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PRODUCTION OF CAPROATE FROM UNDILUTED THIN STILLAGE · [Type here] - 3 - Abstract The search for...
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PRODUCTION OF CAPROATE FROM
UNDILUTED THIN STILLAGE
Aantal woorden: 22.692
Evert Vincent Stamnummer: 01070017
Promotor: Prof. dr. Leen De Gelder
Copromotor: Prof. dr. Korneel Rabaey
Tutors: José Arroyo, Stephen J. Andersen
Masterproef voorgelegd voor het behalen van de graad master in de richting Master of Science in de
industriële wetenschappen: biochemie
Academiejaar: 2016 – 2017
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Acknowledgements
I would like to take this time to express my utmost and most deepfelt gratitude to my tutors; José Arroyo
and Stephen J. Andersen, my promotors Leen De Gelder and Korneel Rabaey, and all the other
wonderful people, the masterstudents, the PhD students, the postdoctorates and the professors of Cmet,
formerly known as Labmet who have made the past year, one of the best years in my life, and certainly
the best year of my academic record.
To my tutors I give an apology, for the amount of work and energy that was invested in me was
disproportionate to what I was able to give back. I cannot pay you back for what you have done, but I
do not forget the debts I owe. I am 95% sure, that if I had ended up with another thesis, with another
tutor, at another place, I doubt that the work I would be able to achieve would be even half of what I
managed to do this past year.
To my fellow masterstudents, to those who could laugh with everything and everyone, who played
pranks with and on me, and got pranked in return, I will miss those days we worked together side by
side.
To the PhD students and postdoctorates, I thank you for keeping me sane, grounded and telling me I
would succeed, even when I was convinced I would never make it to the end.
I would like to express special thanks to my family and loved ones, who did their best to make me
comfortable during sleepless nights, who believed in me, and spent so much effort in order to help me
get where I am today.
Thank you, Thank you, and thank you yet again,
Evert Vincent
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Abstract
The search for green and ecological alternatives to products of petrochemical origins and
products with a large carbon footprint has been thriving for the past few decades, and is still
ongoing, more and more with every day that passes. Stillage, a side-stream from bio refineries
and distilleries is not used as a waste. Through an energy-intensive process it is converted into
a small amount of feed, suitable for ruminants and other livestock. The carboxylate platform,
which forms the basis of this thesis, was formed after the realization that biomass, for example
thin stillage, can be fermented into bio-fuels, chemical building blocks and medicinal
components. Through a process known as chain elongation, substrates such as carbohydrates,
ethanol and lactate, to name a few, can be converted to MCFAs. There are still many barriers
before this technology spreads around the world.
Through a number of experiments, from fed-batch tests to simple batch tests and continuous
fermentation tests, several areas of investigation were explored and studied, from the effect of
hydrogen partial pressure to the influence of hydrolyzation products to in-line extraction. It was
discovered that the presence of solids in fermentation medium lowered ethanol consumption
rates by a factor of five, whilst the solid-free broth of an anaerobic fermentor fed with thin
stillage for the production of caproate almost completely inhibited ethanol consumption. It was
observed that even though at lower pH, as claimed by others, MCFA production is greater, there
is no net-biomass growth in these experiments at a pH of 5.5 or below. Hydrogen partial
pressure was found to inhibit the formation of odd-chain VFAs, whilst simultaneously pushing
chain elongation to the production of MCFAs. And last but not least, there was confirmation
that MCFA production was possible when using thin stillage which did not contain solids.
