and Discussion ............................................................................................................. 46
3.1 Algal Growth and Nutrients Removal............................................................................ 46
3.1.1 Algal Growth and Nutrients Removal in Synthetic Anaerobic Secondary Effluent Polishing ..................................................................................................................... 46
3.1.2 Algal Growth and Nutrients Removal in AnMBR Effluent Polishing ............. 56
3.1.3 Algal Growth and Nutrients Removal in Symbiotic Bioremediation ................ 57
3.2 COD, HEM and LC-OCD Analysis .............................................................................. 58
3.2.1 COD, HEM and LC-OCD Analysis in Synthetic Anaerobic Secondary Effluent Polishing ..................................................................................................................... 58
3.2.2 COD, HEM and LC-OCD Analysis in AnMBR Effluent Polishing ................ 63
3.2.3 COD, HEM and LC-OCD Analysis in Symbiotic Bioremediation ................... 65
3.3 Particle Size Distribution and Critical Flux ................................................................... 67
3.3.1 Critical Flux in Synthetic Effluent Polishing ......................................................... 68
3.3.2 Critical Flux in AnMBR Effluent Polishing .......................................................... 70
3.3.3 Critical Flux in Symbiotic Bioremediation ............................................................. 70
4. Conclusion ................................................................................................................................. 71
5. Appendices ................................................................................................................................. 74
5.1 MATLAB Codes for Data and Plotting ......................................................................... 74
5.2 Summary of Synthetic Anaerobic Secondary Effluent Polishing ............................... 78
5.3 EPA Method 1664A–Extraction of Oil and Grease from Water Samples Using Solid-Phase Extraction (SPE) Cartridge Configuration, Application Note 817 (Eric Francis 2012) ...................................................................................................................................... 80
5.3.1 Introduction ............................................................................................................... 80
5.3.2 Equipment .................................................................................................................. 80
5.3.3 Solvents ....................................................................................................................... 80
8
5.3.4 SPE Material .............................................................................................................. 80
5.3.5 Oil and Grease Cartridge Configuration Method ................................................. 80
5.3.6 Conclusion .................................................................................................................. 82
5.3.7 References .................................................................................................................. 82
5.3.8 List of Manufacturers ............................................................................................... 82
REFERENCES .................................................................................................................................. 84
9
LIST OF ABBREVIATIONS
AnMBR Anaerobic membrane bio-reactor BBM Bold’s basal medium CF Critical flux COD Chemical oxygen demand dCOD The change of COD DO Dissolved oxygen GHGs Greenhouse gases HEM n-Hexane extractable material HRAP High rate algal ponds HRT Hydraulic retention time LCA Life cycle analysis LC-OCD liquid chromatography-organic carbon detection MF Microfiltration MLVSS Mixed liquor volatile suspended solids NH4-N Ammonium nitrogen NO2-N Nitrite nitrogen NO3-N Nitrate nitrogen OD Optical density OND Organic nitrogen detector PBR Photo bio-reactor PO4-P Phosphate-phosphorus PSD Particle size distribution PVDF Polyvinylidene fluoride sCOD Specific COD change rate sN Specific nitrogen removal rate sP Specific phosphorus removal rate SPE Solid phase extraction TAG Triacylglycerol THF Tetrahydrofuran TMP Transmembrane pressure TN Total nitrogen TSS Total suspended solids UF Ultrafiltration UVD UV254 detector
10
LIST OF FIGURES
Fig.1 Schematic presentation of an overall microalgae production, harvesting and recovery process ................................................................................................................................................. 18
Fig. 2 TAG and transesterification .................................................................................................. 19
Fig. 3 Potential algal biomass conversion processes ..................................................................... 26
Fig. 4 MLVSS-OD691.5nm relationship ........................................................................................ 42
Fig. 5 Illustration of improved flux step method .......................................................................... 45
Fig. 6 Final VSS against initial N and initial P for synthetic anaerobic secondary effluent polishing............................................................................................................................................... 47
Fig. 7 Generation time against initial N and initial P for synthetic secondary effluent polishing............................................................................................................................................... 47
Fig. 8 Final N against initial N and initial P for synthetic secondary effluent polishing ......... 49
Fig. 9 Final P against initial N and initial P for synthetic secondary effluent polishing .......... 49
Fig. 10 Specific nitrogen removal rate against initial N and initial P for synthetic secondary effluent polishing ................................................................................................................................ 50
Fig. 11 Specific phosphorus removal rate against initial N and initial P for synthetic secondary effluent polishing ............................................................................................................. 50
Fig. 12 Nutrients removal ratio against initial N and initial P for synthetic secondary effluent polishing............................................................................................................................................... 51
Fig. 13 Relationship between specific nutrients removal rate for synthetic secondary effluent polishing............................................................................................................................................... 51
Fig. 14 Schematic of lipid accumulation triggered by nitrogen deficiency................................. 52
Fig. 15 Nitrogen removal rate against initial N and initial P for synthetic secondary effluent polishing............................................................................................................................................... 54
Fig. 16 Phosphorus removal rate against initial N and initial P for synthetic secondary effluent polishing ................................................................................................................................ 55
Fig. 17 Nutrients removal rate against initial nutrients ratio for synthetic secondary effluent polishing............................................................................................................................................... 56
Fig. 19 Specific COD change against initial N and initial P for synthetic secondary effluent polishing............................................................................................................................................... 60
Fig. 20 HEM for synthetic secondary effluent polishing (measured for batches with initial N=10 mg/L) ....................................................................................................................................... 61
Fig. 21 LC-OCD results for treated synthetic secondary effluent (with initial N=10 mg/L, P=8 mg/L) .......................................................................................................................................... 64
Fig. 22 LC-OCD results for treated AnMBR effluent .................................................................. 66
Fig. 23 LC-OCD results for treated synthetic raw municipal wastewater ................................. 67
Fig. 24 Particle size distribution ....................................................................................................... 68
Fig. 25 Critical flux for synthetic secondary effluent polishing ................................................... 69
11
Fig. 26 Relationship between critical flux and VSS ....................................................................... 69
Fig. 27 Summary of synthetic anaerobic secondary effluent polishing ...................................... 78
12
LIST OF TABLES
Table. 1 Advantages and limitations of PBRs ................................................................................ 33
Table. 2 Recipe of BBM and synthetic anaerobic secondary effluent ........................................ 38
Table. 3 Recipe of synthetic primary effluent ................................................................................ 41
Table. 4 AnMBR effluent Polishing at HRT=8 d， pH=5.2±1，DO=16.2±1 mg/L, Temp=20 ......................................................................................................................................... 57
Table. 5 Symbiotic bioremediation at HRT=8 d，pH=8.6±0.5，DO=9.5±1, Temp=20 ............................................................................................................................................................... 58
Table. 6 n-Hexane Extractable Material (Oil and Grease, in mg/L) .......................................... 62
Table. 7 Summary of synthetic anaerobic secondary effluent polishing .................................... 79
Table. 8 Method for the SPE instrument ....................................................................................... 81
Table. 9 Parameters for the SPE method ....................................................................................... 82
13
1. Introduction
Microalgae have been investigated for over seven decades (Oswald 2003; Burlew 1953), and
is booming in recent years with quantities of bench scale Photo-Bioreactor (PBR) studies.
Algal cultivation in oxidation ponds treating municipal wastewater dated back to the 1950s
(Oswald et al. 1953), while utilization of algae in nutrient remediation to tackle
eutrophication started as early as the 1970s. The concept of algal biofuel production
exploiting wastewater was proposed in a report of the Aquatic Species Program managed
from 1978 to 1996. As carbon dioxide is recognized to be the sole principal culprit for global
warming (Hoyt 1979), together with the formation of UNFCCC in 1992 and Kyoto Protocol
in 1997, carbon capture via PBR has also been popular since the 1990s (Judd et al. 2015).
Phycoremediation, known as an energy saving decentralized alternative approach to
biological nutrients remediation (Oswald 1962; Oswald, Golueke, and Tyler 1967; Oswald
1990; Benemann and Oswald 1996), are also capable of accumulating heavy metals with high
negative charge on cell surface (Mehta and Gaur 2005), along with enhancing pathogen
removal by raising pH, dissolved oxygen (DO), and water temperature (Schumacher, Blume,
and Sekoulov 2003). The sensitivity of microalgae to noxious organic contaminants endorsed
the recommendation as indicators for acute toxicity (Bierkens et al. 1998; Chen and Lin
2006).
Inhibition of heavy metals on photosynthesis (Clijsters and Van Assche 1985) causing
morphological changes on algal cells enabled their usage as test microbes as well (Peña-
Castro et al. 2004; Travieso et al. 1999).
14
In addition to environmental application and biofuel production, microalgae are also
produced at commercial scale for high-value products such as human nutrition source,
animal feed and aquaculture, biofertilizer, poly-unsaturated fatty acids, and recombinant
proteins (Brennan and Owende 2010). The culture of Chlorella as food additive commenced
in the 1960s in Japan and then developed worldwide later (Spolaore et al. 2006). Until 2004,
the microalgae industry had grown to 7000 tons of dry matter production per annum (Pulz
and Gross 2004; Borowitzka 2013; Markou and Nerantzis 2013; Da Silva, Gouveia, and Reis
2014).
This thesis focusing on wastewater polishing and phycoremediation together with oil and
grease release has biodiesel production briefly introduced and cultivation factors extensively
discussed. Detailed information on algal metabolites production and downstream processing
has been elaborated in recent reviews, thence saved here (Brennan and Owende 2010; Scott
et al. 2010; Borowitzka 2013; Markou and Nerantzis 2013; Da Silva, Gouveia, and Reis 2014;
Chauton et al. 2015).
1.1 Microalgae in Wastewater Phycoremediation
The capability of microalgae to grow in nutrient-rich aquatic environments and efficiently
accumulate nitrogen and phosphorus from wastewater (Hoffmann 1998) permits them to be
a potentially low-cost and environmentally friendly nutrient remediation approach compared
to commonly used methods (Oswald et al. 1957; Mallick 2002). Phosphorus removal by algal
processes, which employ lower level technology thus more appealing to developing countries
(Pittman, Dean, and Osundeko 2011), avoids costly chemical remediation (Wang et al. 2008;
Gasperi et al. 2008) with their ability to luxury uptake polyphosphate contributing to
15
efficiency comparable to chemical methods (Hoffmann 1998). PBRs do not require multiple
zones of various DO like in biological nutrients remediation (Judd et al. 2015) hence
mechanical aeration accounting for above 50% of total energy consumption of typical
aerobic plants could be saved (Tchobanoglous and Burton 1991). Moreover, the significant
in-situ photosynthetic oxygenation offered by microalgae leads to effluent with higher DO
(Judd et al. 2015; Mallick 2002), which may bolster treatment of pollutants when combined
with heterotrophic bacteria (Munoz and Guieysse 2006). Phycoremediation is more
environmentally friendly, for generating no additional wastes like sludge by-products
(Pittman, Dean, and Osundeko 2011) and for presenting nutrient recovery solutions via
biomass processing (Wilkie and Mulbry 2002). Besides, wastewater rich in CO2, N and P
performs as a convenient balanced carbon and nutrients source for microalgae growth
(Lundquist 2008).
There has been a significant amount of publication on phycoremediation of wastewater in
recent years, while most of them are focused on treating municipal sewage or agricultural
manure wastewater, and are based on laboratory bench scale or pilot pond scale. They are
mainly aiming at evaluation of nutrients along with heavy metals removal efficiency,
maximizing biomass production and harvesting efficiency (Pittman, Dean, and Osundeko
2011).
Several unicellular microalgae genus, such as Chlorella and Scenedesmus, are found highly
tolerant to wastewater environment and efficient in removing nutrients either in suspended
or immobilized form. Hence, algal cultivation coupled with secondary effluent polishing has
been extensively assessed (Bhatnagar et al. 2010; Ruiz-Marin, Mendoza-Espinosa, and
16
Stephenson 2010; Wang, Li, et al. 2010; Martınez et al. 2000; Zhang et al. 2008). Traditionally,
other than in maturation ponds following urban plants, chlorophytes also play a major role
in decentralized systems including facultative and aerobic ponds (Aziz and Ng 1993;
Abeliovich 1986; Mara and Pearson 1986; Oswald 1988; Oswald 1995). Besides, primary
settled sewage was shown able to support the growth of C. Vulgaris and elimination of
nutrients as well, with biomass yield and removal rate not significantly related to seeding cell
density (Lau, Tam, and Wong 1995). Moreover, recent research based on cultivation in raw
wastewater (Wang, Li, et al. 2010; Wang, Min, et al. 2010) concluded that microalgae may
reach equivalent nutrients or metal ion removal capacity and growth rate either before or
after primary settling. Growth rate under mixotrophic conditions is found to be higher in
comparison with the photoautotrophic one, therefore suggesting potential employment in
raw wastewater treatment (Bhatnagar et al. 2010).
Agricultural wastewater, since derived from animal manure or fertilizer, could be exceedingly
high in nitrogen and phosphorus content (Wilkie and Mulbry 2002). However, chlorophytes
could grow well in agricultural waste and efficiently remove nutrients from manure-based
wastewater (An et al. 2003; González, Cañizares, and Baena 1997; Wilkie and Mulbry 2002).
Algal production rates and nutrient uptake in agricultural effluent fed by semi-continuous
modes were found comparable to those algae grown in municipal wastewater.
Microalgae are also studied for the treatment of industrial effluents, with emphasis on heavy
metal removal and organic pollutants degradation, rather than nutrients removing (Ahluwalia
and Goyal 2007; de-Bashan and Bashan 2010; Mallick 2002). As a result of commonly low N
and P concentration together with high toxin concentrations, algal biomass grow slower in
17
many industrial wastewaters. Nevertheless, given that industrial wastewater quality are varied,
some of them such as carpet mill effluent could still serve as resources for algal production
(Chinnasamy et al. 2010).
Synthetic wastewater is simplified for lab scale research for only taking major components
into consideration. Most recipes are composed of inorganic constituents including high
concentrations of N or P and lack solid organic material and other potential toxins (Aslan
and Kapdan 2006; Lee and Lee 2001; Voltolina et al. 1999). Therefore, using artificial
wastewater for simulation has some downsides. Research has indicated that although
nutrients removal rates are comparable, growth rates are higher in synthetic ones, which is
attributed to the composition, toxicity, competition and predation factors of their
counterparts (Lau, Tam, and Wong 1995; Ruiz-Marin, Mendoza-Espinosa, and Stephenson
2010).
1.2 Microalgae in Biofuel Production
Second generation biofuels developed from microalgae are deemed as a promising
alternative energy source with several entrepreneurs attempting to commercialize (Zhou et al.
2015; Wijffels and Barbosa 2010; Service 2011; Stephens et al. 2010; Singh and Dhar 2011;
Khan et al. 2009). The first generation biofuels that have achieved commercial scale
production are chiefly derived from food and oil crops involving sugar beet, sugarcane,
maize and rapeseed oil combined with animal fats and vegetable oils via conventional
processes, or from lignocellulose agriculture, forest residues and other non-food crop
feedstocks (Organization 2006; Larson 2007). Although it is believed that they should be
carbon neutral and environmentally beneficial when compared with fossil fuel being the
18
main greenhouse gases (GHGs) contributor, it is still controversial whether these terrestrial
crop-based biofuels are economic or threatening food availability (Demirbas 2009; Hill et al.
2006; Rittmann 2008; Pittman, Dean, and Osundeko 2011).
Microalgae has been propositioned as renewable fuel source for a long time (Benemann et al.
1977; Oswald and Golueke 1960), for their competence in significantly higher biomass
productivity (Brennan and Owende 2010), lower cost per yield (Stephens et al. 2010),
availability of all year round production (Schenk et al. 2008), less freshwater demand than
crops (Dismukes et al. 2008; Li, Horsman, Wu, et al. 2008), ability to grow in brackish water
on non-arable land thus less land demand (Searchinger et al. 2008; Rodolfi et al. 2009), high
oil content reaching 20-50% of biomass dry weight (Spolaore et al. 2006), biofixation of
waste CO2 (Chisti 2007), no requirement of herbicides or pesticides (Rodolfi et al. 2009),
feasibility to serve as feed or fertilizer, and their nutrients source could be from wastewater
instead of fertilizer (Cantrell et al. 2008). Therefore, several studies point out that biodiesel
production in combination with wastewater treatment is the most practicable commercial
resolution temporarily (Van Harmelen and Oonk 2006; Benemann 1997).
Fig. 1 Schematic presentation of an overall microalgae production, harvesting and recovery process,
from (Brennan and Owende 2010)
19
A brief introduction of overall microalgae production, harvesting, and processing, is given in
Fig. 1.
As to the cultivation step, quantities of investigation have been done to identify suitable
chlorophyte strains and cultivation conditions to maximize lipid productivity (Griffiths and
Harrison 2009; Hu et al. 2008). Generally, research has demonstrated that even if nutrient
stress (i.e. N or P limitation) may induce high accumulation of neutral lipids (Converti et al.
2009; Dean et al. 2010; Li, Horsman, Wang, et al. 2008; Rodolfi et al. 2009), especially
triacylglycerol (TAG) that is fit for further transesterification (shown in Fig. 2), in algal cells,
biomass productivity would be very low, resulting in a reduced total lipid production rate.
Therefore, environments supporting high cell yield instead may be of optimum total
biodiesel productivity and thus be of research interest (Griffiths and Harrison 2009).
Fig. 2 TAG and transesterification (Sharma and Singh 2009)
Following sections will concisely cover other steps in biodiesel production, with harvesting
part emphasized, for it is a shared phase separation step that is also necessary in wastewater
phycoremediation.
20
1.2.1 Harvesting
Recovery of microalgae biomass encompassing solid-liquid separation steps is a challenging
step in the whole chain and accounts for 20-30% of the total production cost (Wang et al.
2008; Gudin and Thepenier 1986). During microalgae harvesting, biomass are concentrated
from dilute cultivation medium to a concentration that is economically attractive for further
processing (i.e. drying, extraction, etc.), at which the mixed liquor is in paste or slurry state
with 2-7% being total suspended solids (TSS) (Bilad, Arafat, and Vankelecom 2014). Owing
to the low cell densities (typically in the range of 0.3-5 g/L) in culture and the small sizes of
algal cells (usually in the range of 2-40 µm) (Li, Horsman, Wu, et al. 2008), the recovery is
difficult, costly and energy intensive (Sander and Murthy 2010). Wastewater pond treated
effluents containing high TSS would be difficult to meet discharge limits and could be more
severe in treated industrial effluents for the accumulation of hydrophobic organic
compounds or heavy metals (Hoffmann 1998).
Harvesting can be in single or multiple stage process. Single step harvesting via
centrifugation is usually applied for high-value production case where high costs can be
offset (Olaizola 2003). For mass production of low-value output as biofuel, typically two
stage processes are involved. The concentration factor of the primary step is 10-20 times, to
reach 0.2-1.2% suspension via flocculation, flotation, filtration, while the subsequent step
has a concentration factor of 5-10 times, to obtain algal slurry or paste through
centrifugation (Bilad, Arafat, and Vankelecom 2014).
1.2.1.1 Flocculation
21
Flocculation is usually the preparatory step for other harvesting methods such as filtration,
flotation and gravity sedimentation (Grima et al. 2003). The surface of microalgae cells are
negatively charged hence adding flocculants like multivalent cations or cationic polymers
may destabilize and aggregate cells (Grima et al. 2003), though the efficacy of flocculation is
sensitive to pH (Divakaran and Pillai 2002).
For conventional microalgae flocculation, the dosage of cation is relatively high to fulfill
satisfying recovery, which in return generates a large amount of sludge and makes following
dehydration trickier (Chen et al. 2011; Renault et al. 2009). Although residual water could be
recycled, traditional multivalent flocculants may foul the biomass and eventually hazard
product purity (Lee, Lewis, and Ashman 2010). Consequently, researchers now pay more
attention to biodegradable and less toxic polymeric flocculant as an alternative (Renault et al.