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Table of Contents Introduction ............................................................................................................................................. 1
Biomass & forms of biological waste ................................................................................................. 1
Biomass side stream: Stillage .......................................................................................................... 2
Carboxylate platform ....................................................................................................................... 4
Undefined mixed cultures................................................................................................................ 4
Production of SCCAs (C2-C5) ........................................................................................................ 5
Production of MCFAs (C6-C12) ..................................................................................................... 6
Extracting MCFAs ........................................................................................................................... 10
Precipitation ................................................................................................................................... 10
Membrane distillation .................................................................................................................... 11
Liquid/liquid separation................................................................................................................. 11
Electrochemical separation ............................................................................................................ 11
Adsorption ..................................................................................................................................... 12
Context of this thesis ......................................................................................................................... 13
Challenges and hypothesis’s: ........................................................................................................ 13
Materials and methods ........................................................................................................................... 15
Influence of PH2 on ethanol consumption and MCFA production ................................................... 15
Inhibition of ethanol by the feed matrix ............................................................................................ 16
Continuous fermentation of solid-free undiluted thin stillage ........................................................... 17
Analytical methods ............................................................................................................................ 18
Calculations ................................................................................................................................... 19
Results ................................................................................................................................................... 20
Influence of PH2 on MCFA production and ethanol consumption ................................................... 20
Inhibition of ethanol oxidation by the feed matrix ............................................................................ 24
Continuous fermentation of solid free undiluted thin stillage ........................................................... 34
Phase 1: Feeding solid-free undiluted thin stillage ........................................................................ 34
Phase 2: sludge retention ............................................................................................................... 36
Phase 3: Direct electrochemical extraction ................................................................................... 41
Discussion ............................................................................................................................................. 46
Influence of PH2 on MCFA production and ethanol consumption ................................................... 46
Inhibition of ethanol oxidation by the feed matrix ............................................................................ 47
Continuous fermentation of solid-free thin stillage ........................................................................... 49
Phase 1: feeding solid-free undiluted thin stillage......................................................................... 49
Phase 2: Sludge retention .............................................................................................................. 49
Phase 3: Direct electrochemical extraction ................................................................................... 50
Bibliography .......................................................................................................................................... 52
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List of Figures
Figure 1: metabolic pathway for chain elongation of acetate to caproate through ethanol [65] ............. 7
Figure 2: metabolic pathway for chain elongation of acetate to caproate through ethanol [65] ............. 8
Figure 3: Daily maximum hydrogen partial pressure in reactors .......................................................... 20
Figure 4: Concentrations of even-chain RBO products and lactate ...................................................... 21
Figure 5: Product distribution (%-COD) for all reactors ...................................................................... 22
Figure 6: Ethanol production rates (gCOD/L.d) for the four reactors ................................................... 23
Figure 7: average ethanol concentrations (gCOD/L) over time for all series ....................................... 24
Figure 8: maximum ethanol consumption rates (gCOD/L.d) for all series ........................................... 25
Figure 9: VFA concentrations (gCOD/L) shown for all series ............................................................. 26
Figure 10: initial acetate and propionate production rates for all series ............................................... 27
Figure 11: Maximum production and consumption rates after lag phase (gCOD/L.d) for all series .... 28
Figure 12: net consumption/production of substrates and products in gCOD/L ................................... 28
Figure 13: Average ethanol concentrations in each series (gCOD/L) ................................................... 29
Figure 14: Maximum consumption rates of ethanol (gCOD/L.d) in each of the series ........................ 30
Figure 15: Average VFA concentrations (gCOD/L) in each of the batch series for the duration of the
experiment ............................................................................................................................................. 30
Figure 16: Average initial production rates (gCOD/L.d) for acetate and propionate in each of the series
............................................................................................................................................................... 31
Figure 17: Average maximum consumption and production rates of the various VFAs and ethanol ... 32