2009). However, the cationic polymeric flocculant is ineffectual for marine strains, because
of high salinity of seawater. So further development of polymeric flocculant is needed for its
application in harvesting (Bilanovic, Shelef, and Sukenik 1988). Bioflocculation or auto-
flocculation, as a result of EPS production triggered by carbon limitation and DO
deprivation (Schenk et al. 2008; Salehizadeh, Vossoughi, and Alemzadeh 2000), has emerged
as a promising harvesting process. Microbes such as bacteria, fungi and actinomyces are
observed producing EPS as bioflocculant to induce spontaneous aggregation and settling
(Gao et al. 2006). Research on bioflocculation has shown effective recovery rate with cell
wall intact, but sufficient mixing was necessary to realize adequate contact between cells and
EPS (Lee, Lewis, and Ashman 2009).
1.2.1.2 Flotation
22
Flotation does not involve any chemical addition and is based on trapping by micro-bubbles
in dispersion (Wang et al. 2008). It is reported that several strains inclined to float at the
surface of suspension when cell lipid content increased, resulting in a higher recovery by
flotation (Burton et al. 2009). Though floatation has been proposed as possible harvesting
approach, its feasibility remains to be seen (Brennan and Owende 2010).
1.2.1.3 Gravitational and Centrifugal Sedimentation
Gravitational sedimentation utilizing Stokes’ Law (Schenk et al. 2008) is common in
microalgae harvesting succeeding wastewater treatment for the massive volume to treat and
the low value of biomass (Nurdogan and Oswald 1996). However, gravitational
sedimentation only works for large species (circa 70um), for example, spirulina (Munoz and
Guieysse 2006) and Tribonema (Wang, Ji, Wang, et al. 2014).
On the contrary, centrifugation recovery is typically applied to high-value products
(Heasman et al. 2000), since it is highly efficient (cell recovery>95%), energy intensive and
expensive (Grima et al. 2003; Bosma et al. 2003).
1.2.1.4 Filtration
Traditional Media filtration process is proper for the harvesting of large algal cells (>70 µm,
e.g. Coelastrum and Spirulina) but inapplicable for algal cells smaller than 30 µm such as
Scenedesmus, Dunaliella and Chlorella (Mohn 1980). Conventional filtration could operate under
positive or negative pressure, with its efficiency ameliorable by cellulose or diatomaceous
earth (Brennan and Owende 2010).
23
Microfiltration (MF) and ultrafiltration (UF) have been proved valid in the harvesting of algal
cells smaller than 30 µm (Petrusevski et al. 1995), particularly for fragile phytoplankton that
requires low TMP or crossflow velocity (Borowitzka 1997). In some cases processing
volumes less than two tons per day, membrane filtration could be more economical than
centrifugation (MacKay and Salusbury 1988).
1.2.1.5 Immobilization
Immobilization of microalgae prior and during cultivation for easier recovery is also very
effective for wastewater phycoremediation (de-Bashan and Bashan 2010; Mallick 2002).
Algae could be either encapsulated in matrix (enclosure method) or form biofilm on the
surface of carriers (non-enclosure method) (Cui 2013). After immobilization, separation is
performed by scratch and dry for non-enclosure method or extraction from the matrix for
enclosed method, since biomass are accumulated densely inside the reactor (Katarzyna, Sai,
and Singh 2015). The production rate of algal cells by such method could realize grams per
square when cultivating Scenedesmus obliquus (Liu et al. 2013) or Botryococcus braunii (Cheng et al.
2013).
However, it is suggested that though encapsulated cells are easier to control thus fit for
research (Hoffmann 1998), the release of cells is not convenient and will be costly when
scaled up (Laliberte et al. 1994). Instead, by introducing biofilm cultivation, microalgae
bioreactors would have enhanced robustness against biocides, predators and conditions of
the media (such as pH or temperature) that may cause crash of suspended unicellular algal
mass cultivation (Wang, Ji, Wang, et al. 2014).
24
1.2.2 Drying
Harvested microalgae slurry needs to be instantly dehydrated or dried, as the presence of
water will be adverse to downstream processes (Lam and Lee 2012). Commonly applied
methods include sun drying, spray drying (Desmorieux and Decaen 2005), low-pressure shelf
drying (Prakash et al. 1997), drum drying, and freeze drying (Grima et al. 1994).
Although solar drying is the cheapest, it is still criticized as requiring long drying time and
vast footprint, plus possessing the risk of product loss, and is not feasible in regions with
limited sunshine intensity (Lam and Lee 2012). Spray drying is applied for high-value outputs
for being pricey and detrimental to pigments (Desmorieux and Decaen 2005). Freeze drying
could ease extraction of lipids later, despite being expensive for scaling up (Grima et al.
2003).
1.2.3 Extraction
Lipid extraction, ensuing harvesting and drying, is essential towards the production cost
(Ranjan, Patil, and Moholkar 2010), even if energy spent here accounted for a comparatively
small portion (5-10%) (Sander and Murthy 2010; Stephenson et al. 2010). The thick cell wall
of chlorophytes preventing the release of lipid has made extraction quite difficult (Mendes-
Pinto et al. 2001). Usually, biomass is mashed up then treated with a solvent or gets directly
extracted by supercritical fluids to avoid pressing (Lam and Lee 2012).
Supercritical fluid extraction is nonhazardous and protecting products from oxidation or
thermal degradation. However, the high operation cost and special machinery requirements
restrict its application in biodiesel production (Ota et al. 2009). Solvent extraction, exploiting
25
the selectivity and solubility of organic solvents like hexane, methanol, ethanol and
chloroform (Lam and Lee 2012) is therefore widely applied.
While n-hexane is commonly implemented in seed crops oil extraction, it is inefficient for
algal lipid for high contents of unsaturated fatty acid, which may reduce selectivity towards
lipid (Ranjan, Patil, and Moholkar 2010). Instead, modified Bligh and Dyer's method using
mixed methanol-chloroform (2:1 v/v) is favored among the literature for a variety of strains
and exhibits satisfying efficiency compared to other methods (D’Oca et al. 2011; Bligh and
Dyer 1959; Lee et al. 2010).
1.2.4 Downstream Processing
As shown in Fig. 3, the conversion processes for microalgae biomass could be classified into
two categories namely thermochemical and biochemical conversion (Tsukahara and
Sawayama 2005). Consideration for process selection consists of biomass type and quantity,
aimed product or energy form, economic factor and process specific restrictions (McKendry
2002). Some studies proposed that one of the most appealing ways be lipid extraction for
biodiesel production via transesterification together with biogas production (fermentation)
using biomass residual (Brune, Lundquist, and Benemann 2009; Zhou et al. 2015).
26
Fig. 3 Potential algal biomass conversion processes (Brennan and Owende 2010)
1.3 Major Factors in Algal Cultivation
1.3.1 Microalgae Species
The selection of microalgae strains is a crucial aspect in both biodiesel yield and wastewater
treatment (Rosenberg et al. 2008; Burton et al. 2009). It has been commonly raised that the
ideal chlorophyte strain should: 1) be easy to culture and robust to survive shear stress,
competition or grazers and grow fast to predominate in open pond or PBRs; 2) own large
nutrient removal capacity and wide N:P removal ratio range; 3) have high carbon bio-
sequestration rate and high lipid productivity; 4) be tolerant or adaptive to site-specific
fluctuation of pH and temperature both from diurnal or seasonal cycle and water quality
changes (Lam and Lee 2012; Sheehan et al. 1998).
27
Presently, species from Chlorella, Scenedesmus (Garcia, Mujeriego, and Hernandez-Marine 2000;
Ruiz-Marin, Mendoza-Espinosa, and Stephenson 2010), Micractinium (Oswald 2003),
Botryococcus (Lam and Lee 2012), Tribonema (Wang, Ji, Bi, et al. 2014) have shown competence
to be the most suitable for microalgae cultivation.
1.3.2 Carbon Source and Energy Source
Chlorophytes could be cultivated in three conditions: phototrophic, heterotrophic and
mixotrophic. In mixotrophic conditions, algae grow in either phototrophic or heterotrophic
pathway, relying on the given light intensity and organics concentration, typically when
cocultivated with other species including prokaryotes, macrophytes, and fungi (Zhang and
Hu 2012). Although the addition of organic carbon (e.g. glucose, acetate, glycerol) (Mata,
Martins, and Caetano 2010) together with limitation of nitrogen source and light could
gather more lipids in cells (Fernandez and Galvan 2007), and high growth rate up to 14.2g/L
could be realized heterotrophically (Li, Xu, and Wu 2007), only phototrophic method,
utilizing light and inorganic carbon, is industrially applicable in commercial scale, especially
when being outdoor with ample light (Borowitzka 1999).
During photoautotrophic production, microalgae could fix carbon dioxide from air (contains
360 ppmv CO2), flue gases (Hsueh, Chu, and Yu 2007) and soluble carbonates (Huertas et al.
2000; Wang et al. 2008), acting as a superior carbon sink for ability to tolerate up to 150,000
ppmv CO2 (Bilanovic et al. 2009; Chiu et al. 2009). However, heterotrophically CO2 is
released in respiration, which is not functioning solar energy capture or carbon biofixation.
Besides, not all strains can grow in heterotrophic conditions completely. Up to now, only
C.protothecoides (Cheng et al. 2009; Xiong et al. 2008; Xu, Miao, and Wu 2006), C.
28
vulgaris (Liang, Sarkany, and Cui 2009), Crypthecodinium cohnii (Couto et al. 2010)
and Schizochytrium limacinum (Johnson and Wen 2009) are classified as capable to live in
absolute darkness. Moreover, organic carbon source may introduce severe contamination of
other microbes (Chen et al. 2011) and give rise to energy consumption or maintenance cost
(Perez-Garcia et al. 2011; Boyle and Morgan 2009).
1.3.3 Nutrient Source
Some microalgae could uptake nitrogen from air directly (Welsh et al. 2000; Moreno, Vargas,
and Guerrero 2003), while most algae need nitrogen source in a soluble form (Hsieh and Wu
2009). Phosphorus is less needed and is less essential during cultivation (Çelekli,
Yavuzatmaca, and Bozkurt 2009). However, they typically should be supplied in an excess
amount of basic requirement, since metal cations may bond with phosphates making partial
P not available (Chisti 2007).
At the industrial scale, culturing of algal cells is typically performed by adding nitrogen in
nitrate and phosphorus in orthophosphate, with both of them usually from chemicals or
inorganic fertilizers, aiming at reducing contamination and validating sustainable water
recycling. However, life cycle analysis (LCA) reported that half of the total energy
consumption and GHGs release were related to the use of fertilizers(Clarens et al. 2010).
Additionally, cultivation of microalgae requires more nutrients than other oil-bearing crops,
which may incorporate high energy input when using fertilizer (Lam and Lee 2012).
Therefore, other than reusing culture medium, exploiting wastewater as nutrients provider is
also encouraged to improve LCA performance.
29
Ammonia in wastewater, when at high concentration, could significantly inhibit microalgae
growth (Ip et al. 1982; Konig, Pearson, and Silva 1987; Wrigley and Toerien 1990). Algal
cells are sensitive to high NH3 content in medium because ammonia uncouples the electron
transport in Photosystem II and competes with H2O in the oxidation reactions leading to
O2 generation (Azov and Goldman 1982; Abeliovich and Azov 1976; Muñoz, Rolvering, et
al. 2005). High N concentration environment leads to starch production in algal cells for
carbon and energy storage while limited nitrogen medium would have this pathway blocked
with photosynthetic efficiency reduced and redirect fixed carbon into fatty acid and resulting
in higher lipid content in microalgae biomass (Li et al. 2010). However, since chlorophyte
growth will be retarded, algal cells would produce less biomass (Li, Horsman, Wang, et al.
2008) and lipid (Huang et al. 2010) when offered limited nitrogen.
1.3.4 Light Intensity
Available sunlight intensity, as the optimal resource for commercial production (Janssen et al.
2003), fluctuates widely among diurnal and seasonal cycle, and act as generally the limiting
factor for outdoor algae production (Pulz and Scheibenbogen 1998). Microalgae activity rise
with irradiance up to 200-400 μEm−2s−1 as the saturation point (Ogbonna and Tanaka 2000;
Sorokin and Krauss 1958). Photo-inhibition then occurs when sunlight intensity reach 4000
μEm−2s−1, which is more likely to happen at an initial phase when cell density is low and light
intensity is not lessened by mutual shading (Fuentes et al. 1999; Göksan, Durmaz, and
Gökpınar 2003; Contreras-Flores et al. 2003).
30
Periodical low intensity or absence of light may induce a reduction in photosynthesis and
leads to anaerobic conditions in the reactor. However, nutrients removal and photosynthetic
activity resume as long as the light is enough again. Therefore, wastewater stabilization
ponds are invented to increase hydraulic retention time (HRT) and deal with light intensity
fluctuations (Tadesse, Green, and Puhakka 2004) while not compromising the overall
efficiency. Light intensity is also affecting optimum biomass concentration. Cell density
should be optimized to avoid dark respiration and mutual shading at low light intensity,
whereas high biomass concentration is required to defend light inhibition at a strong light
intensity (Munoz and Guieysse 2006).
1.3.5 Cell Density
Biomass concentration controls light utilization efficiency, oxygenation and pollutant
removal rate (Janssen et al. 2000; Muñoz et al. 2004). Since oxygenation rate is maximized
when biomass concentration reaches a critical value, further increase of algal cells density
may only cause mutual shading and dark respiration (Grobbelaar and Soeder 1985). Mutual
shading is helpful to increase the frequency of light/dark cycles that cells are exposed to, and
improve photosynthetic performance (Richmond 2004). However, efficient mixing keeping
cells intact, and suitable surface to volume ratio of PBR (Grima et al. 1999) are required to
maintain high cycle frequency (Hu, Guterman, and Richmond 1996). Besides, the initial
concentration of microalgae in wastewater system is also a principal issue for algal growth
(Lau, Tam, and Wong 1995).
1.3.6 pH
31
Carbon biosequestration by microalgae could raise pH up to 10-11 in high rate algal ponds
(HRAPs) and 9 in some enclosed PBRs using algal-bacterial consortium (Muñoz et al. 2003).
These pH rising is effective in pathogen disinfection, nonetheless, may also decrease
pollutant removal efficiency when in combination with activated sludge process (Oswald
1988; Schumacher, Blume, and Sekoulov 2003) since complete bacterial inhibition is usually
observed at pH higher than 10 in ponds system (Schumacher, Blume, and Sekoulov 2003).
Algal growth is both directly affected by pH and indirectly affected via carbonate or
ammonia equilibrium and phosphorus or heavy metal availability (Laliberte et al. 1994). High
pH up to 9-11 may stimulate ammonium vaporization and phosphate precipitation (Craggs
et al. 1996; Garcia, Hernández-Mariné, and Mujeriego 2000).
1.3.7 Temperature
Algae-based wastewater treatment methods have worse efficiency at low temperatures
(Abeliovich 1986). Studies had shown that removal rate may double when temperature
increased from 25 to 30°C using mixed algal species of C. sorokiniana and R. basilensis (Muñoz
et al. 2004). Algae convert a significant portion of sunlight into heat, increasing temperature
when at high irradiance and biomass density. Temperature control has been proposed by
heat exchangers or water spray with their cost prohibitive (Tredici 2002). The combination
of algal strains with different optimum growth temperature in cultivation has been proved
valid as an alternative (Morita, Watanabe, and Saiki 2001).
1.3.8 Dissolved Oxygen (DO)
32
High dissolved oxygen contents could cause photo-oxidative damage on algal cells (Oswald
1988; Suh and Lee 2003), for example, photo-oxygenation rate may decrease 98% when DO
increased from 0 to 29 mg/L (≈350%) (Matsumoto et al. 1996). Supersaturation of DO in
enclosed PBRs could go up to 400%, triggering severe growth inhibition (Lee and Lee 2003).
Supersaturation of DO in algal-bacterial wastewater treatment processes is helpful for
degradation by heterotrophic bacteria (Guieysse et al. 2002). High DO level could be a good
indicator of removal efficiency in continuous mode thus validating its potential application
for process monitor, control, and optimization (Muñoz et al. 2004).
1.3.9 Biotic Factors
The standing stock of microphytes is related to the density of its grazers, which could
significantly reduce microalgae abundance consequently nutrient uptake (Thrush et al. 2006).
Unexpected process failure of PBR may also derive from infections by parasitic fungi like
Chytridium sp. (Abeliovich and Dikbuck 1977). However, this outcome could be
circumvented by daily maintaining low DO for an hour to suppress aerobic organisms
(Abeliovich 1986). Additionally, other microorganisms may outcompete microalgae for
nutrients, which also potentially hazard system robustness.
1.3.10 Reactor Configuration
The configuration of PBRs for suspended microalgae cultivation could be open (high rate
algal ponds or stabilization ponds) or enclosed (tubular, flat, column). PBRs for wastewater
treatment and microalgal biomass cultivation share the same design criteria: effective light
utilization efficiency, optimal mixing, scalability, operation simplicity, low contamination
33
level, low capital and maintenance cost, suitable hydraulic stress on cells and minimal
footprint requirement (Borowitzka 1999; Lee and Lee 2003; Pulz 2001; Xu et al. 2009).
Common configurations together with their strength and weakness are displayed in Table. 1.
It was found by LCA that open ponds are far more economical than enclosed PBRs (Zhou
et al. 2015). However, due to the long HRT and limited water depth for illuminated
surface/volume ratio, the footprint of open ponds is vast. Therefore, ponds system are
recommended for decentralized applications on a small scale (Crites and Technobanoglous
1998) where land cost may be balanced by lower capital investment, cheaper operation and
management, and potential water reuse.
1.3.11 Mixing
Algal ponds are typically operated as plug-flow systems. However, homogeneous conditions
in the reactor are preferable during the treatment of toxic effluent as pollutant dilution lower
the risk of microalgae inhibition. Mixing also limits the formation of anaerobic zones and
more generally reduce any mass transfer limitations (Grobbelaar 2000). The device used for
mixing
Table. 1 Advantages and limitations of PBRs (Brennan and Owende 2010)
Production system Advantages Limitations
Raceway pond
Relatively cheap Poor biomass productivity
Easy to clean Large area of land required, significant evaporation
Utilizes non-agricultural land Limited to a few strains of algae
Low energy inputs Poor mixing, light and CO2 utilization
Easy maintenance Cultures are easily contaminated, require highly selective environments
Tubular PBR Large illumination surface area Some degree of wall growth
34
Suitable for outdoor cultures Fouling
Relatively cheap Requires large land space
Good biomass productivities Gradients of pH, dissolved oxygen and CO2 along the tubes
Flat plate PBR
High biomass productivities Difficult scale-up
Easy to sterilize Difficult temperature control
Low oxygen build-up Small degree of hydrodynamic stress
Readily tempered Some degree of wall growth
Good light path
Large illumination surface area
Suitable for outdoor cultures
Column PBR
Compact Small illumination area
High mass transfer Expensive compared to open ponds
Low energy consumption Shear stress
Good mixing with low shear stress Sophisticated construction
Easy to sterilize Reduced photoinhibition and photo-oxidation
should be selected to reduce shear stress imposed on the microalgal cells (Barbosa and
Wijffels 2004; Mitsuhashi et al. 1995). An increase of up to 75% in microalgal productivity
was reported when pumps were replaced by an airlift system to suspend the cells (Gudin and
Chaumont 1991). Paddle wheels are also often used for mass algal cultivation in open ponds
and HRAPs as they provide a cost-efficient gentle mixing (Munoz and Guieysse 2006).
1.3.12 Hydraulic Retention Time (HRT)
HRAPs are normally operated at 3-10 d HRT (Alcántara et al. 2015) and similar values have
been reported in enclosed PBRs (Tamer et al. 2006; Muñoz, Jacinto, et al. 2005). Against this,
35
very high removals (>99%) and/or reduced HRTs (16 h) have been reported for the
“advanced” PBR process configurations of column (Marbelia et al. 2014) and parallel plate
biofilm (Tercero et al. 2014)reactors respectively, with further HRT reductions (<8 h)
apparently achievable using immobilized systems (Filippino, Mulholland, and Bott 2015).
1.3.13 Operation Mode
The PBR process may then be operated in either batch or continuous mode, with the algal
biomass being recovered as a useful product. As with conventional sewage treatment,
biomass-water separation then takes place either by simple sedimentation, the predominantly
preferred method (Milledge and Heaven 2013; Şirin, Clavero, and Salvadó 2013), or
occasionally by membrane separation (Gao et al. 2015; Drexler and Yeh 2014; Marbelia et al.