Figure 18: Net consumption/production of the various VFAs and ethanol (gCOD/L) ........................ 33
Figure 19: Feed composition during Phase 1 ........................................................................................ 34
Figure 19: Even-chained RBO substrates and products concentrations in both reactors ...................... 35
Figure 20: Average Concentrations (gCOD/L) and production rates (gCOD/L.d) for both reactors
during days 20-28 .................................................................................................................................. 35
Figure 21: Concentration of suspended solids (gTSS/L) in both reactors ............................................. 36
Figure 22: Composition of the feed during phase 2 .............................................................................. 37
Figure 23: Even-chained RBO substrates and products concentrations (gCOD/L) and production rates
(gCOD/L.d) in R1.................................................................................................................................. 38
Figure 24: Even-chained RBO substrates and products concentrations (gCOD/L) and production rates
(gCOD/L.d) in R2.................................................................................................................................. 38
Figure 25: Concentraction of soluble solids (gTSS/L) in both reactors during phase 2 ........................ 39
Figure 26: Concentrations (gCOD/L) of lactate, glyerol, 1,3 PDO and ethanol in the feed, R1 and R2
during phase 2. ...................................................................................................................................... 40
Figure 27: Composition of feed during phase 3 (gCOD/L) ................................................................... 41
Figure 28: Even-chained RBO substrates and products concentrations (gCOD/L) and production rates
(gCOD/L.d) in R1.................................................................................................................................. 42
Figure 29:Concentration of undissociated and dissociated even-chain VFAs and lactate in the middle
compartment .......................................................................................................................................... 43
Figure 30: Even-chained RBO substrates and products concentrations (gCOD/L) and production rates
(gCOD/L.d) in R2.................................................................................................................................. 44
Figure 31: Concentrations (gCOD/L) of lactate, glyerol, 1,3 PDO and ethanol in the feed, R1 and R2
during phase 3 ....................................................................................................................................... 45
List of Tables Table 1: COD and BOD values of whole stillage from various sources and feed stocks [15, 16] .......... 3
Table 2: Composition of the different waste streams derived from stillage [17] .................................... 3
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Table 3: thermodynamic values and reaction stoichiometry for various SCCAs through multiple
pathways [49, 53, 54] .............................................................................................................................. 5
Table 4: thermodynamic balances for caproate formation through ethanol [37, 47] .............................. 8
Table 5: thermodynamic balances for caproate formation through lactate [65] ...................................... 9
Table 6: Theoretical dissociation concentrations of various VFAs at different pH values ................... 10
Table 7: Compositions of the various feed batches used in: Influence of PH2 on MCFA production and
ethanol consumption .............................................................................................................................. 16
Table 8: Composition of fermentation media of the various series used in: Inhibition of ethanol
oxidation by the feed matrix: primary run ............................................................................................. 16
Table 9: Composition of fermentation media of the various series used in: Inhibition of ethanol
oxidation by the feed matrix .................................................................................................................. 17
Table 10: Composition of SM used for inoculum preparation and fermentation medium in: Inhibition
of ethanol oxidation by the feed matrix ................................................................................................. 17
Table 11: SL-10 composition (1L) used for SM medium ..................................................................... 17
Table 12: 7vit (10x) composition (1L) used for SM medium ............................................................... 17
Table 13: overview of the timeline of experiment 4. Phase 1: days 1-30, Phase 2: days 30-60, Phase 3:
days 60-end ........................................................................................................................................... 18
Table 14: Average concentrations (gCOD/L) for both reactors during days 20-28 .............................. 35
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List of acronyms
A.D. Anaerobic
Digestion
BES of 2-bromoethanesulphonate
BOD Biological Oxygen
Demand
COD Chemical Oxygen Demand
C.R. Complete Retention
CSTR Continuously Stirred Tank Reactor
DDG Dried Distillers’ Grain
DDGS Dried Distillers’ Grain with Solubles
EC Enrichment Culture (inoculum)
HI High hydrogen partial pressure
LCFA Long Chain Fatty Acid
LO Low hydrogen partial pressure
MCFA Medium Chain Fatty Acid
Pi Pilot culture (inoculum)
RBO Reversed-β-Oxidation
SCCA Short Chain Carboxylic Acid
SM Synthetic Medium
SRT Sludge Retention Time
TSS Total Suspended Solids
VFA Volatile Fatty Acid
VLCFA Very Long Chain Fatty Acid
X%S medium with Solids
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Introduction Each year, over 70000 square kilometers of forest are cut down in order to fuel the global farming economy,
heating and cooking, the paper and the wood industry. In 2015, 7,861 million tons of coal were produced,
and by 2030, the global consumption of coal is estimated to increase to 9.05 billion tons [1]). In Belgium, in
2011, almost 185 million m³ of water were used by various industries, and usage is projected to grow
[2].Global consumption increases in all areas, whether we are speaking about food, land, water, energy or
fuels. Day after day, the needs of the global community increase, and the lifespan of our natural resources
decreases. In order to address this issue, increasingly worldwide, countries have started pushing towards
more green alternatives, such as renewable energy, bio-fuels, and recycling. Over the years, these adaptations
have increased in quantity, but the world is still lacking in ability to close the cycle and be able to recover as
much from waste as is necessary for consumption. The drive for recovery of resources from waste includes
(focuses on?) the fact that there is a rapid depletion of sources of minerals, some estimated to be fully depleted
within the next 50 years [3], the need to provide clean water for drinking and domestic use, and many more.