2014). For steady-state systems, the hydraulic and solids (or biomass) retention times, and
thus the algal biomass concentration in the reactor, can be controlled prior to harvesting of
the algae, which accounts for 20–30% of the total costs (Barros et al. 2015). However, the
research has predominantly been based on batch systems.
1.4 Scope of this Research
Bench scale bubbling column reactors were operated in batch modes to test wastewater
treatment capacity and involved factors. Polishing of either a series of synthetic anaerobic
secondary effluent or one AnMBR effluent exploited Chlorella Vulgaris UTEX-259. A
symbiotic batch fed with synthetic primary effluent seeded with both C.Vulgaris and
activated sludge was also experimented. Since the unicellular microalgae were characterized
36
as oleaginous and might hazard effluent quality by releasing metabolites, COD together with
oil and grease were also measured. Finally, ultrafiltration, adopted as phase separation step,
was studied via critical flux and LC-OCD analysis.
37
2. Materials and Methods
2.1 Inoculum Preparation
Chlorella vulgaris (UTEX 259), as a unicellular microalgae who was tolerant to various sewage
conditions, efficient in nutrients removal from wastewater (Valderrama et al. 2002; Tuantet
et al. 2014), highly productive in biodiesel production (Liu, Wang, and Zhou 2008) or carbon
dioxide assimilation (Concas et al. 2012), able to dominant in open pond or controlled
symbiotic conditions (Alcántara et al. 2015), and thus most frequently used by researchers
(Oswald 2003), was selected for this study. Microalgae stock was prepared by batch
cultivation in Bold’s Basal Medium (BBM, from Sigma-Aldrich) with the recipe shown in
Table. 2 and membrane filtration (pore size=30 nm) was employed prior to seeding to
minimize contamination from BBM. Activated sludge used for the later symbiotic system
was taken from the MBR wastewater treatment plant in King Abdullah University of Science
and Technology, then stored for a month at 4 to avoid grazer effect (Kesaano and Sims
2014), and reactivated in synthetic primary wastewater aerobically for two weeks before
inoculum.
2.2 Reactor Design and HRT
Experiments were set in batch mode at 20, as previous research has been predominantly
based on it (Judd et al. 2015). HRT was 8 days, within the range of 3-10 days that applied in
HRAPs (Alcántara et al. 2015).
Treatment was performed in graduated glass cylinders (Kimble™ KIMAX™ Class B Mixing
Cylinders, 2000 mL) with aerators to mimic bubbling column PBR, since lower shear stress
38
and higher microalgal productivity were met when fulfilled mixing by bubbling rather than
pumps (Gudin and Chaumont 1991; Brennan and Owende 2010; Barbosa and Wijffels 2004).
Table. 2 Recipe of BBM and synthetic anaerobic secondary effluent Component (mg/L) Formula BBM Syn.An.Eff.
Ammonium Chloride NH4Cl 0.000 - Boric Acid H3BO3 11.420 11.420
Calcium Chloride Dihydrate CaCl2 · 2H2O 25.000 25.000
Cobalt Nitrate Hexahydrate CoNO3 · 6H2O 0.490 0.000
Cupric Sulfate Pentahydrate CuSO4 · 5H2O 1.570 1.570
EDTA (free acid) C10H16N2O8,
(HO2CCH2)2NCH2-CH2N(CH2CO2H)2
50.000 0.000
Ferrous Sulfate Heptahydrate FeSO4 · 7H2O 4.980 4.980 Magnesium Sulfate Anhydrous MgSO4 36.628 36.628
Manganese Chloride Tetrahydrate MnCl2 · 4H2O 1.440 1.440 Molybdenum Trioxide MoO3 0.710 0.000
Nickel Sulfate Hexahydrate NiSO4 · 6H2O 0.003 0.003 Potassium Hydroxide KOH 31.000 31.000
Potassium Iodate KIO3 0.004 0.004 Potassium Phosphate Monobasic KH2PO4 175.000 -
Potassium Phosphate Dibasic K2HPO4 75.000 - Sodium Chloride NaCl 25.000 25.000 Sodium Nitrate NaNO3 250.000 0.000 Sodium Selenite Na2SeO3 0.002 0.000 Stannic Chloride SnCl4 0.001 0.000
Vanadium Sulfate Trihydrate VOSO4 0.002 0.000 Zinc Chloride ZnCl2 4.181 4.181
More than offering the most efficient mixing and the highest mass transfer rate, column
PBR being compact and easy to operate (Eriksen 2008), could provide the best controllable
growth conditions permitting a culture of single species with a lower risk of contamination
(Chisti 2007).
2.3 Carbon Source and pH
39
Carbon source and pH are interrelated critical factors in algal cultivation. It has been
reported that CO2 bubbling could accelerate 5.2 times higher biomass productivity
compared to NaHCO3. When NaHCO3 is supplied, C. vulgaris prefer to utilized free
CO2 molecules at acidic cultivation condition (pH=4) instead of bicarbonate ions at alkaline
conditions (pH=8.5) (Lam and Lee 2013). However, carbon biosequestration by microalgae
could raise pH up to 10-11 in HRAPs or 9 in enclosed PBRs (Muñoz et al. 2003), which may
affect carbonate or ammonia equilibrium and phosphorus availability.
Therefore, room air containing 360 ppmv CO2 (Cuellar-Bermudez et al. 2015) was aerated
via diffuser at a rate of 1vvm (2L air volume/2L reactor volume/min) controlled by
flowmeters (Dwyer Instruments RMA-21-SSV) to render excessive carbon source and
sufficient mixing, while balancing pH at the same time.
2.4 Energy Source and Light Intensity
Although the addition of organic carbon as chemotrophic energy source (e.g. glucose,
acetate, glycerol) (Mata, Martins, and Caetano 2010) together with limitation of nitrogen
source and light could gather more lipids in cells (Fernandez and Galvan 2007), and high
growth rate up to 14.2g/L could be realized heterotrophically (Li, Xu, and Wu 2007),
photoautotrophic cultivation, utilizing light and inorganic carbon, is the only feasible method
on commercial scale, especially when being outdoor with ample light. Moreover, organic
carbon sources may introduce severe contamination of other microbes.
In this experiment, a convenient set of cool daylight fluorescent lamps (Philips TL-D
36W/54-765) offering 24h illumination within 400-700 nm was installed as photon source,
40
with light intensity measured by lux meter (Testo 435) and converted to a photosynthetically
active radiation value equal to 14 μmol m−2 s−1. Saturation point of cell density premeasured
by batch cultivation at this light intensity was around 650 mg/L in mixed liquor volatile
suspended solids (MLVSS or VSS).
2.5 Feed Water Composition and Seeding
The recipe modified from BBM shown in Table. 2 simulates anaerobic secondary effluent
since anaerobic digested wastewater is the preferred pretreatment (Olguín 2000; Marchant
2001; Wang, Min, et al. 2010). Ammonium nitrogen was set at 10, 20, 30, 40 and 50 mg/L
with phosphate phosphorus at 2, 4, 6, 8 and 10 mg/L in combinations while 40 mg/L of
C.vulgaris was seeded into mentioned batches.
Besides, real anaerobic membrane bioreactor (AnMBR) effluent with COD=25.04 mg/L,
NH4-N=28.59 mg/L, PO4-P=10.00 mg/L was employed for another one
phycoremediation experiment, together with an inoculum of 40 mg/L C.vulgaris.
A symbiotic bioremediation batch was operated by feeding synthetic primary effluent (recipe
shown in Table. 3) with COD= 282.35 mg/L, TN= 47.57 mg/L, NH4-N=26.18 mg/L,
PO4-P=26.24 mg/L, and is seeded by 200 mg/L C.vulgaris plus 20 mg/L activated sludge.
This 10:1 seeding ratio was reported to exhibit maximum COD and ammonium removal
efficiency, faster algal growth and as a whole keeps the reactor showing algal behavior
(Roudsari et al. 2014)
41
2.6 Water Quality Characterization
The value of pH was recorded daily by CyberScan pH 6000 while DO was daily by inoLab®
Multi 9430 IDS. MLVSS was measured following protocols in Standard Methods for the
Examination of Water and Wastewater, 20th ed. (Clescerl, Greenberg, and Eaton 1999).
Algal biomass in pure culture was calculated by optical density (OD) measured at 691.5nm
by UV-Vis spectrophotometer (Shimadzu UV-2550) via pre-calibrated MLVSS-OD691.5
relationship (shown in Fig. 4).
Chemical oxygen demand (COD), total nitrogen (TN), ammonium nitrogen (NH4-N),
nitrite nitrogen (NO2-N), nitrate nitrogen (NO3-N), phosphate phosphorus (PO4-P) were
measured by HACH kits (TNT 821, 826, 827, 830, 832, 840, 835, 836, 843, 845) and UV-Vis
spectrophotometer DR5000 according to suitable assay range.
Table. 3 Recipe of synthetic primary effluent
Component Conc. (mg/L)
Urea 76.46 NH4Cl 6.38
MgHPO4·3H2O 14.50 KH2PO4 33.10
FeSO4·7H2O 2.90 Milk powder 58.09
Starch 61.00 Yeast 26.12
Peptone 8.70 Na-Acetate 39.66
Cr(NO3)3·9H2O 0.26
42
CuCl2·2H2O 0.27 MnSO4·H2O 0.05 NiSO4·6H2O 0.17
Fig. 4 MLVSS-OD691.5nm relationship
Hexane extractable material (HEM), as a parameter characterizing oil and grease in water,
was measured at the end of batches for those treating synthetic anaerobic secondary effluent
initiated at N=10 mg/L. This was based on the knowledge that, though high N
concentration environment led to starch production in algal cells for carbon and energy
storage, limited nitrogen medium would have this pathway blocked with photosynthetic
efficiency reduced and redirect fixed carbon into fatty acid, especially TAG, and resulting in
higher lipid content in microalgae biomass, (Li et al. 2010) which may lead to lipid release
and HEM rising. HEM was also measured in other two treatment scenarios as a potential
limit-exceeding parameter.
HEM was analyzed gravimetrically by following modified EPA method 1664 (EPA 2010)
and vendor validated solid phase extraction (SPE) protocol (Eric Francis 2012) (see
appendix for details), in which Dionex™ SolEx™ C18 silica-based SPE cartridges installed
y = 361.2423 x R² = 0.9976
0
100
200
300
400
500
600
0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600
MLV
SS m
g/L
Abs.
43
on Dionex™ AutoTrace™ 280 was conditioned by hexane, methanol and acidified water
(pH=2), then loaded with sample of 1 L, dried by gas, eluted by hexane as fraction #1,
washed by methane, eluted by hexane-tetrahydrofuran (hexane:THF, v/v=1:1) as fraction
#2. Then both fractions were evaporated at 70 for later gravimetric determination by
Mettler Toledo Balance XP2U with weighting precision down to 0.1 mg. Based on the
preparation step, HEM was further classified as total HEM, dissolved HEM and HEM in
the UF (pore size of 30 nm) filtrate. Total HEM is derived by firstly acidification of sample
to pH=2 then filtered by 0.45 µm Whatman® glass microfiber syringe filters for later SPE,
while dissolved HEM is firstly filtered by 0.45 µm filter then acidified to pH=2, and HEM in
the UF (pore size of 30 nm) filtrate was directly acidified to pH=2 prior analysis.
It is notable that n-hexane is commonly applied in seed crops oil extraction, but is not
efficient in algal lipids extraction when compared with methanol-chloroform (v/v=2:1, Bligh
and Dyer method) (Lam and Lee 2012), because high contents of unsaturated fatty acid in
microalgae lipid reduces selectivity towards lipid (Ranjan, Patil, and Moholkar 2010).
Therefore, n-hexane extraction process probably would underestimate oil and grease in
microalgae treated wastewater.
Liquid chromatography with online organic carbon detection (LC-OCD) installed with
UV254 detector (UVD), and organic nitrogen detector (OND) was utilized for detailed
composition analysis. The instrument was installed with Toyopearl size exclusion
chromatography column HW65S + HW50S, and was operated at a flow rate of 1.1 mL/min.
44
Particle size distribution (PSD) was performed by Malvern Mastersizer MicroPlus for feed
before filtration since PSD was considered as an important factor affecting membrane
permeability (Castaing et al. 2011) and reversible fouling (Ladner, Vardon, and Clark 2010).
2.7 Filtration Process and Critical Flux
Membrane filtration which involves least biomass washout or chemical contamination
(Boonchai and Seo 2015) is chosen in the separation step for following reasons. The
ecological niche provided by cultivation media and designed for microalgae is also fit for
some toxic microalgae or cyanobacteria, implying potential toxin kept in biomass in real
application if not removed to some extent (Castaing et al. 2010; Guzzon et al. 2008; Rier,
Stevenson, and LaLiberte 2006; Besemer et al. 2007). Besides, chlorophyte cells are
demonstrated to survive chlorine residual of 5 mg/L whereas E.coli would be sufficiently
destroyed at 1 mg/L in chlorination during the disinfection preceding discharge (Oswald
2003). Moreover, microalgae must be removed from pond effluent that is to be disinfected
with ultraviolet lights due to their light absorption (Oswald 2003).
Dead-end filtration mode is usually used instead of tangential flow owing to its lower
specific energy consumption and less exposure to shear stress in pumping systems (Castaing
et al. 2011; Bilad et al. 2012b). Although MF and UF both have been shown valid and
economic (MacKay and Salusbury 1988) in harvesting of fragile phytoplankton cells smaller
than 30 µm (Petrusevski et al. 1995; Borowitzka 1997), UF has shown superiority over MF
with less adsorption and irreversible fouling in microalgae harvesting (Rossignol et al. 1999;
Rossi et al. 2004). Thus, a polyvinylidene fluoride (PVDF) hollow fiber membrane with a
pore size of 30 nm and a surface area of 48.38cm2 was selected for filtration. Bubbling by
45
diffuser was kept at 1vvm equal to the same flow rate as above mentioned to mitigate
reversible fouling by cell deposition, which may perform as a major foulant (Saint Cyr et al.
2014).
Critical flux (CF) was measured as a permeability assessment, by using a hollow fiber of the
same type and same surface area as mentioned ahead, with the critical transmembrane
pressure (TMP) increase set at 20 Pa/min. The improved flux-step method with relaxation
between each step as illustrated in Fig. 5, was adopted by following literature (van der Marel
et al. 2009; Bilad et al. 2012b).
Fig. 5 Illustration of improved flux step method (Bilad et al. 2012a)
2.8 Data Processing
OD-MLVSS relationship presented above as well as PSD and LC-OCD results were edited
by Microsoft Excel 2013. All other plots and contours were achieved by MATLAB R2015a
with codes attached in the appendix.
46
3. Results and Discussion
3.1 Algal Growth and Nutrients Removal
3.1.1 Algal Growth and Nutrients Removal in Synthetic Anaerobic Secondary Effluent
Polishing
During the whole process of synthetic anaerobic secondary effluent treatment by pure algal
species, pH in all batches were maintained at 5.2±1 by aeration, as a suitable acidic condition
for abundant carbon dioxide supply (Alcántara et al. 2015; Lam and Lee 2013) as well as
minimizing potential ammonia toxicity (Abeliovich and Azov 1976; Peccia et al. 2013), free
from ammonia stripping and phosphate precipitation (Ruiz-Martinez et al. 2014), since at
this acidic pH carbonate equilibrium was shifted towards carbon dioxide, ammonia was
transformed into ammonium and phosphorus was kept in dissolved state. Therefore,
nutrients removal mechanisms here should be relying on cell assimilation.
DO values were kept at 16.2±1 mg/L, higher than saturation point measured as 9.0 mg/L
(at 20), but still lower than 20 mg/L being a common threshold reported in photo-
oxygenation studies. This could be attributed to photo-oxygenation balanced with sufficient
bubbling, mixing thus stripping at an aeration flow up to 1vvm.
Algal growth is depicted by Fig. 6, 7, in which final VSS (sensitive at high growth rate) and
generation time (suitable at low growth rate) are contoured against initial nutrients
concentration. Algal cells amount in the range from below 60 to over 360 mg/L, together
with generation time varying between three and ten days, showed susceptibility of growth
rate to initial nutrients concentration.
47
Fig. 6 Final VSS against initial N and initial P for synthetic anaerobic secondary effluent polishing
Fig. 7 Generation time against initial N and initial P for synthetic secondary effluent polishing
48
Phosphorus was a sensitive limiting factor when P<5 mg/L while nitrogen was in charge of
the rest region though less powerful than P in terms of generation time. However, when
ammonium nitrogen, as a positive stimulating factor, exceeded 40 mg/L, final VSS decreased,
probably owing to ammonium toxicity or inhibition at high concentration (Markou 2015).
Similarly, phosphate was positively correlated with algal biomass production when below 8
mg/L, especially at higher initial N concentration, but may weaken growth when further
added, in which case, nitrogen would be limited again.
The final nitrogen concentration ranged from 5 to 45 mg/L, with most area in Fig. 8 lower
than 15 mg/L, showing satisfying outcome. Initial P content was an important factor when
N>20 mg/L and P<5 mg/L, accompanied with N as a secondary one in this region.
The final phosphorus concentration ranged from 2 to 9 mg/L, with most area in Fig. 9
poorly higher than 2 mg/L, was dominated by initial P concentration, even if nitrogen
functioning as a negatively related argument when N exceeded 25 mg/L.
49
Fig. 8 Final N against initial N and initial P for synthetic secondary effluent polishing
Fig. 9 Final P against initial N and initial P for synthetic secondary effluent polishing
50
Fig. 10 Specific nitrogen removal rate against initial N and initial P for synthetic secondary effluent polishing
Fig. 11 Specific phosphorus removal rate against initial N
and initial P for synthetic secondary effluent polishing
51
Fig. 12 Nutrients removal ratio against initial N and initial P for synthetic secondary effluent polishing
Fig. 13 Relationship between specific nutrients removal rate for synthetic secondary effluent polishing
52
Specific nitrogen removal rate (nitrogen removal per VSS increase, sN) fluctuating from 8 to
20%, is depicted in Fig. 10. In general, sN was positively affected by initial N, albeit slightly
decreased where N=40 mg/L. Comparable studies using nitrate at pH=8 showed the same
positive trends but hampered at higher N=50 mg/L (Beuckels, Smolders, and Muylaert
2015), likely due to postponed ammonium toxicity based on required metabolism conversion
from nitrate to nitrite then ammonium as shown in Fig. 14 (Shen et al. 2015).
Fig. 14 Schematic of lipid accumulation triggered by nitrogen deficiency. (TCA cycle, PPP, GAP, and G6P refer to tricarboxylic acid cycle, pentose phosphate pathway, glyceraldehyde 3-phosphate and glucose 6-phosphate, respectively)
Interestingly, a concave formed at N=40 mg/L, centered on P=6 mg/L in Fig. 10. This
recession was close to the peak of VSS, implying that shading might happen then causing
lower energy input, lower metabolic activity thus lower sN, because synthesis of protein was
less activated (Ruiz-Martinez et al. 2014). Besides, the amount of final nitrogen might also
take effect when approaching the end of long HRT=8 days, since it has been reported that
53
low nitrogen concentration may decrease cell growth, chlorophyll content (Lin et al. 2011)
and protein synthesis (Geider and La Roche 2002; Loladze and Elser 2011).
Phosphate conditions helped to modify the curve of sN at different initial ammonium levels,
with the optimum P concentration for local highest sN at various N concentration shifted
from P=6 to 10 mg/L for N<35 mg/L, and jumped to P=5 mg/L at N=50 mg/L.
Phosphate uptake has been reported to be helpful in the absorption of N (Ruiz-Martinez et
al. 2014). Consequently, the two peaks in sN contour might be a combined outcome from
both shading via VSS and two neighboring peaks of specific phosphorus removal rate as
shown in Fig. 11.
Specific phosphorus removal rate (phosphorus removal per VSS increase, sP) varied from
0.4 to 1.4% is plotted in Figure 11. Since the maximum value was still below 3% as a critical
value for luxury uptake (Reynolds 2006; Ruiz-Martinez et al. 2014), phosphorus removal
mechanism should be ascribed to assimilation. This contour was largely following the trend
in ones of VSS and generation time: controlled by initial P content when below 5 mg/L,
then by initial N in the rest region. Since nitrogen has a positive effect on the accumulation
of P (Beuckels, Smolders, and Muylaert 2015), and the mentioned shading effect would also
reduce cellular ribosomal RNA content, and P removal (Sterner and Elser 2002), the two
peaks in sP might be shaped by both VSS and sN.
Nutrients removal ratio (sN/sP) is contoured in figure 12, where the value between 14~38:1
is within the range from literature (Boelee et al. 2012; Klausmeier et al. 2004; Klausmeier et
al. 2008). Initial P was a limiting negatively correlated parameter when less than 5 mg/L,
54
with the convoluted rest area more affected by initial nitrogen concentration together with
shading.
The relationship between specific removal rates (nitrogen removed versus phosphorus
removed, or sN vs. sP) is presented in Fig. 13, showing an overall weak positive correlation,
hinting cooperative removal process mentioned ahead for Fig. 10 and 11.
Fig. 15 Nitrogen removal rate against initial N and initial P for synthetic secondary effluent polishing
Nitrogen removal rate is given in Fig. 15. The contour was similar to ones of VSS (Fig. 6),
generation time (Fig. 7), final nitrogen (Fig. 8), but not sN (Fig. 10). Therefore, nitrogen
removal rate might be determined by growth rate along with final nitrogen concentration
rather than cell content of nitrogen. Removal efficiency ranged from 10 to 90%,
demonstrating the sensitivity of system performance based on assimilation mechanism
because cell growth characterized by generation time was responsive to initial conditions.
55
Fig. 16 Phosphorus removal rate against initial N and initial P for synthetic secondary effluent polishing
Phosphorus removal rate is shown in Fig. 16. This contour was resembling the one of sP
(Fig. 11), and the ones of VSS (Fig. 6) or generation time (Fig. 7), but not final phosphorus
(Fig. 9). It could be inferred that removal performance of P, poorly ranging from 5 to 50%,
was regulated by both cell content of P and growth rate. Therefore, phosphorus removal
efficiency would be compromised when nutrients ratio was not optimized in the influent.
Nutrients removal rate against initial nutrients ratio is given in Fig. 17. With boundary value
(at P=2 mg/L) excluded, removal rates accumulate in regions surrounded by dashed lines.
Phosphorus removal efficiency was roughly improved with the increased ratio, while this
effect on nitrogen removal rate was not evident, which confirmed that P removal was subtle
to influent nutrients ratio while N removal was less affected. Adjustment of wastewater for
nutrients could be a solution, such as sodium nitrate or industrial urea addition (Christenson
56
and Sims 2012; Boelee et al. 2012). The strong limitation for both removal rates at initial
P=2 mg/L was revealed, as a reasonable outcome since initial P<5 mg/L was already
restricting cell activity.
Fig. 17 Nutrients removal rate against initial nutrients ratio for synthetic secondary effluent polishing
3.1.2 Algal Growth and Nutrients Removal in AnMBR Effluent Polishing
Batch treatment results are presented in Table. 4, together with interpolated data derived
from synthetic anaerobic secondary effluent with similar nutrients content to AnMBR
effluent. DO and pH in the reactor was still within the range from synthetic effluent
polishing, validating this comparison. It could be found that algal growth was overestimated.
Besides, ammonium removal was higher than anticipation, while phosphorus removal was
slightly lower. However, both specific removal rates were underestimated. Inhibitory
compounds produced by AnMBR might be present in the feed
57
(Holmstrom and Kjelleberg 2000; Rivas, Vargas, and Riquelme 2010), leading to lower
biomass production and more complicated metabolic pathway thence more specific
nutrients uptake.
Table. 4 AnMBR effluent Polishing at HRT=8 d，pH=5.2±1，DO=16.2±1 mg/L, Temp=20
NH4-N PO4-P MLVSS Generation
Time sN sP COD sCOD CF
mg/L mg/L mg/L days % % mg/L % LMH
Initial 28.59 10.00 40.00 - - - 25.04 - - Final 1.86 8.89 166.28 3.8919 21.17 0.88 64.73 0.31 28
Syn. Final 3.50 8.85 183.15 3.6447 18.51 0.80 49.47 0.17 26
3.1.3 Algal Growth and Nutrients Removal in Symbiotic Bioremediation
Symbiotic bioremediation results are given in Table. 5. Without adding buffer or alkali, pH in
this scenario increased to alkaline region unfavoring CO2 but bicarbonate, as it has been
reported in cases using algal-bacterial consortium (Muñoz et al. 2003). DO dropped to 9.5±1
mg/L, but was still near measured saturation point (DO=9.0 mg/L) and higher than the
concentration in the mixed liquor where sludge was pre-cultivated (DO=3.5 mg/L).
The anticipated faster mixotrophic growth of microalgae did not seem significant since
generation time of total biomass was approaching 10 d though seeded with activated sludge,
for which shading together with undesirable carbon or nitrogen source might be responsible.
Nitrite and nitrate were accumulated in the reactor, showing bacteria oxidizing activity and
competition with chlorophytes assimilation since ammonium was preferred nitrogen source
of C.Vulgaris and utilization of nitrate was a detour metabolic pathway (shown in Fig. 14),
plus elevated DO was also supporting nitrogen oxidation (Xin et al. 2010; Shen et al. 2015).
58
Therefore, a relatively low sN=14.11% was reached, giving total nitrogen removal rate of
48.83%.
Though total phosphorus removal rate was as low as 30.22%, specific phosphorus removal
rate realized 4.82%, indicating the occurrence of luxury uptake, adsorption or phosphate
precipitation (Eixler, Karsten, and Selig 2006; Powell et al. 2009; Di Termini et al. 2011;
Wang and Lan 2011).
Table. 5 Symbiotic bioremediation at HRT=8 d，pH=8.6±0.5，DO=9.5±1, Temp=20
Algae Sludge Total
VSS Gen. Time TN NH4-N NO2-N NO3-N PO4-P sN sP COD CF
mg/L mg/L mg/L days mg/L mg/L mg/L mg/L % % mg/L LMH
Initial 200.0 20.0 220.00 - 47.57 1.67 - - 26.24 - - 282.35 - Final - - 384.60 9.9273 24.34 4.29 0.87 12.85 18.31 14.11 4.82 121.03 17
3.2 COD, HEM and LC-OCD Analysis
3.2.1 COD, HEM and LC-OCD Analysis in Synthetic Anaerobic Secondary Effluent
Polishing
The change of COD (dCOD) is plotted in Fig. 18. All values were positive, ranging from 10
to 60 mg/L. dCOD increased with initial phosphate concentration, but fluctuated along
nitrogen addition, resulting in two peaks located close to ones of VSS and final nitrogen.
This could be inferred from a common rule that when nutrients become limiting, microalgae
accumulated C-rich compounds including carbohydrates and lipids (González-Fernández
and Ballesteros 2012), which could be released into water and give rise to COD. Also, it has
been concluded that for lipid production, the growth rate of biomass is more important than
cellular lipid content (Weldy and Huesemann 2007). However, when shading effect occurred,
individual cells were receiving less photon, and might produce less lipid per unit biomass as
59
proved in Fig. 19 (Courchesne et al. 2009). Therefore, dCOD extremes were centered where
nitrogen was depleting, and VSS peaked, with the one possessing higher VSS contrarily
showing relatively lower dCOD.
Specific COD change rate (sCOD), varying from 0.1 to 0.8% is contoured in Fig. 19.
Primarily, sCOD was negatively ruled by N concentration, conforming to the general
application of nitrogen limitation on lipids production (Shen et al. 2015; Beuckels, Smolders,
and Muylaert 2015; Weldy and Huesemann 2007). Nonetheless, a decrease of sCOD along
nitrogen was revealed at N=15-10 mg/L, probably owing to restricted photosynthesis results
from extreme nitrogen limitation causing inadequate protein and chlorophyll production
(Lin et al. 2011). Moreover, phosphate concentration might also be negatively influential to
sCOD when below 5 mg/L, which could be related to carbohydrate production when
extracellular phosphorus was deprived (Beuckels, Smolders, and Muylaert 2015; Brányiková
et al. 2011; Dragone et al. 2011).
The endpoint of effluent polishing is usually required to have low nutrients concentration, at
which C-rich metabolites like carbohydrates and lipids could be accumulated and released
into treated water, potentially exceeding the COD limit for discharge. Since biomass
production is the driving force in nutrients removal by assimilation and hence inevitably,
further research on a solution to minimize sCOD is probably necessitated.
60
Fig. 18 COD change against initial N and initial P for synthetic secondary effluent polishing
Fig. 19 Specific COD change against initial N and initial P for synthetic secondary effluent polishing
61
Fig. 20 HEM for synthetic secondary effluent polishing (measured for batches with initial N=10 mg/L)
HEM tests performed for batches with initial N=10 mg/L, as a nitrogen limiting condition
for potential lipid accumulation, are delineated in Fig. 20, with data shown in Table. 6. HEM
in the 30nm filtrate composed the major part of dissolved HEM (in 0.45µm filtrate), while
dissolved HEM occupied more than half of total HEM, with an exception at P=10 mg/L
where nearly 90% of total HEM was dissolved.
The tendency of three types of HEM amounts similarly to dCOD (at N=10 mg/L) that also
peaked at P=8 mg/L. The drop of total HEM at P=10 mg/L was mainly precipitated by the
particulate fraction or cellular content. Since the bottom of nutrients removal ratio (Fig. 12)
was in proximity where P=10 mg/L, together with the peak of dCOD and generation time,
this phenomenon might be induced by the metabolic shifting of algal cells. It has been
62
reported that under nutrients limitation, algal cells will replenish their internal carbohydrate
reservoirs, which could be later secreted as EPS (Barranguet et al. 2005; De Brouwer and
Stal 2002; Markou 2015, 2012). Moreover, when nutrients stress is more severe,
photosynthetic products would switch to lipids, as well as internally stored carbohydrate
would be converted to lipids (Li, Fei, and Deng 2012; Rodolfi et al. 2009; Roessler 1990), as
shown in Fig. 14. Therefore, microalgae that actually experienced nitrogen deficiency for
having lowest nutrients removal ratio (sN/sP) might have decreased cellular carbohydrate
content but maintained lipid content which was more soluble than its precursor.
Considering hexane as a nonpolar solvent is not effective in algal lipids extraction (Ranjan,
Patil, and Moholkar 2010), analysis of each fraction of HEM in the same bar may not be
meaningful for their solubility difference in both washing and eluting solvents. However, it
should be noted that even if algal lipids counted in oil and grease are less extractable by
hexane, for most cases filtered or dissolved HEM still exceed 5 mg/L as a discharge limit
(PME 2001), hazarding the applicability of effluent polishing via oleaginous microalgae or
conceptual wastewater treatment process combined with biodiesel production.
Table. 6 n-Hexane Extractable Material (Oil and Grease, in mg/L)
Syn. Batches Initiated with N=10 mg/L AnMBR.Eff Sym.
Initial P mg/L 2.00 4.00 6.00 8.00 10.00 10.00 26.24
Total 7.280 10.220 14.380 14.850 8.010 10.277 5.082
Dissolved 4.080 5.960 8.470 8.319 7.193 4.540 3.581
30nm Filtrate 3.890 5.770 8.040 8.190 7.011 2.174 3.156
LC-OCD results for treated synthetic secondary effluent with initial N=10 mg/L, P=8
mg/L is presented in Fig. 21, where 0.45 µm permeate from parallel batches showed good
similarity. By comparison, 30nm UF membrane showed partial rejection for humics, and
63
significant rejection for the biopolymers monopeak that mainly consisted of polysaccharides
for intense OCD but weak OND signal. A peak for low molecular weight acids was
reproduced in 0.45 µm permeate. This might be attributed to TAG (around 850 Dalton)
release by cell disruption during manual filtration by syringe filter, since shear stress around
1Pa could largely affect microalgae viability (Michels et al. 2010; Leupold et al. 2013).
3.2.2 COD, HEM and LC-OCD Analysis in AnMBR Effluent Polishing
From Table. 4, both COD and sCOD in the effluent of AnMBR polishing were higher than
those in synthetic anaerobic secondary effluent polishing, while the ratio of filtered HEM
over dissolved HEM in Table. 6 decreased if compared with those fed with N equals to 10
mg/L. Microalgae cultivated in real wastewater is often observed having higher C-rich
metabolites content than those treating synthetic wastewater (Órpez et al. 2009). Thus, this
case might be enhanced by lower VSS and less shading. The elevated secretion of EPS
composed mainly of polysaccharide might have happened as a response to inhibitory
compounds generated by AnMBR (Holmstrom and Kjelleberg 2000).
LC-OCD results for treated AnMBR effluent is presented in Fig. 22. After cultivation carried
on with ammonium removal, 0.45 µm filtrate contained more biopolymers (mainly
polysaccharides with wider spectrum, hinting more complicated metabolic pathways as
mentioned ahead) and humics than original AnMBR effluent, while this fraction was rejected
by 30 nm UF. Again, a peak of low molecular weight acid probably representing TAG
occurred.
64
Fig. 21 LC-OCD results for treated synthetic secondary effluent (with initial N=10 mg/L, P=8 mg/L)
65
3.2.3 COD, HEM and LC-OCD Analysis in Symbiotic Bioremediation
From Table. 5, COD was reduced from 282.35 to 121.03 mg/L during the given 8days,
revealing inefficiency of this symbiotic system in COD removal as a balance between
microalgae and activated sludge. From Table. 6, HEMs were relatively lower, which could be
related to ample residual N and P that were not stimulating HEM production.
LC-OCD results for treated synthetic primary effluent is presented in Fig. 23. The peak of
nitrate induced by nitrification was observed. Biopolymers (consisted of both
polysaccharides and proteins as indicated by OCD and OND) together with fractional
humics remained in the 0.45 µm permeate could be rejected by 30 nm UF. Signal of
polymers are also multimodal and different from the one of pure algal cultivation, showing
algal metabolism affected by seeded sludge or EPS produced by bacteria. The suspected
TAG peak didn’t appear here, which was in accordance with HEM results because lipid
production was not stimulated in this batch.
66
Fig. 22 LC-OCD results for treated AnMBR effluent
Bio
poly
mer
s
Hum
ics
Bui
ldin
g B
lock
s
LMW
Aci
ds a
nd H
S
LMW Neutrals
Nitr
ate
Ure
a
Am
mon
ium
Byp
ass
0
1
2
3
4
5
6
7
8
9
10
11
12
0 30 60 90 120 150 180
rel.
Sig
nal R
espo
nse
Retention Time in Minutes
---An.Treated,0.03 ---An.Treated,0.45 ---AnMBR.Eff
OND
UVDX10
OCDX5
67
Fig. 23 LC-OCD results for treated synthetic raw municipal wastewater
3.3 Particle Size Distribution and Critical Flux
Particle size distribution of pure algae culture, activated sludge and symbiotic mixed liquor
are shown in Fig. 24. Their PSD were all above one micron, justifying complete cell removal
of microalgae and activated sludge by UF. Besides, the bimodal shape curve in symbiotic
68
system indicated that either mixed flocs, namely algal/bacterial aggregates expected to be
larger than 500 µm (Alcantara et al. 2015), didn’t appear, or flocs were still dominated by
activated sludge.
Fig. 24 Particle size distribution
3.3.1 Critical Flux in Synthetic Effluent Polishing
Critical flux was contoured in Fig. 25, ranging from 18-39 LMH strongly resembling Fig. 6
of VSS. Critical Flux was limited majorly by phosphorus when P<5 mg/L, and by nitrogen
in the rest region, with the bottom located around N=40, P=7 mg/L.
A plot between critical flux and VSS is drawn in Fig. 26, demonstrating a strong relationship.
It was noted that for short term UF, microalgae cell was the major resistance contributor
acting as a reversible fouling layer (Castaing et al. 2011).
0
2
4
6
8
10
12
14
16
0.1 1 10 100 1000
Vol
ume
%
Diamter µm
Algae&Sludge
Algae
Sludge
69
Fig. 25 Critical flux for synthetic secondary effluent polishing
Fig. 26 Relationship between critical flux and VSS
y = -0.0745x + 42.107 R² = 0.9134
10
15
20
25
30
35
40
45
50
50 100 150 200 250 300 350 400
Crit
ical
flux
LM
H
VSS mg/L
Critical Flux vs VSS
70
3.3.2 Critical Flux in AnMBR Effluent Polishing
From Table 4, critical flux reached 28 LMH, slightly deviating from the value in synthetic
cultivation as 26 LMH, which was reasonable since VSS as 166.28 mg/L lower than
predicted value as 183.15 mg/L.
3.3.3 Critical Flux in Symbiotic Bioremediation
From Table 5, critical flux reached 17 LMH at VSS=384.6 mg/L, though the one calculated
via linear fitted equation in Fig. 26 was 13 LMH. Accordingly, the presence of activated
sludge instead of pure algal cells might be helpful in alleviating fouling, or larger particulates
tend to form less dense cake layer in such short term UF thus allowing higher flux.
71
4. Conclusion
In the synthetic anaerobic secondary effluent polishing scenario, nutrients remediation by
microalgae was dominated by assimilation. Nitrogen removal rate was directly affected by
biomass production rate instead of specific nitrogen removal rate, while phosphorus removal
rate was influenced by both cell growth and specific phosphorus removal rate. Cell growth
was limited by initial P when it's less than 5 mg/L, or by initial N in the rest region though
higher concentration may lead to ammonia toxicity. Specific nutrients removal rate were
interrelated sign of cell activity, and were indirectly affected by cell production, rendering a
nutrients removal ratio restricted by initial P when lower than 5 mg/L, or by initial N and
shading effect when P>5 mg/L. As a whole, residual nitrogen was largely below 15 mg/L
and was controlled by initial P only when N>20 mg/L, P<5 mg/L. However, residual
phosphorus was largely over 2 mg/L and was affected by initial N only when N>25 mg/L,
suggesting weakness of C.Vulgaris in phosphorus removal (Beuckels, Smolders, and Muylaert
2015). COD increase as consistent with HEM analysis in synthetic anaerobic secondary
effluent polishing was clearly related to VSS and sCOD, while the type of carbon-rich
metabolites shifted according to nutrients stress facing.
AnMBR effluent polishing batch test displayed comparable results to batch feeding synthetic
secondary effluent, with the former possessing lower cell production, higher specific
nutrients removal and higher sCOD, which might be attributed to inhibitory compounds in
AnMBR effluent and metabolic pathway change.
72
Symbiotic bioremediation yielded lower cell production and nitrogen source competition
between microalgae and activated sludge. Nonetheless, the specific phosphorus removal rate
was improved to be higher than luxury uptake threshold of microalgae.
Critical flux was closely related to algal cell concentration. UF with a pore size of 30nm
achieved complete cell removal (as endorsed by PSD results) and rejection of most
biopolymers (as detailed by LC-OCD results), though a suspecting TAG peak occurred due
to cell disruption during manual MF.
Since assimilation is the primary mechanism for nutrients removal in algal processes,
maximizing cell productivity, optimization of sN, sP in balance with minimization of sCOD
are worthy of further research. HEM results, though underestimating lipid concentration,
imply that effluent polishing via oleaginous microalgae or conceptual wastewater treatment
process combined with biodiesel production may deserve reconsideration.
As LCA favors open pond in which symbiosis with bacteria is inevitable, treatment of COD
by activated sludge may be sensible. Further experiments would utilize rotating biological
contactor to minimize aeration and footprint while enhancing system robustness via biofilm
process (Katarzyna, Sai, and Singh 2015; Blanken et al. 2014). Adapted species would be
collected from real secondary clarifier walls (Olguín 2012; Jimenez-Perez et al. 2004). Diurnal
changes would be incorporated for microalgal treatment capacity varies with light availability
(Boelee et al. 2012).