Now, research on resource recovery from waste is being done across the world, not only for reducing
pollution, but because advances in science and technology now have changed waste management from being
a cost, to being a potential additional source of income for those industries who produce a waste stream which
is not being used to the utmost of its capabilities. Companies are now actively searching and funding research
for the technology and application of product recovery, whether it is stripping spare circuit boards for gold,
silver and copper [4], harvesting biogas from organic waste [5], or filtering proteins out of waste water from
the food and drink industry [6].
The valorization of a resource is defined as the increase of value of a substance or product through artificial
means. Of the many valorizing techniques used in the world, perhaps the resource with the widest arrange of
applications is biomass. Valorization of biomass does not follow the same line of thought as the treatment of
biomass, even though these two often go hand in hand. The largest difference between the two is the
difference in economic value. Treatment is a cost which is a necessity, whilst valorization is a source of
profit. When talking about the valorization of biomass, one typically speaks about valorizing a biomass waste
stream, i.e. a byproduct of an industrial process in which bio-based goods are made or processed. Such
streams which are readily valorized include, amongst others, whey to produce proteins and lactate, sludge
from wastewater treatment plants for biogas, cellulose and chitin-rich biomass for bio-polymers [7]. One of
the key factors that plays a driving role in developing more bio-based valorization is the need to reduce
fossil fuel usage, due to the costly nature of fossil fuels, the non-renewable aspect of it, as well as the
environmental impact.
Biomass & forms of biological waste Biomass is defined as the sum total of organic fuel derived from living or deceased organisms[8]. Industries
that produce raw biomass are typically logging industries and agricultural industries, the latter which can be
sub-divided into different branches, such as industries for the production of food, feed, textiles and oils.
Processed biomass is most often in the form of food or drink, but also in the form of furniture, bio-fuels,
bioplastics, and more recently, a whole range of bio-based chemicals. And then there is the biomass waste,
which is produced by the industries which produce or process raw biomass. Biomass waste has as many
different forms as there are companies in the various branches of the bio-based world. It can range from
specific aqueous streams, such as whey from the cheese industry, stillage from bio-refineries, sludge from
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treatment plants and sawdust from the paper industry. Each of these different streams have different qualities
and properties, which largely determine what can be done with them.
Municipal solid waste (MSW) is a complex type of waste, produced by individuals, families, governments
and public institutions, as well as commercial industries. MSW contains, amongst other things, food wastes,
paper, wood plastics, glass and metal [9]. MSW in the United States consists of 29.6% organics, 35.5% paper,
13.2% plastics, and an assortment of other, nonorganic wastes. On average, the potential for MSW with this
composition to produce methane was measured to be at 78.2m³ per ton [10]. In the end, apart from sorting
MSW into the different components and recycling, the tendency remains to simply landfill the organic
fractions, and harvest the methane produced during the anaerobic fermentation that spontaneously occurs
within said landfills [11].
Another biomass waste stream is agricultural waste. The waste streams from agriculture have a tendency to
be completely used as either animal, fertilizer, or other direct applications. Because of the organic nature of
this waste stream, no or little treatment is needed prior to use in the various areas. However, to say every bit
of the waste stream is used in such manners would be false. In fact, there are many parts to the agricultural
waste streams that are not reused. In 1996 in the Netherlands, more than 73% of agricultural wastes did not
find its way to recycling [12].
Wood waste streams, which are mainly produced as the results of commercial forestry, pruning of public
parks, or industrial wood processing. In 2012, 52.9 million tons of wood were treated in the EU. 46% of this
waste stream was incinerated with recovery of energy, whilst 51% was recycled [12]. The remainder is either
landfilled, or incinerated without energy recovery [13].
Sludges and liquid waste, derived from waste treatment, are mainly landfilled (over 50%) or recovered in
other ways (42.6%). Typical sludge recovery consists of elemental recovery, such as of phosphor (0.5-0.7%
total solids), nitrogen (2.5-5% total solids) and carbon, the latter being extracted in the form of energy through
incineration or more commonly, anaerobic digestion. Other than elemental recovery, sludge can also be used
to produce organic molecules such as polyhydroxyalkanoates, proteins, enzymes and volatile fatty acids
(VFAs). Although 50% of sludge produced in Europe is landfilled, this mainly occurs in countries where
sludge is treated as waste. In other countries, such as Belgium and the UK, sludge is more often seen as a
valuable resource, due to its nutritional value [14].
Biomass side stream: Stillage
Stillage is an organic side stream produced as a by-product by the distillation of fermented grains from both
the bio-ethanol industry as well as the food and drink industry. Stillage is produced regardless of which
substrate is fermented, whether it is rice, wheat, corn or sugar crops such as beet. When the substrates, after
the starch is broken down into small saccharides by enzymes, are fermented, the resulting fermentation beer
is then distilled. Thus, the fermentation beer is split into two different fractions; a fraction with a high ethanol
concentration (the desired product), and the stillage. As a result, the stillage is mainly composed of leftover
saccharides, husks and other unused components. A rough estimate indicates that up to 20L of stillage may
be produced for every liter of ethanol produced through distillation [15]. The fact that produced stillage can
have chemical oxygen demand (COD) concentrations as high as 100g/L means that this side stream must
undergo a strenuous treatment to reduce its impact on the environment in the case of disposal or reuse in
other sectors. However, the actual composition of stillage differs greatly depending on the feedstock, the
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process used and the final product. In table 1 a representation of the biological oxygen demand (BOD) and
COD of a few sources of stillage is shown.
Component Brewery
Guinness
Brewery
fermencam
Winery
Sofavinc
Corn
distillery
Beet
fermentation
Beet
molasses
COD (mg/L) 1118 26907.33 457.67 56000 65000 91100
BOD (mg/L) 337.67 25044.67 40 37000 38000 44900
Table 1: COD and BOD values of whole stillage from various sources and feed stocks [15, 16]
The first and most influential way in which the BOD/COD can be decreased from the stream, is by
centrifuging the whole stillage into two fractions called thin stillage (aqueous), and wet distillers’ grain
(WDG). Thin stillage can then undergo evaporation after which it becomes known as syrup. Both WDG and
syrup are used in animal feed, separate or together, in which case they combine to form dried distillers grain
with solubles (DDGS). DDGS is seen as a high-quality feedstock for animals, and is heavily scrutinized in
terms of nutritional value, digestibility and other important characteristics. In table 2, a summary is shown
of some of the average characteristics of the three fractions which are obtained from whole stillage.
DDGS (g/L) WDG (g/L) Thin stillage (g/L)
Dry mass 888 353 77
Soluble components 219 31
Hydrophobic
components
103 34
Proteins 221 129 1
Carbohydrates (starch) 308 (46) 139 (21) 2 (0.4)
Glucose 0.7
Lactic acid 13
Glycerol 11
Ethanol 0.5
Table 2: Composition of the different waste streams derived from stillage [17]
From table 2 it is possible to see that the composition of these three side products is very different, and due
to these inherent differences, different fields of application are open for further processing of these streams.