74
5. Appendices
5.1 MATLAB Codes for Data and Plotting
data =[ %Init.N Init.P Init.COD Init.VSS Fnl.N Fnl.P Fnl.COD Fnl.VSS Fnl.CF
T.HEM, 45.HEM, 03.HEM 10.0000 2.0000 26.2400 40.0000 7.2900 1.9100 41.5800 69.1000 39.4300
7.2800 4.0800 3.8900 10.0000 4.0000 27.5200 40.0000 4.6400 3.7200 55.9100 96.4000 34.2600
10.2200 5.9600 5.7700 10.0000 6.0000 28.2200 40.0000 0.4800 5.3200 71.6000 134.1000 32.6200
14.3800 8.4700 8.0400 10.0000 8.0000 27.6600 40.0000 0.7700 7.2200 75.3700 137.1000 30.1900
14.8500 8.3190 8.1900 10.0000 10.0000 31.0000 40.0000 3.1300 9.4400 74.7700 132.8000 30.6000
8.0100 7.1930 7.0110 20.0000 2.0000 28.1400 40.0000 16.0900 1.8900 59.4500 75.9000 37.7300
NaN NaN NaN 20.0000 4.0000 31.5500 40.0000 7.1300 3.4100 79.3800 138.2000 30.4800
NaN NaN NaN 20.0000 6.0000 29.1000 40.0000 4.5200 5.2300 88.9200 142.0000 30.1200
NaN NaN NaN 20.0000 8.0000 31.6600 40.0000 5.9000 7.0300 94.8700 140.5000 31.0700
NaN NaN NaN 20.0000 10.0000 25.9300 40.0000 8.3800 9.3000 88.0000 139.3000 31.4100
NaN NaN NaN 30.0000 2.0000 24.2600 40.0000 25.0300 1.8700 39.6800 80.8000 38.2500
NaN NaN NaN 30.0000 4.0000 28.4800 40.0000 12.7200 3.2100 48.8300 157.2000 30.1100
NaN NaN NaN 30.0000 6.0000 31.0500 40.0000 5.0000 4.5300 60.4700 205.2000 24.5600
NaN NaN NaN 30.0000 8.0000 29.3500 40.0000 0.1200 5.8400 46.6800 228.3000 23.1300
NaN NaN NaN 30.0000 10.0000 25.5200 40.0000 3.5500 8.8000 47.7300 189.4000 25.4200
NaN NaN NaN 40.0000 2.0000 29.4000 40.0000 34.5800 1.8600 36.4900 82.2000 37.3900
NaN NaN NaN 40.0000 4.0000 26.3100 40.0000 11.8900 2.3500 57.9700 272.8000 25.6800
NaN NaN NaN 40.0000 6.0000 29.3700 40.0000 4.1700 3.0100 69.1800 338.6000 16.6300
NaN NaN NaN 40.0000 8.0000 29.5600 40.0000 1.0500 4.3600 73.4900 370.9000 17.6700
NaN NaN NaN 40.0000 10.0000 24.5400 40.0000 6.1900 7.4400 54.5000 295.6000 22.8800
NaN NaN NaN
75
50.0000 2.0000 30.2400 40.0000 45.4900 1.8800 45.4600 75.9000 39.4700 NaN NaN NaN
50.0000 4.0000 29.4000 40.0000 17.2400 2.5300 32.2900 198.1000 26.2100 NaN NaN NaN
50.0000 6.0000 24.0500 40.0000 11.8300 3.4800 34.6800 232.5000 22.2800 NaN NaN NaN
50.0000 8.0000 28.8200 40.0000 9.2500 4.5100 34.8000 272.8000 20.2600 NaN NaN NaN
50.0000 10.0000 27.0900 40.0000 9.8900 7.6500 35.2200 251.9000 22.7000 NaN NaN NaN
]; n=9:1:51;p=1.8:.2:10.2;[xq,yq]=meshgrid(n,p); Init.N=data(:,1);Init.P=data(:,2);Init.COD=data(:,3);Init.VSS=data(:,4); Fnl.N=data(:,5);Fnl.P=data(:,6);Fnl.COD=data(:,7);Fnl.VSS=data(:,8); Fnl.CF=data(:,9); %Mesh Contour for Fnl.VSS,GenerationTime, %Fnl.VSS zq=griddata(Init.N,Init.P,Fnl.VSS,xq,yq,'v4'); figure();hold on;grid on; contour(xq,yq,zq,60:30:360,'ShowText','on','LineColor','k','LineWidth',1); title('Final VSS mg/L');xlabel('Initial N mg/L');ylabel('Initial P mg/L'); %Gen.Time GT=8*log(2)./(log(data(:,8))-log(data(:,4))); zq=griddata(Init.N,Init.P,GT,xq,yq,'v4'); figure();hold on;grid on; contour(xq,yq,zq,2:1:11,'ShowText','on','LineColor','k','LineWidth',1); title('Generation Time d');xlabel('Initial N mg/L');ylabel('Initial P mg/L'); %Mesh Contour for Fnl.N,NR=dN/Init.N,Fnl.P,PR=dP/Init.P %Fnl.N zq=griddata(Init.N,Init.P,Fnl.N,xq,yq,'v4'); figure();hold on;grid on; contour(xq,yq,zq,0:5:50,'ShowText','on','LineColor','k','LineWidth',1); title('Final N mg/L');xlabel('Initial N mg/L');ylabel('Initial P mg/L'); %NR=dN/Init.N NR=100.*(Init.N-Fnl.N)./Init.N; zq=griddata(Init.N,Init.P,NR,xq,yq,'v4'); figure();hold on;grid on; contour(xq,yq,zq,0:10:100,'ShowText','on','LineColor','k','LineWidth',1); title('N Removal %');xlabel('Initial N mg/L');ylabel('Initial P mg/L'); %Fnl.P zq=griddata(Init.N,Init.P,Fnl.P,xq,yq,'v4'); figure();hold on;grid on; contour(xq,yq,zq,0:1:10,'ShowText','on','LineColor','k','LineWidth',1); title('Final P mg/L');xlabel('Initial N mg/L');ylabel('Initial P mg/L'); %PR=dP/Init.P PR=100.*(Init.P-Fnl.P)./Init.P;
76
zq=griddata(Init.N,Init.P,PR,xq,yq,'v4'); figure();hold on;grid on; contour(xq,yq,zq,0:5:100,'ShowText','on','LineColor','k','LineWidth',1); title('P Removal %');xlabel('Initial N mg/L');ylabel('Initial P mg/L'); %Mesh Contour for sN=dN/dVSS,sP=dP/dVSS,dN/dP %sN=dN/dVSS sN=(Init.N-Fnl.N).*100./(Fnl.VSS-Init.VSS); zq=griddata(Init.N,Init.P,sN,xq,yq,'v4'); figure();hold on;grid on; contour(xq,yq,zq,0:2:22,'ShowText','on','LineColor','k','LineWidth',1); title('sN %');xlabel('Initial N mg/L');ylabel('Initial P mg/L'); %sP=dP/dVSS sP=(Init.P-Fnl.P).*100./(Fnl.VSS-Init.VSS); zq=griddata(Init.N,Init.P,sP,xq,yq,'v4'); figure();hold on;grid on; contour(xq,yq,zq,0:.2:2,'ShowText','on','LineColor','k','LineWidth',1); title('sP %');xlabel('Initial N mg/L');ylabel('Initial P mg/L'); %dN/dP NPR=(Init.N-Fnl.N)./(Init.P-Fnl.P); zq=griddata(Init.N,Init.P,NPR,xq,yq,'v4'); figure();hold on;grid on; contour(xq,yq,zq,10:4:38,'ShowText','on','LineColor','k','LineWidth',1); title('sN/sP');xlabel('Initial N mg/L');ylabel('Initial P mg/L'); %Mesh Contour for dCOD,dCOD/dVSS,Crit.Flux %dCOD dCOD=Fnl.COD-Init.COD; zq=griddata(Init.N,Init.P,dCOD,xq,yq,'v4'); figure();hold on;grid on; contour(xq,yq,zq,0:10:70,'ShowText','on','LineColor','k','LineWidth',1); title('dCOD mg/L');xlabel('Initial N mg/L');ylabel('Initial P mg/L'); %sCOD=dCOD/dVSS sCOD=dCOD./(Fnl.VSS-Init.VSS); zq=griddata(Init.N,Init.P,dCOD./(Fnl.VSS-Init.VSS),xq,yq,'v4'); figure();hold on;grid on; contour(xq,yq,zq,0:.1:1,'ShowText','on','LineColor','k','LineWidth',1); title('sCOD');xlabel('Initial N mg/L');ylabel('Initial P mg/L'); %Crit.Flux zq=griddata(Init.N,Init.P,Fnl.CF,xq,yq,'v4'); figure();hold on;grid on; contour(xq,yq,zq,0:3:40,'ShowText','on','LineColor','k','LineWidth',1); title('Critical Flux LMH');xlabel('Initial N mg/L');ylabel('Initial P mg/L'); %Plot Init.Ratio vs dN/Inf.N,dP/Inf.P; dN/dVSS vs dP/dVSS; Crit.Flux vs VSS; %Init.Ratio vs NR,PR figure();
77
Init.Ratio=Init.N./Init.P; plot(Init.Ratio,NR./100,'^r'); hold on; plot(Init.Ratio,PR./100,'vb'); title('Nutrients Removal vs Initial Nutrients Ratio'); xlabel('Initial N/P Ratio');ylabel('Removal Rate');legend('N Removal Rate','P Removal
Rate'); hold on;grid on; plot([.9;14;1;.9],[1.1;.65;.5;1.1],'--r'); plot([5;13.5;.58;5],[.52;.37;.02;.52],'--b'); %dN/dVSS vs dP/dVSS figure(); x=(Init.N-Fnl.N).*100./(Fnl.VSS-Init.VSS); y=(Init.P-Fnl.P).*100./(Fnl.VSS-Init.VSS); plot(x,y,'sk'); [sN_sPfit,gof]=fit(x,y,'poly1'); display(sN_sPfit);display(gof); lsline;hold on;grid on;
annotation('textarrow',[.38,.5],[.76,.47],'String','y=0.05703*x+0.006252,R^2=0.3678','FontSize',15);
title('sP vs sN'); xlabel('sN %');ylabel('sP %'); %Crit.Flux vs VSS figure(); plot(Fnl.VSS,Fnl.CF,'dk'); [CF_VSSfit,gof]=fit(Fnl.VSS,Fnl.CF,'poly1'); display(CF_VSSfit);display(gof); lsline;hold on;grid on; annotation('textarrow',[.56,.46],[.7,.46],'String','y=-
0.07451*x+42.11,R^2=0.9134','FontSize',15); title('Critical Flux vs VSS'); xlabel('VSS mg/L');ylabel('Critical Flux LMH'); axis([50,400,15,45]); %BarPlot Oil&Grease(HEM) O_T_45=data(1:5,10)-data(1:5,11); O_45_03=data(1:5,11)-data(1:5,12); figure(); bar([O_T_45,O_45_03,data(1:5,12)],'stacked'); xlabel('Initial P mg/L');ylabel('HEM mg/L'); legend('>0.45\mum','0.03-0.45\mum','<0.03\mum'); set(gca,'xticklabel',2:2:10,'ytick',0:3:15); %Summary Boxplot figure(); boxplot([GT,data(:,[1,2,5,6]),NR,PR,sN,sP,Init.Ratio,NPR,data(:,[3,7]),sCOD.*100,data(:,9
)]);
78
title('Summary Boxplot'); set(gca,'XLim',[.5,15.5],'YLim',[0,100],'xticklabel',... 'Gen.Time','Init.N','Init.P','Fnl.N','Fnl.P',... 'Rmvl.N','Rmvl.P','sN','sP','Init.Ratio','Rmvl.Ratio',... 'Init.COD','Fnl.COD','sCOD','Fnl.CF','YMinorTick','on');
5.2 Summary of Synthetic Anaerobic Secondary Effluent Polishing
Fig. 27 Summary of synthetic anaerobic secondary effluent polishing
79
Table. 7 Summary of synthetic anaerobic secondary effluent polishing
Init VSS mg/L
Fnl VSS mg/L
Gen Time
d
Init N mg/L
Init P mg/L
Fnl N mg/L
Fnl P
mg/L
N Rmvl %
P Rmvl % sN % sP % Init
Ratio Rmvl Ratio
Init COD mg/L
Fnl COD mg/L
sCOD Fnl. CF
LMH
T. HEM mg/L
0.45 HEM mg/L
0.03 HEM mg/L
40.00 69.10 10.14 10.00 2.00 7.29 1.91 27.10 4.50 9.31 0.31 5.00 30.11 26.24 41.58 0.53 39.43 7.280 4.080 3.890 40.00 96.40 6.30 10.00 4.00 4.64 3.72 53.60 7.00 9.50 0.50 2.50 19.14 27.52 55.91 0.50 34.26 10.220 5.960 5.770 40.00 134.10 4.58 10.00 6.00 0.48 5.32 95.20 11.33 10.12 0.72 1.67 14.00 28.22 71.60 0.46 32.62 14.380 8.470 8.040 40.00 137.10 4.50 10.00 8.00 0.77 7.22 92.30 9.75 9.51 0.80 1.25 11.83 27.66 75.37 0.49 30.19 14.850 8.319 8.190 40.00 132.80 4.62 10.00 10.00 3.13 9.44 68.70 5.60 7.40 0.60 1.00 12.27 31.00 74.77 0.47 30.60 8.010 7.193 7.011 40.00 75.90 8.66 20.00 2.00 16.09 1.89 19.55 5.50 10.89 0.31 10.00 35.55 28.14 59.45 0.87 37.73 - - - 40.00 138.20 4.47 20.00 4.00 7.13 3.41 64.35 14.75 13.11 0.60 5.00 21.81 31.55 79.38 0.49 30.48 - - - 40.00 142.00 4.38 20.00 6.00 4.52 5.23 77.40 12.83 15.18 0.75 3.33 20.10 29.10 88.92 0.59 30.12 - - - 40.00 140.50 4.41 20.00 8.00 5.90 7.03 70.50 12.13 14.03 0.97 2.50 14.54 31.66 94.87 0.63 31.07 - - - 40.00 139.30 4.44 20.00 10.00 8.38 9.30 58.10 7.00 11.70 0.70 2.00 16.60 25.93 88.00 0.63 31.41 - - - 40.00 80.80 7.89 30.00 2.00 25.03 1.87 16.57 6.50 12.18 0.32 15.00 38.23 24.26 39.68 0.38 38.25 - - - 40.00 157.20 4.05 30.00 4.00 12.72 3.21 57.60 19.75 14.74 0.67 7.50 21.87 28.48 48.83 0.17 30.11 - - - 40.00 205.20 3.39 30.00 6.00 5.00 4.53 83.33 24.50 15.13 0.89 5.00 17.01 31.05 60.47 0.18 24.56 - - - 40.00 228.30 3.18 30.00 8.00 0.12 5.84 99.60 27.00 15.87 1.15 3.75 13.83 29.35 46.68 0.09 23.13 - - - 40.00 189.40 3.57 30.00 10.00 3.55 8.80 88.17 12.00 17.70 0.80 3.00 22.04 25.52 47.73 0.15 25.42 - - - 40.00 82.20 7.70 40.00 2.00 34.58 1.86 13.55 7.00 12.84 0.33 20.00 38.71 29.40 36.49 0.17 37.39 - - - 40.00 272.80 2.89 40.00 4.00 11.89 2.35 70.28 41.25 12.07 0.71 10.00 17.04 26.31 57.97 0.14 25.68 - - - 40.00 338.60 2.60 40.00 6.00 4.17 3.01 89.58 49.83 12.00 1.00 6.67 11.98 29.37 69.18 0.13 16.63 - - - 40.00 370.90 2.49 40.00 8.00 1.05 4.36 97.38 45.50 11.77 1.10 5.00 10.70 29.56 73.49 0.13 17.67 - - - 40.00 295.60 2.77 40.00 10.00 6.19 7.44 84.53 25.60 13.23 1.00 4.00 13.21 24.54 54.50 0.12 22.88 - - - 40.00 75.90 8.66 50.00 2.00 45.49 1.88 9.02 6.00 12.56 0.33 25.00 37.58 30.24 45.46 0.42 39.47 - - - 40.00 198.10 3.47 50.00 4.00 17.24 2.53 65.52 36.75 20.72 0.93 12.50 22.29 29.40 32.29 0.02 26.21 - - - 40.00 232.50 3.15 50.00 6.00 11.83 3.48 76.34 42.00 19.83 1.31 8.33 15.15 24.05 34.68 0.06 22.28 - - - 40.00 272.80 2.89 50.00 8.00 9.25 4.51 81.50 43.63 17.50 1.50 6.25 11.68 28.82 34.80 0.03 20.26 - - - 40.00 251.90 3.01 50.00 10.00 9.89 7.65 80.22 23.50 18.93 1.11 5.00 17.07 27.09 35.22 0.04 22.70 - - -
80
5.3 EPA Method 1664A–Extraction of Oil and Grease from Water Samples Using
Solid-Phase Extraction (SPE) Cartridge Configuration, Application Note 817 (Eric Francis
2012)
5.3.1 Introduction
EPA Method 1664, Revision A, n-Hexane Extractable Material (HEM; Oil and Grease) and Silica Gel-Treated n-Hexane Extractable Material (SGT-HEM; Non-polar Material) by Extraction and Gravimetry, describes the determination of oil and grease from liquid (typically water) samples. Liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are both proven techniques that meet the requirements of EPA 1664A. Although generally effective, LLE is a very time-consuming, labor-intensive technique that uses large amounts of solvent. SPE is a good alternative to liquid-liquid extraction. The SPE technique provides a more reliable, less labor-intensive solution to liquid extraction, and uses far less solvent than LLE. Also, emulsion formation is eliminated by using SPE. The Thermo Scientific™ Dionex™ AutoTrace™ 280 Solid-Phase Extraction instrument automates the conditioning, rinsing, loading, drying, and extraction steps and also can simultaneously process 1 to 6 samples. The Dionex AutoTrace 280 instrument is available in both disk and cartridge configurations to suit the needs of every laboratory. 5.3.2 Equipment
• Dionex AutoTrace 280 Automated Large Volume Solid-Phase Extraction instrument for a 6 mL cartridge (P/N 071385) • pH paper 5.3.3 Solvents
• Hexane, pesticide-grade or equivalent (Fisher Scientific) • Methanol (CH3OH), pesticide-grade or equivalent (Fisher Scientific) • Water acidified to pH 2, 18 MMilli-Q® water system (Millipore) • Tetrahydrofuran (THF), pesticide-grade or equivalent (Fisher Scientific) • Water, 18 MΩ, Milli-Q water system (Millipore) • Hydrochloric acid (HCL) 5.3.4 SPE Material
Thermo Scientific™ Dionex™ SolEx™ SPE cartridge (P/N 074410), 6 mL, C18 Dionex SolEx cartridge with 1.0 g of packing 5.3.5 Oil and Grease Cartridge Configuration Method
Place required number of samples (1–6) in the sample vial rack. Allow all samples to reach room temperature and adjust the pH of each sample to 2 using 6 M HCL. Insert sample lines into each sample bottle. Collection:
81
Label the required number of collection vials (1–6) and place these into the collection rack. Solvents: Add methanol to solvent bottle 1, water (pH=2) to solvent bottle 2, hexane/THF (1:1) to solvent bottle 3, hexane to solvent bottle 4, and water to solvent bottle 5. Place these solvent bottles to the left side of the Dionex AutoTrace instrument and insert the solvent lines into the corresponding bottle (up to five different solvents can be used with the Dionex AutoTrace instrument). SPE Media: Insert SPE cartridges onto the Dionex AutoTrace instrument (see Dionex (now part of Thermo Scientific) AutoTrace 280 Operation Manual for details1) and secure the cartridge into place using the cartridge holder. The green LED will be illuminated when the cartridge is locked into place. Standard Solutions: Standard stock solution: 400 mg each of hexadecane and stearic acid into 100 mL acetone. Standard acidification solution: equal parts concentrated HCl (12 M) and water to obtain 6 M HCl. Sample Preparation (six samples): Dispense 1 l of water from the Milli-Q water system. Add 5 mL of the standard stock solution and mix gently. Add 10 mL of CH3OH and mix gently. Add 3 mL of 6 M HCl and mix gently. Program, download, save, and run the following method to the Dionex AutoTrace 280 Solid-Phase Extraction instrument, and enter the following parameters for the extraction method under the Params. Tab:
Table. 8 Method for the SPE instrument No. Method User Intervention 1 Process 6 samples using the following method steps 2 Wash syringe with 3.0 mL CH3OH 3 Condition cartridge with 5.0 mL hexane into solvent waste 4 Condition cartridge with 5.0 mL hexane into solvent waste 5 Dry cartridge with gas for 5.0 min 6 Condition cartridge with 5.0 mL CH3OH into solvent waste 7 Condition cartridge with 5.0 mL water, pH 2 into aqueous waste 8 Load 1200.0 mL of sample onto cartridge 9 Dry cartridge with gas for 60.0 min 10 Manually rinse sample container with 30.0 mL to collect Rinse sample bottles with 10 mL hexane 11 Pause and alert operator, resume when CONTinue is pressed Fraction#1. Place new vials in collection rack 12 Concentrate sample with gas for 10.0 min 13 Wash syringe with 3.0 mL CH3OH 14 Soak and collect 4.0 mL fraction using hexane THF (1:1) 15 Pause for 2.0 min 16 Collect 3.0 mL fraction into sample tube using hexane/THF (1:1) 17 Soak and collect 3.0 mL fraction using hexane/THF (1:1) 18 Pause for 2.0 min 19 Collect 3.0 mL fraction into sample tube using hexane/THF (1:1) Fraction #2. Combine fractions 1 and 2. Evaporate
solvent for gravimetric determination.