Starting from whole stillage, studies have been made to use this as feed for the production of lactate, after
which the remnants could be valorized to the form of animal feed [18, 19] or for the production of biogas by
manner of anaerobic digestion (A.D.) [20]. WDG only sees use as an animal feed[21]. DDGS, like WDG, is
mostly used for animal feed, and depending on its source it is supposedly better for some animals than others
[22, 23]. The difference is that there also exist novel adaptations of DDGS, more specifically for the
production of various solvents such as butanol and acetone [24], and more interesting approaches such as
reusing DDGS for a second fermentation cycle to produce more ethanol [25]. For whole stillage, DDGS and
WDG, before any fermentation for the production of new chemicals or products can take place, there is an
intense need to hydrolyze the leftover carbohydrates into a fermentable state, in the form of physical,
chemical or enzymatic processing, which adds to the cost of production. In the end, it is the thin stillage that
has the widest field of applications in industry. These applications include the more mundane such as
production of biogas [26, 27] or recovery of energy, but a large spread of uses based on the fact that lactate
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and glycerol can be produced in thin stillage. Glycerol and lactic acid can be produced in quantities up to
47g/L of glycerol and 4g/L of lactic acid through repeated ethanol fermentation [28], although more
optimized fermentation processes can produce lactic acid at concentrations of above 42g/L [29]. A simple
method of extraction for example is to harvest the lactic acid and glycerol through filtration [30], but by using
the present components, being lactate and glycerol, amongst others, as substrate for fermentation, it is
possible to produce a wide array of economically interesting products including but not limited to, ethanol,
single cell oils and general chemicals [31]. For example, the glycerol present in thin stillage can easily be
fermented to butanol, with higher production rates compared to using synthetic media [32, 33]. It has also
been shown that by using genetically engineered strains of bacteria, it is possible to convert the vast amount
of glycerol present into more ethanol, thereby increasing the yield [34]. In another study, G. lucidum, a
fungus, was used to convert the soluble and remaining substrates in thin stillage into easily harvestable
polysaccharides[35]. From these examples, it can be shown that thin stillage is an excellent substrate
provider for many different fermentation processes, and it is for this reason that thin stillage was the waste
stream chosen to produce the desired products of this study.
Carboxylate platform
The carboxylate platform is the term given to mixed-culture fermentation of biomass where the desirable end
products are carboxylates, which can be further modified to bio-fuels [36]. Currently, success has been made
in producing carboxylates with a 2-8carbon chain [37] from waste streams, although further accomplishments
in the field could allow for the production of carboxylates with longer chain lengths. The carboxylate platform
is still in development, but could eventually reach the same degree of importance as the sugar platform and
the syngas platform. The carboxylate platform is a process identical to anaerobic digestion, in which biomass
is anaerobically fermented to a methane/carbon dioxide mixture, with the aim being to produce as much
methane as possible. The difference is that the carboxylate platform does not aim for the production of
methane, but cuts off at the previous step, the acetogenesis step. From biomass waste, the complex polymers
are first hydrolyzed, producing monomers. After this come the acidogenesis and acetogenesis steps, where
carbohydrates, proteins and lipids function as electron donors, and internal molecules serve the purpose as
electron acceptors, with the electron-accepting molecules, the carboxylates, being the desirable end-product
of the carboxylate platform. Carboxylates are molecules with at least one COO- tail, and although there are
many different substances which contain one or more carboxyl groups, the carboxylate platforms focus on
the simple straight chain molecules whose main defining characteristic is the carboxyl group. The length of
the carbon chains of all carboxylates vary from 2 (acetate) to 26 (hexacosanoic acid). The collection of
carboxylic acids with a carbon chain length of 2-5 are referred to as short chain carboxylic acids (SCCA), a
carbon chain length of 6-12 is called a medium chain fatty acid (MCFA), and for longer carbon chains the
terms can be long chain fatty acids (LCFA) or very long chain fatty acids (VLCFA). All of these molecules
can be produced by organisms, although more complex organisms are (currently) required to produce LCFAs
and VLCFAs [38].
Undefined mixed cultures
A major advantage of the carboxylate platform is the possibility of applying undefined mixed cultures. This
is in stark contrast to for example the sugar platform, which requires sterilized feed substrate, and
fermentation reactors with pure cultures. The various advantages that mixed cultures bring with them are
plentiful. For one, it removes the need for sterilization, which saves time, energy and capital. This also
includes the feed, which can be raw, non-heat-treated materials, except in the case that pretreatment is
required to hydrolyze the larger and more stable compounds into fermentable monomers. Then there is also
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the ability to utilize impure streams, such as waste. Mixed cultures are more stable in response to shifts in
organic loading rate, composition of feed or other such shifts in parameters.. Furthermore, the efficiency with
which the available organic matter is converted to useful products is greater in the carboxylate platform when
compared to the sugar platform, or indeed, compared to using pure cultures due to a reduction of the amount
of oxidation to gaseous substances which occurs in the aerobic sugar platform [39], and due mixed cultures
suffer less from inhibitory components in mixed streams, which means a greater organic matter conversion
efficiency when fermenting mixed feed streams [40].