82
20 End
Table. 9 Parameters for the SPE method Flow Rates SPE Parameters
Cond Flow 10 mL/min Push Delay 5.0 s Load Flow 10 mL/min Air Factor 1.0 Rinse Flow 10 mL/min Autowash Vol. 2.00 mL Elute Flow 5 mL/min
Cond Air Push 10 mL/min Rinse Air Push 10 mL/min Elute Air Push 5 mL/min
Estimated time: 4 h 12 min (for 6 samples), Estimated solvent used: 25 mL
Save and download this program on the Dionex AutoTrace instrument for future use. Note:
Up to 24 different programs can be stored on the Dionex AutoTrace instrument. These
methods can be written and transferred to the Dionex AutoTrace instrument using a
computer. Once the desired methods are stored on the Dionex AutoTrace instrument, there
is no further need for the Dionex AutoTrace instrument to be connected to the computer.
5.3.6 Conclusion
The Dionex AutoTrace 280 Solid-Phase Extraction instrument with the cartridge
configuration provides an automated, solid-phase extraction solution that replaces traditional
liquid-liquid extraction and meets the requirements of EPA 1664, Revision A.
5.3.7 References
1. Dionex Corporation (now part of Thermo Scientific), Dionex AutoTrace 280 Operator’s
Manual, Doc. 065330-01, Sunnyvale, CA.
5.3.8 List of Manufacturers
Dionex Corporation (now part of Thermo Scientific), 1228 Titan Way, P.O. Box 3603,
Sunnyvale, CA 94088-3603, http://www.dionex.com
83
Fisher Scientific, 2000 Park Lane, Pittsburgh, PA, 15275-1126, Tel: 800-766-7000,
www.fishersci.com
84
REFERENCES
Abeliovich, A. 1986. 'Algae in wastewater oxidation ponds', Handbook of Microalgal Mass Culture, 35: 331-38.
Abeliovich, AHARON, and Yosef Azov. 1976. 'Toxicity of ammonia to algae in sewage oxidation ponds', Applied and environmental microbiology, 31: 801-06.
Abeliovich, Aharon, and Sarah Dikbuck. 1977. 'Factors affecting infection of Scenedesmus obliquus by a Chytridium sp. in sewage oxidation ponds', Applied and environmental microbiology, 34: 832-36.
Ahluwalia, Sarabjeet Singh, and Dinesh Goyal. 2007. 'Microbial and plant derived biomass for removal of heavy metals from wastewater', Bioresource technology, 98: 2243-57.
Alcantara, C., J. M. Dominguez, D. Garcia, S. Blanco, R. Perez, P. A. Garcia-Encina, and R. Munoz. 2015. 'Evaluation of wastewater treatment in a novel anoxic-aerobic algal-bacterial photobioreactor with biomass recycling through carbon and nitrogen mass balances', Bioresour Technol, 191: 173-86.
Alcántara, Cynthia, Jesús M Domínguez, Dimas García, Saúl Blanco, Rebeca Pérez, Pedro A García-Encina, and Raúl Muñoz. 2015. 'Evaluation of wastewater treatment in a novel anoxic–aerobic algal–bacterial photobioreactor with biomass recycling through carbon and nitrogen mass balances', Bioresource technology, 191: 173-86.
An, Jin-Young, Sang-Jun Sim, Jin Suk Lee, and Byung Woo Kim. 2003. 'Hydrocarbon production from secondarily treated piggery wastewater by the green alga Botryococcus braunii', Journal of Applied Phycology, 15: 185-91.
Aslan, Sebnem, and Ilgi Karapinar Kapdan. 2006. 'Batch kinetics of nitrogen and phosphorus removal from synthetic wastewater by algae', Ecological Engineering, 28: 64-70.
Aziz, MA, and WJ Ng. 1993. 'Industrial wastewater treatment using an activated algae-reactor', Water science and technology, 28: 71-76.
Azov, Y, and Joel C Goldman. 1982. 'Free ammonia inhibition of algal photosynthesis in intensive cultures', Applied and environmental microbiology, 43: 735-39.
Barbosa, Maria J, and René H Wijffels. 2004. 'Overcoming shear stress of microalgae cultures in sparged photobioreactors', Biotechnology and bioengineering, 85: 78-85.
Barranguet, Christiane, Bart Veuger, Sebastien AM Van Beusekom, Peter Marvan, Jan J Sinke, and Wim Admiraal. 2005. 'Divergent composition of algal-bacterial biofilms developing under various external factors', European Journal of Phycology, 40: 1-8.
Barros, Ana I, Ana L Gonçalves, Manuel Simões, and José CM Pires. 2015. 'Harvesting techniques applied to microalgae: A review', Renewable and sustainable energy reviews, 41: 1489-500.
85
Benemann, John R. 1997. 'CO 2 mitigation with microalgae systems', Energy Conversion and Management, 38: S475-S79.
Benemann, John R, and William J Oswald. 1996. "Systems and economic analysis of microalgae ponds for conversion of CO sub 2 to biomass. Final report." In.: California Univ., Berkeley, CA (United States). Dept. of Civil Engineering.
Benemann, John R, Joseph C Weissman, Ben L Koopman, and William J Oswald. 1977. 'Energy production by microbial photosynthesis', Nature, 268: 19-23.
Besemer, Katharina, Gabriel Singer, Romana Limberger, Ann-Kathrin Chlup, Gerald Hochedlinger, Iris Hödl, Christian Baranyi, and Tom J Battin. 2007. 'Biophysical controls on community succession in stream biofilms', Applied and environmental microbiology, 73: 4966-74.
Beuckels, Annelies, Erik Smolders, and Koenraad Muylaert. 2015. 'Nitrogen availability influences phosphorus removal in microalgae-based wastewater treatment', Water research, 77: 98-106.
Bhatnagar, Ashish, Monica Bhatnagar, Senthil Chinnasamy, and KC Das. 2010. 'Chlorella minutissima—a promising fuel alga for cultivation in municipal wastewaters', Applied biochemistry and biotechnology, 161: 523-36.
Bierkens, J, G Klein, P Corbisier, R Van Den Heuvel, L Verschaeve, R Weltens, and G Schoeters. 1998. 'Comparative sensitivity of 20 bioassays for soil quality', Chemosphere, 37: 2935-47.
Bilad, M. R., D. Vandamme, I. Foubert, K. Muylaert, and I. F. Vankelecom. 2012a. 'Harvesting microalgal biomass using submerged microfiltration membranes', Bioresour Technol, 111: 343-52.
Bilad, MR, Hassan A Arafat, and Ivo FJ Vankelecom. 2014. 'Membrane technology in microalgae cultivation and harvesting: a review', Biotechnology advances, 32: 1283-300.
Bilad, Muhammad Roil, Dries Vandamme, Imogen Foubert, Koenraad Muylaert, and Ivo FJ Vankelecom. 2012b. 'Harvesting microalgal biomass using submerged microfiltration membranes', Bioresource technology, 111: 343-52.
Bilanovic, D, G Shelef, and A Sukenik. 1988. 'Flocculation of microalgae with cationic polymers—effects of medium salinity', Biomass, 17: 65-76.
Bilanovic, Dragoljub, Arsema Andargatchew, Tim Kroeger, and Gedaliahu Shelef. 2009. 'Freshwater and marine microalgae sequestering of CO 2 at different C and N concentrations–response surface methodology analysis', Energy Conversion and Management, 50: 262-67.
Blanken, W, M Janssen, M Cuaresma, Z Libor, T Bhaiji, and RH Wijffels. 2014. 'Biofilm growth of Chlorella sorokiniana in a rotating biological contactor based photobioreactor', Biotechnology and bioengineering, 111: 2436-45.
86
Bligh, E Graham, and W Justin Dyer. 1959. 'A rapid method of total lipid extraction and purification', Canadian journal of biochemistry and physiology, 37: 911-17.
Boelee, Nadine C, Hardy Temmink, Marcel Janssen, Cees JN Buisman, and René H Wijffels. 2012. 'Scenario analysis of nutrient removal from municipal wastewater by microalgal biofilms', Water, 4: 460-73.
Boonchai, Rawiwan, and Gyutae Seo. 2015. 'Microalgae membrane photobioreactor for further removal of nitrogen and phosphorus from secondary sewage effluent', Korean Journal of Chemical Engineering, 32: 2047-52.
Borowitzka, Michael A. 1997. 'Microalgae for aquaculture: opportunities and constraints', Journal of Applied Phycology, 9: 393-401.
———. 1999. 'Commercial production of microalgae: ponds, tanks, tubes and fermenters', Journal of biotechnology, 70: 313-21.
———. 2013. 'High-value products from microalgae—their development and commercialisation', Journal of Applied Phycology, 25: 743-56.
Bosma, Rouke, Wim A van Spronsen, Johannes Tramper, and René H Wijffels. 2003. 'Ultrasound, a new separation technique to harvest microalgae', Journal of Applied Phycology, 15: 143-53.
Boyle, Nanette R, and John A Morgan. 2009. 'Flux balance analysis of primary metabolism in Chlamydomonas reinhardtii', BMC systems biology, 3: 1.
Brányiková, Irena, Barbora Maršálková, Jiří Doucha, Tomáš Brányik, Kateřina Bišová, Vilém Zachleder, and Milada Vítová. 2011. 'Microalgae—novel highly efficient starch producers', Biotechnology and bioengineering, 108: 766-76.
Brennan, Liam, and Philip Owende. 2010. 'Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and co-products', Renewable and sustainable energy reviews, 14: 557-77.
Brune, DE, TJ Lundquist, and JR Benemann. 2009. 'Microalgal biomass for greenhouse gas reductions: potential for replacement of fossil fuels and animal feeds', Journal of Environmental Engineering, 135: 1136-44.
Burlew, John S. 1953. 'Algal culture', From Laboratory to Pilot Plant, Carnegie Inst. Washington Publ, 600.
Burton, Tom, Henry Lyons, Yannick Lerat, Michele Stanley, and Michael Bo Rasmussen. 2009. "A review of the potential of marine algae as a source of biofuel in Ireland." In.: Dublin: Sustainable Energy Ireland-SEI.
Cantrell, Keri B, Thomas Ducey, Kyoung S Ro, and Patrick G Hunt. 2008. 'Livestock waste-to-bioenergy generation opportunities', Bioresource technology, 99: 7941-53.
87
Castaing, J-B, A Massé, M Pontié, V Séchet, J Haure, and P Jaouen. 2010. 'Investigating submerged ultrafiltration (UF) and microfiltration (MF) membranes for seawater pre-treatment dedicated to total removal of undesirable micro-algae', Desalination, 253: 71-77.
Castaing, Jean-Baptiste, A Massé, Veronique Sechet, N-E Sabiri, M Pontié, Joel Haure, and P Jaouen. 2011. 'Immersed hollow fibres microfiltration (MF) for removing undesirable micro-algae and protecting semi-closed aquaculture basins', Desalination, 276: 386-96.
Çelekli, Abuzer, Mehmet Yavuzatmaca, and Hüseyin Bozkurt. 2009. 'Modeling of biomass production by Spirulinaplatensis as function of phosphate concentrations and pH regimes', Bioresource technology, 100: 3625-29.
Chauton, Matilde S, Kjell Inge Reitan, Niels Henrik Norsker, Ragnar Tveterås, and Hans T Kleivdal. 2015. 'A techno-economic analysis of industrial production of marine microalgae as a source of EPA and DHA-rich raw material for aquafeed: Research challenges and possibilities', Aquaculture, 436: 95-103.
Chen, Chun-Yen, Kuei-Ling Yeh, Rifka Aisyah, Duu-Jong Lee, and Jo-Shu Chang. 2011. 'Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: a critical review', Bioresource technology, 102: 71-81.
Chen, Chung-Yuan, and Jui-Ho Lin. 2006. 'Toxicity of chlorophenols to Pseudokirchneriella subcapitata under air-tight test environment', Chemosphere, 62: 503-09.
Cheng, Pengfei, Bei Ji, Lili Gao, Wei Zhang, Junfeng Wang, and Tianzhong Liu. 2013. 'The growth, lipid and hydrocarbon production of Botryococcus braunii with attached cultivation', Bioresource technology, 138: 95-100.
Cheng, Yun, Wenguang Zhou, Chunfang Gao, Kenneth Lan, Yang Gao, and Qingyu Wu. 2009. 'Biodiesel production from Jerusalem artichoke (Helianthus Tuberosus L.) tuber by heterotrophic microalgae Chlorella protothecoides', Journal of Chemical Technology and Biotechnology, 84: 777-81.
Chinnasamy, Senthil, Ashish Bhatnagar, Ryan W Hunt, and KC Das. 2010. 'Microalgae cultivation in a wastewater dominated by carpet mill effluents for biofuel applications', Bioresource technology, 101: 3097-105.
Chisti, Yusuf. 2007. 'Biodiesel from microalgae', Biotechnology advances, 25: 294-306.
Chiu, Sheng-Yi, Chien-Ya Kao, Ming-Ta Tsai, Seow-Chin Ong, Chiun-Hsun Chen, and Chih-Sheng Lin. 2009. 'Lipid accumulation and CO 2 utilization of Nannochloropsis oculata in response to CO 2 aeration', Bioresource technology, 100: 833-38.
Christenson, Logan B, and Ronald C Sims. 2012. 'Rotating algal biofilm reactor and spool harvester for wastewater treatment with biofuels by‐products', Biotechnology and bioengineering, 109: 1674-84.
88
Clarens, Andres F, Eleazer P Resurreccion, Mark A White, and Lisa M Colosi. 2010. 'Environmental life cycle comparison of algae to other bioenergy feedstocks', Environmental science & technology, 44: 1813-19.
Clescerl, Lenore S, Arnold E Greenberg, and Andrew D Eaton. 1999. 'Standard methods for examination of water & wastewater'.
Clijsters, H, and F Van Assche. 1985. 'Inhibition of photosynthesis by heavy metals', Photosynthesis Research, 7: 31-40.
Concas, Alessandro, Giovanni Antonio Lutzu, Massimo Pisu, and Giacomo Cao. 2012. 'Experimental analysis and novel modeling of semi-batch photobioreactors operated with Chlorella vulgaris and fed with 100%(v/v) CO 2', Chemical engineering journal, 213: 203-13.
Contreras-Flores, C, JM Pena-Castro, LB Flores-Cotera, and RO Canizares-Villanueva. 2003. 'Advances in conceptual design of photobioreactors for microalgal culture', Interciencia, 28: 450-+.
Converti, A., A. A. Casazza, E. Y. Ortiz, P. Perego, and M. Del Borghi. 2009. 'Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production', Chemical Engineering and Processing, 48: 1146-51.
Courchesne, Noémie Manuelle Dorval, Albert Parisien, Bei Wang, and Christopher Q Lan. 2009. 'Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches', Journal of biotechnology, 141: 31-41.
Couto, Ricardo Miguel, Pedro Calado Simoes, Alberto Reis, Teresa Lopes Da Silva, Vítor Hugo Martins, and Yolanda Sánchez‐Vicente. 2010. 'Supercritical fluid extraction of lipids from the heterotrophic microalga Crypthecodinium cohnii', Engineering in Life Sciences, 10: 158-64.
Craggs, Rupert J, Walter H Adey, Kyle R Jenson, Matthais S St John, F Bailey Green, and William J Oswald. 1996. 'Phosphorus removal from wastewater using an algal turf scrubber', Water science and technology, 33: 191-98.
Crites, Ron, and George Technobanoglous. 1998. Small and decentralized wastewater management systems (McGraw-Hill).
Cuellar-Bermudez, Sara P, Jonathan S Garcia-Perez, Bruce E Rittmann, and Roberto Parra-Saldivar. 2015. 'Photosynthetic bioenergy utilizing CO 2: an approach on flue gases utilization for third generation biofuels', Journal of Cleaner Production, 98: 53-65.
Cui, Yan. 2013. Fundamentals in Microalgae Harvesting: From Flocculation to Self-attachment.
D’Oca, Marcelo G Montes, Carolina V Viêgas, Juliana S Lemoes, Eriksen K Miyasaki, Joaquín A Morón-Villarreyes, Ednei G Primel, and Paulo C Abreu. 2011.
89
'Production of FAMEs from several microalgal lipidic extracts and direct transesterification of the Chlorella pyrenoidosa', biomass and bioenergy, 35: 1533-38.
Da Silva, Teresa Lopes, Luísa Gouveia, and Alberto Reis. 2014. 'Integrated microbial processes for biofuels and high value-added products: the way to improve the cost effectiveness of biofuel production', Applied microbiology and biotechnology, 98: 1043-53.
de-Bashan, Luz E, and Yoav Bashan. 2010. 'Immobilized microalgae for removing pollutants: review of practical aspects', Bioresource technology, 101: 1611-27.
De Brouwer, Jody FC, and Lucas J Stal. 2002. 'Daily fluctuations of exopolymers in cultures of the benthic diatoms cylindrotheca closterium and nitzschia SP.(Bacillariophyceae) 1', Journal of Phycology, 38: 464-72.
Dean, Andrew P, David C Sigee, Beatriz Estrada, and Jon K Pittman. 2010. 'Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae', Bioresource technology, 101: 4499-507.
Demirbas, Ayhan. 2009. 'Biofuels securing the planet’s future energy needs', Energy Conversion and Management, 50: 2239-49.
Desmorieux, Hélène, and Nadège Decaen. 2005. 'Convective drying of spirulina in thin layer', Journal of food engineering, 66: 497-503.
Di Termini, Ilaria, Annalisa Prassone, Claudia Cattaneo, and Mauro Rovatti. 2011. 'On the nitrogen and phosphorus removal in algal photobioreactors', Ecological Engineering, 37: 976-80.
Dismukes, G Charles, Damian Carrieri, Nicholas Bennette, Gennady M Ananyev, and Matthew C Posewitz. 2008. 'Aquatic phototrophs: efficient alternatives to land-based crops for biofuels', Current opinion in biotechnology, 19: 235-40.
Divakaran, Ravi, and VN Sivasankara Pillai. 2002. 'Flocculation of algae using chitosan', Journal of Applied Phycology, 14: 419-22.
Dragone, Giuliano, Bruno D Fernandes, Ana P Abreu, António A Vicente, and José A Teixeira. 2011. 'Nutrient limitation as a strategy for increasing starch accumulation in microalgae', Applied energy, 88: 3331-35.
Drexler, Ivy LC, and Daniel H Yeh. 2014. 'Membrane applications for microalgae cultivation and harvesting: a review', Reviews in Environmental Science and Bio/Technology, 13: 487-504.
Eixler, Sebastian, Ulf Karsten, and Uwe Selig. 2006. 'Phosphorus storage in Chlorella vulgaris (Trebouxiophyceae, Chlorophyta) cells and its dependence on phosphate supply', Phycologia, 45: 53-60.
90
EPA, U.S. 2010. 'Method 1664, Revision B: n-Hexane Extractable Material (HEM; Oil and Grease) and Silica Gel Treated n-Hexane Extractable Material (SGT-HEM; Non-polar Material) by Extraction and Gravimetry': 1-35.
Eric Francis, David Knowles, and Bruce Richter. 2012. ' EPA Method 1664A--Extraction of Oil and Grease from Water Samples Using AutoTrace 280 Solid-Phase Extraction Cartridge Configuration (DIONEX AN 817)': 3.
Eriksen, Niels T. 2008. 'The technology of microalgal culturing', Biotechnology letters, 30: 1525-36.
Fernandez, Emilio, and Aurora Galvan. 2007. 'Inorganic nitrogen assimilation in Chlamydomonas', Journal of experimental botany, 58: 2279-87.