Production of SCCAs (C2-C5)
SCCAs are commonly produced products, with applications ranging from preservatives [41], production of
industrial polymers such as polyvinyl alcohol [42] and cellulose acetate butyrate [43] and pharmaceuticals
such as betamethasone valerate [44]. Typically, acetate and propionate are produced through chemical
means [45], but can also be produced by fermentation of sugars [46] or glycerol [47]. SCCAs are water
soluble components, with no upper limit on their solubility. Apart from the solubility, each SCCA has
another difficulty which prevents low cost extraction. Acetate, for example, is produced as a salt, in which
form it is difficult to separate from fermentation broth. Propionate has low added value when produced
through fermentation, where the used substrate can account for up to 50% of the product cost, which means
in order for an economically viable production, high yields of propionate are required. As for butyrate and
valerate, in order for down-stream processing techniques to have a lower financial impact, high titers of
product are required. However, due to the fact that acetate and propionate are produced as by-products
during the fermentation of butyrate and valerate respectively, it is difficult to produce high purity products
due to their similar nature. One way of overcoming this problem is through the use of artificial electron
carriers such as methyl viologen [45]. SCCAs can be produced both through anabolic (Table 3) and
catabolic metabolism, where in the case of the latter, carboxylic acids can be formed through diamide
pathway fragmentation of proteins after acid hydrolysis [48]. Examples of anabolic production of SCCAs
include the autotrophic production of acetate and propionate from biogas containing either a CO/H2
mixture [49, 50] or a CO2/CH4 mixture [51]. As for the larger SCCAs, table 3 shows that butyrate is
produced mostly by using other organic molecules as a substrate, mainly ethanol, lactate and acetate. One
study has shown that butyrate can also be produced using only CO2 as the carbon source with a microbial
electro synthesis technique [52].
Substrate Product ΔGr0 (kJ/mol)
Glucose + H2O Acetic acid + Ethanol + 2H2 + 2CO2
ethanol + H2O Acetic acid + H+ + 2H2 +10.11
lactic acid + H2O Acetic acid + 2H2 + CO2 28.51
4H2 + 2CO2 Acetic acid + H+ + 2H2O -86.78
4CO + 2H2O Acetic acid + 2CO2 -154.6
2CO + 2H2O Acetic acid -114.5
Acetic acid + H2O + CO2 + 3H2 Propionic acid + 3H2O -76.5
3HCO3- + 2H+ + 7H2 Propionic acid + 7H2O -181.1
Lactic acid + H2 Propionic acid + H2O -43.32
ethanol + acetate n-butyrate_ + H2O -40.34
2 acetate + H+ + 2H2 n-butyrate_ + 2H2O -47.55
lactic acid + acetic acid n-butyric acid + H2O + CO2 −57.52
Table 3: thermodynamic values and reaction stoichiometry for various SCCAs through multiple pathways
[49, 53, 54]
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Production of MCFAs (C6-C12)
MCFAs are carboxylic acids where the aliphatic tail has between 6-12 carbons. The longer the tail is, the
lower the oxygen: carbon ratio is, which means that the hydrophobicity is increased, but also their usefulness
as fuels increases as shown in table 2. Currently, caproate (C6), caprylate (C8) and capric acid (C10) are
mainly produced from palm oil and other animal fats[53, 54]. This is inefficient, as can be seen from the
composition of palm oil (caproate: 0.2%, caprylate: 3.3%, capric acid: 3.5%) [55]. Caproate can, however be
extracted or produced through a few non-biological pathways, such as conversion of ethylene, and from
propanal or other hydrocarbons through oxidation [56]. However, due to the rise in petroleum costs, and the
environmental impact of petroleum, fermentation is rapidly becoming the preferred manner of production
over the petrochemical route. Caproate not only has potential as a possible bio-fuel, but it also has an
antimicrobial effect and is as such used as an antimicrobial agent [57, 58]. It is also used as a drug to lower
the chances of preterm delivery in pregnancy [59]and for the production of organic chemicals. But these are
not the main reasons why producing caproate is seen as a high-worth fermentation product. The crux of the
matter is that extraction of caproate from fermentation broth or other aqueous solutions is remarkably easy.