Filippino, Katherine C, Margaret R Mulholland, and Charles B Bott. 2015. 'Phycoremediation strategies for rapid tertiary nutrient removal in a waste stream', Algal Research, 11: 125-33.
Fuentes, MM Rebolloso, JL Garcıa Sánchez, JM Fernández Sevilla, FG Acién Fernández, JA Sánchez Pérez, and E Molina Grima. 1999. 'Outdoor continuous culture of Porphyridium cruentum in a tubular photobioreactor: quantitative analysis of the daily cyclic variation of culture parameters', Journal of biotechnology, 70: 271-88.
Gao, Feng, Zhao-Hui Yang, Chen Li, Guang-Ming Zeng, Dan-Hui Ma, and Li Zhou. 2015. 'A novel algal biofilm membrane photobioreactor for attached microalgae growth and nutrients removal from secondary effluent', Bioresource technology, 179: 8-12.
Gao, Jie, Hua-ying Bao, Ming-xiu Xin, Yuan-xia Liu, Qian Li, and Yan-fen Zhang. 2006. 'Characterization of a bioflocculant from a newly isolated Vagococcus sp. W31', Journal of Zhejiang University Science B, 7: 186-92.
Garcia, J, R Mujeriego, and M Hernandez-Marine. 2000. 'High rate algal pond operating strategies for urban wastewater nitrogen removal', Journal of Applied Phycology, 12: 331-39.
Garcia, Joan, Mariona Hernández-Mariné, and Rafael Mujeriego. 2000. 'Influence of phytoplankton composition on biomass removal from high-rate oxidation lagoons by means of sedimentation and spontaneous flocculation', Water environment research: 230-37.
Gasperi, Johnny, Stéphane Garnaud, Vincent Rocher, and Régis Moilleron. 2008. 'Priority pollutants in wastewater and combined sewer overflow', Science of the total environment, 407: 263-72.
Geider, Richard, and Julie La Roche. 2002. 'Redfield revisited: variability of C: N: P in marine microalgae and its biochemical basis', European Journal of Phycology, 37: 1-17.
91
Göksan, Tolga, Yaşar Durmaz, and Şevket Gökpınar. 2003. 'Effects of light path lengths and initial culture density on the cultivation of Chaetoceros muelleri (Lemmermann, 1898)', Aquaculture, 217: 431-36.
González-Fernández, Cristina, and Mercedes Ballesteros. 2012. 'Linking microalgae and cyanobacteria culture conditions and key-enzymes for carbohydrate accumulation', Biotechnology advances, 30: 1655-61.
González, Luz Estela, Rosa Olivia Cañizares, and Sandra Baena. 1997. 'Efficiency of ammonia and phosphorus removal from a Colombian agroindustrial wastewater by the microalgae Chlorella vulgaris and Scenedesmus dimorphus', Bioresource technology, 60: 259-62.
Griffiths, Melinda J, and Susan TL Harrison. 2009. 'Lipid productivity as a key characteristic for choosing algal species for biodiesel production', Journal of Applied Phycology, 21: 493-507.
Grima, E Molina, E-H Belarbi, FG Acién Fernández, A Robles Medina, and Yusuf Chisti. 2003. 'Recovery of microalgal biomass and metabolites: process options and economics', Biotechnology advances, 20: 491-515.
Grima, E Molina, FG Acién Fernández, F Garcıa Camacho, and Yusuf Chisti. 1999. 'Photobioreactors: light regime, mass transfer, and scaleup', Journal of biotechnology, 70: 231-47.
Grima, E Molina, A Robles Medina, A Giménez Giménez, JA Sánchez Pérez, F Garcia Camacho, and JL García Sánchez. 1994. 'Comparison between extraction of lipids and fatty acids from microalgal biomass', Journal of the American Oil Chemists’ Society, 71: 955-59.
Grobbelaar, Johan U. 2000. 'Physiological and technological considerations for optimising mass algal cultures', Journal of Applied Phycology, 12: 201-06.
Grobbelaar, Johan U, and Carl J Soeder. 1985. 'Respiration losses in planktonic green algae cultivated in raceway ponds', Journal of Plankton Research, 7: 497-506.
Gudin, C, and C Thepenier. 1986. 'Bioconversion of solar energy into organic chemicals by microalgae', Advances in biotechnological processes (USA).
Gudin, Claude, and Daniel Chaumont. 1991. 'Cell fragility—the key problem of microalgae mass production in closed photobioreactors', Bioresource technology, 38: 145-51.
Guieysse, Benoit, Xavier Borde, Raul Muñoz, Rajni Hatti-Kaul, Caroline Nugier-Chauvin, Henri Patin, and Bo Mattiasson. 2002. 'Influence of the initial composition of algal-bacterial microcosms on the degradation of salicylate in a fed-batch culture', Biotechnology letters, 24: 531-38.
92
Guzzon, Antonella, Andreas Bohn, Marco Diociaiuti, and Patrizia Albertano. 2008. 'Cultured phototrophic biofilms for phosphorus removal in wastewater treatment', Water research, 42: 4357-67.
Heasman, M, J Diemar, W O'connor, T Sushames, and L Foulkes. 2000. 'Development of extended shelf‐ life microalgae concentrate diets harvested by centrifugation for bivalve molluscs–a summary', Aquaculture Research, 31: 637-59.
Hill, Jason, Erik Nelson, David Tilman, Stephen Polasky, and Douglas Tiffany. 2006. 'Environmental, economic, and energetic costs and benefits of biodiesel and ethanol biofuels', Proceedings of the National Academy of Sciences, 103: 11206-10.
Hoffmann, James P. 1998. 'Wastewater treatment with suspended and nonsuspended algae', Journal of Phycology, 34: 757-63.
Holmstrom, Carola, and Staffan Kjelleberg. 2000. 'Bacterial interactions with marine fouling organisms', Biofilms: recent advances in their study and control. Harwood Academic Publisher, Amsterdam, 101: 115.
Hoyt, Douglas V. 1979. 'An empirical determination of the heating of the Earth by the carbon dioxide greenhouse effect'.
Hsieh, Chih-Hung, and Wen-Teng Wu. 2009. 'Cultivation of microalgae for oil production with a cultivation strategy of urea limitation', Bioresource technology, 100: 3921-26.
Hsueh, HT, Hsin Chu, and SHIH-TSUNG Yu. 2007. 'A batch study on the bio-fixation of carbon dioxide in the absorbed solution from a chemical wet scrubber by hot spring and marine algae', Chemosphere, 66: 878-86.
Hu, Q., M. Sommerfeld, E. Jarvis, M. Ghirardi, M. Posewitz, M. Seibert, and A. Darzins. 2008. 'Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances', Plant Journal, 54: 621-39.
Hu, Qiang, Hugo Guterman, and Amos Richmond. 1996. 'A flat inclined modular photobioreactor for outdoor mass cultivation of photoautotrophs', Biotechnology and bioengineering, 51: 51-60.
Huang, GuanHua, Feng Chen, Dong Wei, XueWu Zhang, and Gu Chen. 2010. 'Biodiesel production by microalgal biotechnology', Applied energy, 87: 38-46.
Huertas, I Emma, Brian Colman, George S Espie, and Luis M Lubian. 2000. 'Active transport of CO2 by three species of marine microalgae', Journal of Phycology, 36: 314-20.
Ip, SY, JS Bridger, CT Chin, WRB Martin, and WGC Raper. 1982. 'Algal growth in primary settled sewage: the effects of five key variables', Water research, 16: 621-32.
Janssen, Marcel, Matthias Janssen, Marcel de Winter, Johannes Tramper, Luuc R Mur, Jan Snel, and René H Wijffels. 2000. 'Efficiency of light utilization of Chlamydomonas
93
reinhardtii under medium-duration light/dark cycles', Journal of biotechnology, 78: 123-37.
Janssen, Marcel, Johannes Tramper, Luuc R Mur, and Rene H Wijffels. 2003. 'Enclosed outdoor photobioreactors: Light regime, photosynthetic efficiency, scale‐up, and future prospects', Biotechnology and bioengineering, 81: 193-210.
Jimenez-Perez, MV, P Sanchez-Castillo, O Romera, D Fernandez-Moreno, and C Pérez-Martınez. 2004. 'Growth and nutrient removal in free and immobilized planktonic green algae isolated from pig manure', Enzyme and Microbial Technology, 34: 392-98.
Johnson, Michael B, and Zhiyou Wen. 2009. 'Production of biodiesel fuel from the microalga Schizochytrium limacinum by direct transesterification of algal biomass', Energy & Fuels, 23: 5179-83.
Judd, Simon, Leo JP van den Broeke, Mohamed Shurair, Yussuf Kuti, and Hussein Znad. 2015. 'Algal remediation of CO 2 and nutrient discharges: A review', Water research, 87: 356-66.
Katarzyna, Ledwoch, Gu Sai, and Oinam Avijeet Singh. 2015. 'Non-enclosure methods for non-suspended microalgae cultivation: literature review and research needs', Renewable and sustainable energy reviews, 42: 1418-27.
Kesaano, Maureen, and Ronald C Sims. 2014. 'Algal biofilm based technology for wastewater treatment', Algal Research, 5: 231-40.
Khan, Shakeel A, Mir Z Hussain, S Prasad, and UC Banerjee. 2009. 'Prospects of biodiesel production from microalgae in India', Renewable and sustainable energy reviews, 13: 2361-72.
Klausmeier, Christopher A, E Litchman, T Daufresne, and SA Levin. 2008. 'Phytoplankton stoichiometry', Ecological Research, 23: 479-85.
Klausmeier, Christopher A, Elena Litchman, Tanguy Daufresne, and Simon A Levin. 2004. 'Optimal nitrogen-to-phosphorus stoichiometry of phytoplankton', Nature, 429: 171-74.
Konig, A, HW Pearson, and Salomao Anselmo Silva. 1987. 'Ammonia toxicity to algal growth in waste stabilization ponds', Water science and technology, 19: 115-22.
Ladner, David A, Derek R Vardon, and Mark M Clark. 2010. 'Effects of shear on microfiltration and ultrafiltration fouling by marine bloom-forming algae', Journal of Membrane Science, 356: 33-43.
Laliberte, Gilles, Daniel Proulx, Niels De Pauw, and J De La Noue. 1994. 'Algal technology in waste water treatment. Chapt. 11', Algae and water pollution. LC Rai, JP Gaur & CJ Soeder (Eds). E. Schweizerbart'sche Verlagsbuchhandlung (Nagele u. Obermiller), Stuttgart: 283-302.
94
Lam, Man Kee, and Keat Teong Lee. 2012. 'Microalgae biofuels: a critical review of issues, problems and the way forward', Biotechnology advances, 30: 673-90.
———. 2013. 'Effect of carbon source towards the growth of Chlorella vulgaris for CO 2 bio-mitigation and biodiesel production', International Journal of Greenhouse Gas Control, 14: 169-76.
Larson, Eric D. 2007. Sustainable bioenergy: A framework for decision makers (UN-Energy).
Lau, PS, NFY Tam, and YS Wong. 1995. 'Effect of algal density on nutrient removal from primary settled wastewater', Environmental Pollution, 89: 59-66.
Lee, Andrew K, David M Lewis, and Peter J Ashman. 2009. 'Microbial flocculation, a potentially low-cost harvesting technique for marine microalgae for the production of biodiesel', Journal of Applied Phycology, 21: 559-67.
———. 2010. 'Energy requirements and economic analysis of a full-scale microbial flocculation system for microalgal harvesting', Chemical Engineering Research and Design, 88: 988-96.
Lee, Jae-Yon, Chan Yoo, So-Young Jun, Chi-Yong Ahn, and Hee-Mock Oh. 2010. 'Comparison of several methods for effective lipid extraction from microalgae', Bioresource technology, 101: S75-S77.
Lee, Jin-Suk, and Joon-Pyo Lee. 2003. 'Review of advances in biological CO 2 mitigation technology', Biotechnology and Bioprocess Engineering, 8: 354-59.
Lee, Kwangyong, and Choul-Gyun Lee. 2001. 'Effect of light/dark cycles on wastewater treatments by microalgae', Biotechnology and Bioprocess Engineering, 6: 194-99.
Leupold, Marco, Stefan Hindersin, Giselher Gust, Martin Kerner, and Dieter Hanelt. 2013. 'Influence of mixing and shear stress on Chlorella vulgaris, Scenedesmus obliquus, and Chlamydomonas reinhardtii', Journal of Applied Phycology, 25: 485-95.
Li, Xiufeng, Han Xu, and Qingyu Wu. 2007. 'Large‐ scale biodiesel production from microalga Chlorella protothecoides through heterotrophic cultivation in bioreactors', Biotechnology and bioengineering, 98: 764-71.
Li, Yajun, Xiaowen Fei, and Xiaodong Deng. 2012. 'Novel molecular insights into nitrogen starvation-induced triacylglycerols accumulation revealed by differential gene expression analysis in green algae Micractinium pusillum', biomass and bioenergy, 42: 199-211.
Li, Yanqun, Mark Horsman, Bei Wang, Nan Wu, and Christopher Q Lan. 2008. 'Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans', Applied microbiology and biotechnology, 81: 629-36.
Li, Yanqun, Mark Horsman, Nan Wu, Christopher Q Lan, and Nathalie Dubois‐Calero. 2008. 'Biofuels from microalgae', Biotechnology progress, 24: 815-20.
95
Li, Yantao, Danxiang Han, Guangrong Hu, Milton Sommerfeld, and Qiang Hu. 2010. 'Inhibition of starch synthesis results in overproduction of lipids in Chlamydomonas reinhardtii', Biotechnology and bioengineering, 107: 258-68.
Liang, Yanna, Nicolas Sarkany, and Yi Cui. 2009. 'Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions', Biotechnology letters, 31: 1043-49.
Lin, Lin, Zhou Cunshan, Saritporn Vittayapadung, Shen Xiangqian, and Dong Mingdong. 2011. 'Opportunities and challenges for biodiesel fuel', Applied energy, 88: 1020-31.
Liu, Tianzhong, Junfeng Wang, Qiang Hu, Pengfei Cheng, Bei Ji, Jinli Liu, Yu Chen, Wei Zhang, Xiaoling Chen, and Lin Chen. 2013. 'Attached cultivation technology of microalgae for efficient biomass feedstock production', Bioresource technology, 127: 216-22.
Liu, Zhi-Yuan, Guang-Ce Wang, and Bai-Cheng Zhou. 2008. 'Effect of iron on growth and lipid accumulation in Chlorella vulgaris', Bioresource technology, 99: 4717-22.
Loladze, Irakli, and James J Elser. 2011. 'The origins of the Redfield nitrogen‐ to‐phosphorus ratio are in a homoeostatic protein‐to‐rRNA ratio', Ecology letters, 14: 244-50.
Lundquist, Tryg J. 2008. "Production of algae in conjunction with wastewater treatment." In Renewable Energy Laboratory and Air Force Office of Scientific Research Workshop: Algal Oil for Jet Fuel Production, Arlington, VA, Feb, 19-21.
MacKay, D, and T Salusbury. 1988. 'Choosing between centrifugation and crossflow microfiltration', Chemical engineer: 45-50.
Mallick, Nirupama. 2002. 'Biotechnological potential of immobilized algae for wastewater N, P and metal removal: a review', Biometals, 15: 377-90.
Mara, DD, and H Pearson. 1986. 'Artificial freshwater environment: waste stabilization ponds', Biotechnology, 8: 177-206.
Marbelia, Lisendra, Muhammad Roil Bilad, Iason Passaris, Valerie Discart, Dries Vandamme, Annelies Beuckels, Koenraad Muylaert, and Ivo FJ Vankelecom. 2014. 'Membrane photobioreactors for integrated microalgae cultivation and nutrient remediation of membrane bioreactors effluent', Bioresource technology, 163: 228-35.
Marchant, Roger. 2001. 'Environmental biotechnology and cleaner bioprocesses. Edited by Eugenia J Olguin, Gloria Sanchez and Elizabeth Hernandez, Taylor & Francis London, 2000, 319 pp, price UK $60.00, ISBN 0 7484 0729‐4', Journal of Chemical Technology and Biotechnology, 76: 106-06.
96
Markou, Giorgos. 2012. 'Alteration of the biomass composition of Arthrospira (Spirulina) platensis under various amounts of limited phosphorus', Bioresource technology, 116: 533-35.
———. 2015. 'Fed-batch cultivation of Arthrospira and Chlorella in ammonia-rich wastewater: optimization of nutrient removal and biomass production', Bioresource technology, 193: 35-41.
Markou, Giorgos, and Elias Nerantzis. 2013. 'Microalgae for high-value compounds and biofuels production: a review with focus on cultivation under stress conditions', Biotechnology advances, 31: 1532-42.
Martınez, ME, S Sanchez, JM Jimenez, F El Yousfi, and L Munoz. 2000. 'Nitrogen and phosphorus removal from urban wastewater by the microalga Scenedesmus obliquus', Bioresource technology, 73: 263-72.
Mata, Teresa M, Antonio A Martins, and Nidia S Caetano. 2010. 'Microalgae for biodiesel production and other applications: a review', Renewable and sustainable energy reviews, 14: 217-32.
Matsumoto, Hiroyo, Akihiro Hamasaki, Norio Shioji, and Yoshiaki Ikuta. 1996. 'Influence of dissolved oxygen on photosynthetic rate of microalgae', Journal of chemical engineering of Japan, 29: 711-14.
McKendry, Peter. 2002. 'Energy production from biomass (part 1): overview of biomass', Bioresource technology, 83: 37-46.
Mehta, SK, and JP Gaur. 2005. 'Use of algae for removing heavy metal ions from wastewater: progress and prospects', Critical reviews in biotechnology, 25: 113-52.
Mendes-Pinto, MM, MFJ Raposo, J Bowen, AJ Young, and R Morais. 2001. 'Evaluation of different cell disruption processes on encysted cells of Haematococcus pluvialis: effects on astaxanthin recovery and implications for bio-availability', Journal of Applied Phycology, 13: 19-24.
Michels, Michiel HA, Atze J van der Goot, Niels-Henrik Norsker, and René H Wijffels. 2010. 'Effects of shear stress on the microalgae Chaetoceros muelleri', Bioprocess and biosystems engineering, 33: 921-27.
Milledge, John J, and Sonia Heaven. 2013. 'A review of the harvesting of micro-algae for biofuel production', Reviews in Environmental Science and Bio/Technology, 12: 165-78.
Mitsuhashi, S, K Hosaka, E Tomonaga, H Muramatsu, and K Tanishita. 1995. 'Effects of shear flow on photosynthesis in a dilute suspension of microalgae', Applied microbiology and biotechnology, 42: 744-49.
Mohn, FH. 1980. 'Experiences and strategies in the recovery of biomass from mass cultures of microalgae', Algae biomass: production and use/[sponsored by the National Council for
97
Research and Development, Israel and the Gesellschaft fur Strahlen-und Umweltforschung (GSF), Munich, Germany]; editors, Gedaliah Shelef, Carl J. Soeder.
Moreno, José, M Ángeles Vargas, and Miguel G Guerrero. 2003. 'Outdoor cultivation of a nitrogen-fixing marine cyanobacterium, Anabaena sp. ATCC 33047', Biomolecular engineering, 20: 191-97.
Morita, M, Y Watanabe, and H Saiki. 2001. 'Instruction of microalgal biomass production for practically higher photosynthetic performance using a photobioreactor', Food and bioproducts processing, 79: 176-83.
Munoz, Raul, and Benoit Guieysse. 2006. 'Algal–bacterial processes for the treatment of hazardous contaminants: a review', Water research, 40: 2799-815.
Muñoz, Raul, Marco Jacinto, Benoit Guieysse, and Bo Mattiasson. 2005. 'Combined carbon and nitrogen removal from acetonitrile using algal–bacterial bioreactors', Applied microbiology and biotechnology, 67: 699-707.
Muñoz, Raul, Claudia Köllner, Benoit Guieysse, and Bo Mattiasson. 2003. 'Salicylate biodegradation by various algal-bacterial consortia under photosynthetic oxygenation', Biotechnology letters, 25: 1905-11.
———. 2004. 'Photosynthetically oxygenated salicylate biodegradation in a continuous stirred tank photobioreactor', Biotechnology and bioengineering, 87: 797-803.
Muñoz, Raul, C Rolvering, Benoit Guieysse, and Bo Mattiasson. 2005. 'Photosynthetically oxygenated acetonitrile biodegradation by an algal-bacterial microcosm: a pilot-scale study', Water science and technology, 51: 261-65.
Nurdogan, Yakup, and William J Oswald. 1996. 'Tube settling of high-rate pond algae', Water science and technology, 33: 229-41.
Ogbonna, James C, and Hideo Tanaka. 2000. 'Light requirement and photosynthetic cell cultivation–Development of processes for efficient light utilization in photobioreactors', Journal of Applied Phycology, 12: 207-18.
Olaizola, Miguel. 2003. 'Commercial development of microalgal biotechnology: from the test tube to the marketplace', Biomolecular engineering, 20: 459-66.
Olguín, EJ. 2000. 'The cleaner production strategy applied to animal production', Environment biotech. and cleane processes. London: 227-41.
Olguín, Eugenia J. 2012. 'Dual purpose microalgae–bacteria-based systems that treat wastewater and produce biodiesel and chemical products within a Biorefinery', Biotechnology advances, 30: 1031-46.
Organization, Agriculture. 2006. The State of Food and Agriculture, 2006: Food Aid for Food Security? (Food & Agriculture Org.).
98
Órpez, Rafael, Ma Eugenia Martínez, Gassan Hodaifa, Farida El Yousfi, Nohman Jbari, and Sebastián Sánchez. 2009. 'Growth of the microalga Botryococcus braunii in secondarily treated sewage', Desalination, 246: 625-30.
Oswald, William J. 1962. 'The coming industry of controlled photosynthesis', American Journal of Public Health and the Nations Health, 52: 235-42.
———. 1988. 'Micro-algae and waste-water treatment'.
———. 1990. "Advanced integrated wastewater pond systems." In Proceedings of the ASCE convention: Supplying water and saving the environment for six billion people, 73-80.
———. 2003. 'My sixty years in applied algology', Journal of Applied Phycology, 15: 99-106.
Oswald, William J, and Claeence G Golueke. 1960. 'Biological transformation of solar energy', Adv. Appl. Microbiol, 2: 223-62.
Oswald, William J, Clarence G Golueke, and Robert W Tyler. 1967. 'Integrated pond systems for subdivisions', Journal (Water Pollution Control Federation): 1289-304.
Oswald, William J, HB Gotaas, Harvey F Ludwig, and Victoria Lynch. 1953. 'Algae symbiosis in oxidation ponds: III. Photosynthetic oxygenation', Sewage and Industrial Wastes: 692-705.
Oswald, WJ. 1995. 'Ponds in the twenty-first century', Water science and technology, 31: 1-8.
Oswald, WJ, HB Gotaas, CG Golueke, WR Kellen, EF Gloyna, and ER Hermann. 1957. 'Algae in waste treatment [with discussion]', Sewage and Industrial Wastes, 29: 437-57.
Ota, Masaki, Hiromoto Watanabe, Yoshitaka Kato, Masaru Watanabe, Yoshiyuki Sato, Richard Lee Smith, and Hiroshi Inomata. 2009. 'Carotenoid production from Chlorococcum littorale in photoautotrophic cultures with downstream supercritical fluid processing', Journal of separation science, 32: 2327-35.
Peccia, Jordan, Berat Haznedaroglu, James Gutierrez, and Julie B Zimmerman. 2013. 'Nitrogen supply is an important driver of sustainable microalgae biofuel production', Trends in biotechnology, 31: 134-38.
Peña-Castro, Julián Mario, Fernando Martı nez-Jerónimo, Fernando Esparza-Garcıa, and Rosa Olivia Cañizares-Villanueva. 2004. 'Phenotypic plasticity in Scenedesmus incrassatulus (Chlorophyceae) in response to heavy metals stress', Chemosphere, 57: 1629-36.
Perez-Garcia, Octavio, Froylan ME Escalante, Luz E de-Bashan, and Yoav Bashan. 2011. 'Heterotrophic cultures of microalgae: metabolism and potential products', Water research, 45: 11-36.
Petrusevski, B, G Bolier, AN Van Breemen, and GJ Alaerts. 1995. 'Tangential flow filtration: a method to concentrate freshwater algae', Water research, 29: 1419-24.
99
Pittman, Jon K, Andrew P Dean, and Olumayowa Osundeko. 2011. 'The potential of sustainable algal biofuel production using wastewater resources', Bioresource technology, 102: 17-25.
PME. 2001. 'Kingdom of Saudi Arabia National Environmental Standard Industrial and Municipal Wastewater Discharges', PME Reference: 17.
Powell, Nicola, Andy Shilton, Yusuf Chisti, and Steven Pratt. 2009. 'Towards a luxury uptake process via microalgae–defining the polyphosphate dynamics', Water research, 43: 4207-13.
Prakash, J, B Pushparaj, P Carlozzi, G Torzillo, E Montaini, and R Materassi. 1997. 'MICROALGAL BIOMASS DRYING BY A SIMPLE SOLAR DEVICE∗', International journal of solar energy, 18: 303-11.
Pulz, Otto. 2001. 'Photobioreactors: production systems for phototrophic microorganisms', Applied microbiology and biotechnology, 57: 287-93.
Pulz, Otto, and Wolfgang Gross. 2004. 'Valuable products from biotechnology of microalgae', Applied microbiology and biotechnology, 65: 635-48.
Pulz, Otto, and Karl Scheibenbogen. 1998. 'Photobioreactors: design and performance with respect to light energy input.' in, Bioprocess and algae reactor technology, apoptosis (Springer).
Ranjan, Amrita, Chetna Patil, and Vijayanand S Moholkar. 2010. 'Mechanistic assessment of microalgal lipid extraction', Industrial & Engineering Chemistry Research, 49: 2979-85.
Renault, François, Bertrand Sancey, P-M Badot, and Grégorio Crini. 2009. 'Chitosan for coagulation/flocculation processes–an eco-friendly approach', European Polymer Journal, 45: 1337-48.
Reynolds, Colin S. 2006. The ecology of phytoplankton (Cambridge University Press).
Richmond, Amos. 2004. 'Principles for attaining maximal microalgal productivity in photobioreactors: an overview.' in, Asian Pacific Phycology in the 21st Century: Prospects and Challenges (Springer).
Rier, Steven T, R Jan Stevenson, and Gina D LaLiberte. 2006. 'PHOTO‐ACCLIMATION RESPONSE OF BENTHIC STREAM ALGAE ACROSS EXPERIMENTALLY MANIPULATED LIGHT GRADIENTS: A COMPARISON OF GROWTH RATES AND NET PRIMARY PRODUCTIVITY1', Journal of Phycology, 42: 560-67.
Rittmann, Bruce E. 2008. 'Opportunities for renewable bioenergy using microorganisms', Biotechnology and bioengineering, 100: 203-12.
Rivas, Mariella O, Pedro Vargas, and Carlos E Riquelme. 2010. 'Interactions of Botryococcus braunii cultures with bacterial biofilms', Microbial ecology, 60: 628-35.
100
Rodolfi, Liliana, Graziella Chini Zittelli, Niccolò Bassi, Giulia Padovani, Natascia Biondi, Gimena Bonini, and Mario R Tredici. 2009. 'Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low ‐ cost photobioreactor', Biotechnology and bioengineering, 102: 100-12.
Roessler, Paul G. 1990. 'Environmental control of glycerolipid metabolism in microalgae: commercial implications and future research directions', Journal of Phycology, 26: 393-99.
Rosenberg, Julian N, George A Oyler, Loy Wilkinson, and Michael J Betenbaugh. 2008. 'A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution', Current opinion in biotechnology, 19: 430-36.
Rossi, N, P Jaouen, P Legentilhomme, and I Petit. 2004. 'Harvesting of cyanobacterium Arthrospira platensis using organic filtration membranes', Food and bioproducts processing, 82: 244-50.
Rossignol, N, L Vandanjon, P Jaouen, and F Quemeneur. 1999. 'Membrane technology for the continuous separation microalgae/culture medium: compared performances of cross-flow microfiltration and ultrafiltration', Aquacultural Engineering, 20: 191-208.
Roudsari, Fatemeh Pourasgharian, Mohammad Reza Mehrnia, Akram Asadi, Zohreh Moayedi, and Reza Ranjbar. 2014. 'Effect of microalgae/activated sludge ratio on cooperative treatment of anaerobic effluent of municipal wastewater', Applied biochemistry and biotechnology, 172: 131-40.
Ruiz-Marin, Alejandro, Leopoldo G Mendoza-Espinosa, and Tom Stephenson. 2010. 'Growth and nutrient removal in free and immobilized green algae in batch and semi-continuous cultures treating real wastewater', Bioresource technology, 101: 58-64.
Ruiz-Martinez, A, J Serralta, M Pachés, A Seco, and J Ferrer. 2014. 'Mixed microalgae culture for ammonium removal in the absence of phosphorus: Effect of phosphorus supplementation and process modeling', Process Biochemistry, 49: 2249-57.
Saint Cyr, D de Gouvion, C Wisniewski, L Schrive, E Farhi, and C Rivasseau. 2014. 'Feasibility study of microfiltration for algae separation in an innovative nuclear effluents decontamination process', Separation and Purification Technology, 125: 126-35.
Salehizadeh, H, M Vossoughi, and I Alemzadeh. 2000. 'Some investigations on bioflocculant producing bacteria', Biochemical engineering journal, 5: 39-44.
Sander, Kyle, and Ganti S Murthy. 2010. 'Life cycle analysis of algae biodiesel', The International Journal of Life Cycle Assessment, 15: 704-14.
Schenk, Peer M, Skye R Thomas-Hall, Evan Stephens, Ute C Marx, Jan H Mussgnug, Clemens Posten, Olaf Kruse, and Ben Hankamer. 2008. 'Second generation biofuels: high-efficiency microalgae for biodiesel production', Bioenergy research, 1: 20-43.
101
Schumacher, G, T Blume, and I Sekoulov. 2003. 'Bacteria reduction and nutrient removal in small wastewater treatment plants by an algal biofilm', Water science and technology, 47: 195-202.
Scott, Stuart A, Matthew P Davey, John S Dennis, Irmtraud Horst, Christopher J Howe, David J Lea-Smith, and Alison G Smith. 2010. 'Biodiesel from algae: challenges and prospects', Current opinion in biotechnology, 21: 277-86.
Searchinger, Timothy, Ralph Heimlich, Richard A Houghton, Fengxia Dong, Amani Elobeid, Jacinto Fabiosa, Simla Tokgoz, Dermot Hayes, and Tun-Hsiang Yu. 2008. 'Use of US croplands for biofuels increases greenhouse gases through emissions from land-use change', Science, 319: 1238-40.
Service, RF. 2011. 'Algae's second try', Science (New York, NY), 333: 1238.
Sharma, YC, and B Singh. 2009. 'Development of biodiesel: current scenario', Renewable and sustainable energy reviews, 13: 1646-51.
Sheehan, John, Terri Dunahay, John Benemann, and Paul Roessler. 1998. A look back at the US Department of Energy's Aquatic Species Program: Biodiesel from algae (National Renewable Energy Laboratory Golden).
Shen, Qiao-Hui, Yu-Peng Gong, Wen-Zhe Fang, Zi-Cheng Bi, Li-Hua Cheng, Xin-Hua Xu, and Huan-Lin Chen. 2015. 'Saline wastewater treatment by Chlorella vulgaris with simultaneous algal lipid accumulation triggered by nitrate deficiency', Bioresource technology, 193: 68-75.
Singh, Nirbhay Kumar, and Dolly Wattal Dhar. 2011. 'Microalgae as second generation biofuel. A review', Agronomy for Sustainable Development, 31: 605-29.
Şirin, Sema, Ester Clavero, and Joan Salvadó. 2013. 'Potential pre-concentration methods for Nannochloropsis gaditana and a comparative study of pre-concentrated sample properties', Bioresource technology, 132: 293-304.
Sorokin, Constantine, and Robert W Krauss. 1958. 'The Effects of Light Intensity on the Growth Rates of Green Algae', Plant physiology, 33: 109.
Spolaore, Pauline, Claire Joannis-Cassan, Elie Duran, and Arsène Isambert. 2006. 'Commercial applications of microalgae', Journal of bioscience and bioengineering, 101: 87-96.
Stephens, Evan, Ian L Ross, Zachary King, Jan H Mussgnug, Olaf Kruse, Clemens Posten, Michael A Borowitzka, and Ben Hankamer. 2010. 'An economic and technical evaluation of microalgal biofuels', Nature biotechnology, 28: 126-28.
Stephenson, Anna L, Elena Kazamia, John S Dennis, Christopher J Howe, Stuart A Scott, and Alison G Smith. 2010. 'Life-cycle assessment of potential algal biodiesel production in the United Kingdom: a comparison of raceways and air-lift tubular bioreactors', Energy & Fuels, 24: 4062-77.
102
Sterner, Robert Warner, and James J Elser. 2002. Ecological stoichiometry: the biology of elements from molecules to the biosphere (Princeton University Press).
Suh, In Soo, and Choul-Gyun Lee. 2003. 'Photobioreactor engineering: design and performance', Biotechnology and Bioprocess Engineering, 8: 313-21.
Tadesse, I, FB Green, and JA Puhakka. 2004. 'Seasonal and diurnal variations of temperature, pH and dissolved oxygen in advanced integrated wastewater pond system® treating tannery effluent', Water research, 38: 645-54.
Tamer, E, MA Amin, ET Ossama, M Bo, and G Benoit. 2006. 'Biological treatment of industrial wastes in a photobioreactor', Water science and technology, 53: 117-25.
Tchobanoglous, George, and Franklin L Burton. 1991. 'Wastewater engineering', Management, 7: 1-4.
Tercero, EA Ramos, E Sforza, M Morandini, and A Bertucco. 2014. 'Cultivation of Chlorella protothecoides with urban wastewater in continuous photobioreactor: biomass productivity and nutrient removal', Applied biochemistry and biotechnology, 172: 1470-85.
Thrush, Simon F, Judi E Hewitt, Max Gibbs, Carolyn Lundquist, and Alf Norkko. 2006. 'Functional role of large organisms in intertidal communities: community effects and ecosystem function', Ecosystems, 9: 1029-40.
Travieso, L, RO Canizares, R Borja, F Benitez, AR Dominguez, R Dupeyrón y, and V Valiente. 1999. 'Heavy metal removal by microalgae', Bulletin of environmental contamination and toxicology, 62: 144-51.
Tredici, Mario R. 2002. 'Bioreactors, photo', Encyclopedia of Bioprocess Technology.
Tsukahara, Kenichiro, and Shigeki Sawayama. 2005. 'Liquid fuel production using microalgae', J Jpn Pet Inst, 48: 251.
Tuantet, Kanjana, Hardy Temmink, Grietje Zeeman, Marcel Janssen, René H Wijffels, and Cees JN Buisman. 2014. 'Nutrient removal and microalgal biomass production on urine in a short light-path photobioreactor', Water research, 55: 162-74.
Valderrama, Luz T, Claudia M Del Campo, Claudia M Rodriguez, Luz E de-Bashan, and Yoav Bashan. 2002. 'Treatment of recalcitrant wastewater from ethanol and citric acid production using the microalga Chlorella vulgaris and the macrophyte Lemna minuscula', Water research, 36: 4185-92.
van der Marel, Perry, Arie Zwijnenburg, Antoine Kemperman, Matthias Wessling, Hardy Temmink, and Walter van der Meer. 2009. 'An improved flux-step method to determine the critical flux and the critical flux for irreversibility in a membrane bioreactor', Journal of Membrane Science, 332: 24-29.
103
Van Harmelen, T, and H Oonk. 2006. 'Microalgae biofixation processes: applications and potential contributions to greenhouse gas mitigation options', TNO Built Environment and Geosciences, Apeldoorn, The Netherlands: 56.
Voltolina, Domenico, Beatriz Cordero, Mario Nieves, and Lilia P Soto. 1999. 'Growth of Scenedesmus sp. in artificial wastewater', Bioresource technology, 68: 265-68.
Wang, Bei, and Christopher Q Lan. 2011. 'Biomass production and nitrogen and phosphorus removal by the green alga Neochloris oleoabundans in simulated wastewater and secondary municipal wastewater effluent', Bioresource technology, 102: 5639-44.
Wang, Bei, Yanqun Li, Nan Wu, and Christopher Q Lan. 2008. 'CO2 bio-mitigation using microalgae', Applied microbiology and biotechnology, 79: 707-18.
Wang, Hui, Bei Ji, Junfeng Wang, Fajin Guo, Wenjun Zhou, Lili Gao, and Tian Zhong Liu. 2014. 'Growth and biochemical composition of filamentous microalgae Tribonema sp. as potential biofuel feedstock', Bioprocess and biosystems engineering, 37: 2607-13.
Wang, Hui, Chunli Ji, Shenglei Bi, Peng Zhou, Lin Chen, and Tianzhong Liu. 2014. 'Joint production of biodiesel and bioethanol from filamentous oleaginous microalgae Tribonema sp', Bioresource technology, 172: 169-73.
Wang, Liang, Yecong Li, Paul Chen, Min Min, Yifeng Chen, Jun Zhu, and Roger R Ruan. 2010. 'Anaerobic digested dairy manure as a nutrient supplement for cultivation of oil-rich green microalgae Chlorella sp', Bioresource technology, 101: 2623-28.
Wang, Liang, Min Min, Yecong Li, Paul Chen, Yifeng Chen, Yuhuan Liu, Yingkuan Wang, and Roger Ruan. 2010. 'Cultivation of green algae Chlorella sp. in different wastewaters from municipal wastewater treatment plant', Applied biochemistry and biotechnology, 162: 1174-86.
Weldy, Chad Share, and MICHAEL Huesemann. 2007. 'Lipid production by Dunaliella salina in batch culture: effects of nitrogen limitation and light intensity', US Department of Energy Journal of Undergraduate Research, 7: 115-22.
Welsh, David T, Marco Bartoli, Daniele Nizzoli, Giuseppe Castaldelli, Stéphane A Riou, and Pierluigi Viaroli. 2000. 'Denitrification, nitrogen fixation, community primary productivity and inorganic-N and oxygen fluxes in an intertidal Zostera noltii meadow', Marine Ecology Progress Series, 208.
Wijffels, René H, and Maria J Barbosa. 2010. 'An outlook on microalgal biofuels', Science(Washington), 329: 796-99.
Wilkie, Ann C, and Walter W Mulbry. 2002. 'Recovery of dairy manure nutrients by benthic freshwater algae', Bioresource technology, 84: 81-91.
Wrigley, TJ, and DF Toerien. 1990. 'Limnological aspects of small sewage ponds', Water research, 24: 83-90.
104
Xin, Li, Hu Hong-ying, Gan Ke, and Yang Jia. 2010. 'Growth and nutrient removal properties of a freshwater microalga Scenedesmus sp. LX1 under different kinds of nitrogen sources', Ecological Engineering, 36: 379-81.
Xiong, Wei, Xiufeng Li, Jinyi Xiang, and Qingyu Wu. 2008. 'High-density fermentation of microalga Chlorella protothecoides in bioreactor for microbio-diesel production', Applied microbiology and biotechnology, 78: 29-36.
Xu, Han, Xiaoling Miao, and Qingyu Wu. 2006. 'High quality biodiesel production from a microalga Chlorella protothecoides by heterotrophic growth in fermenters', Journal of biotechnology, 126: 499-507.
Xu, Ling, Pamela J Weathers, Xue‐Rong Xiong, and Chun‐Zhao Liu. 2009. 'Microalgal bioreactors: challenges and opportunities', Engineering in Life Sciences, 9: 178-89.
Zhang, Endong, Bing Wang, Qihua Wang, Shubiao Zhang, and Budiao Zhao. 2008. 'Ammonia–nitrogen and orthophosphate removal by immobilized Scenedesmus sp. isolated from municipal wastewater for potential use in tertiary treatment', Bioresource technology, 99: 3787-93.
Zhang, Jianguo, and Bo Hu. 2012. 'A novel method to harvest microalgae via co-culture of filamentous fungi to form cell pellets', Bioresource technology, 114: 529-35.
Zhou, Xinping, Shuo Yuan, Ranchi Chen, and Reccab M Ochieng. 2015. 'Sustainable production of energy from microalgae: Review of culturing systems, economics, and modelling', Journal of Renewable and Sustainable Energy, 7: 012701.