This is due to the low solubility of caproic acid (and caprylic acid). As a consequence, an oil phase separation
takes place when the concentration reaches 10.08g/L (0.79g/L for caprylic acid)[60]. This means that any
produced MCFAs do not need to be extracted by means of extensive downstream processing, but can be
extracted with simple procedures.
MCFAs are produced by micro-organisms under strictly anaerobic conditions as a sink for reducing
equivalents, which are formed when, for example, ethanol is oxidized to acetate for ATP production, where
the reducing equivalents would otherwise accumulate. The process by which medium chain carboxylates are
produced is the reverse-beta oxidation (RBO), also known as the chain elongation cycle. During this cycle,
a two carbon atom, acetyl CoA (reducing equivalent), is added to one of the SCCAs with each cycle. This
way, acetate is elongated to butyrate. Butyrate is elongated to caproate, and caproate to caprylate. If the
starting SCCA is propionate, then similarly, the elongated products will also have odd-numbered chains.
Because the production of even-chain elongation and odd-chain elongation depends mostly on the starting
substrate, the focus will be placed on the production of even-chained MCFAs, primarily due to the fact that,
when starting from sugars, more acetate is formed rather than propionate, where propionate is the starting
molecule for odd-chain RBO. Seeing as how thin stillage contains a large amount of sugars and
carbohydrates[61] as well as ethanol, thin stillage forms an almost ideal feed for even chained RBO.
However, acetate and ethanol can also be provided from an external source, produced by autotrophic
organisms, or obtained by the catabolism, hydrolyzation or oxidation[62] of larger molecules, as stated in
Production of SCCAs. An example of alternative RBO-substrate supply is when a reactor was fed with
hydrogen and carbon dioxide, and successfully produced MCFAs[63, 64]. The hydrogen and carbon dioxide
are fixated by homoacetogenic bacteria to form acetate, and then other acetogenic bacteria can further convert
the acetate to ethanol, using hydrogen as the electron donor. However, this type of setup is subpar compared
to simply feeding a reactor directly with ethanol. In terms of anabolic production of acetate, the pathway
taken varies depending on the type of substrate, and the thermodynamic and stoichiometric models have
already been represented in table 3. Below we will discuss the RBO of even chained MCFAs from ethanol,
lactate and acetate.
7
The figure below is a representation of the double cycle required to go from acetate to caproate using ethanol
as the source for reducing equivalents and energy.
Figure 1: metabolic pathway for chain elongation of acetate to caproate through ethanol [65]
From figure 1, it can be seen that the only place where ATP is produced is when ethanol, is oxidized to acetic
acid. This conversion produces 2 NADH molecules, which would normally accumulate and cause the
metabolism to enter dormancy. In order to continue growing and metabolizing, an extra 5 molecules of
ethanol are oxidized to acetyl CoA for every ATP molecule generated through oxidation of ethanol to acetate.
In order to stop these acetyl CoA molecules from accumulating, they either combine with only each other to
form butyrate, or combine with acetate to the same effect. During the conversion of crotonyl CoA to butyryl
CoA, a ferredoxin redox complex, the bifurcation system (not pictured above) allows for the regeneration of
NADH to NAD+, which also produces molecular hydrogen from protons. In this manner, the microorganisms
can sustain their metabolic activity by excreting their waste product, namely butyrate and the longer-chained
carboxylic acids [65].
Substrate Product ΔGr0 (kJ/mol) Repetitions Total ΔGr
0 (kJ/mol)
ATP generation Ethanol + H2O Acetate + H+ +
2H2
+10.11/+7.22 1 +10.11/+7.22
Cycle 1 Ethanol +
Acetate
n-Butyrate + H2O -38.62/-40.32 5 -193.10/-201.68
Cycle 2 Ethanol + n-
Butyrate
n-Caproate + H2O -38.74/-38.00 5 -193.70/-190.00
8
Overall caproate
formation
12 ethanol + 3
Acetate
5 n-caproate +
4H2 + 8H2O
-30.55 -183.59/-182.78
Table 4: thermodynamic balances for caproate formation through ethanol [37, 47]
The RBO cycle for production of caproate through lactate (Fig. 2) is very similar RBO through ethanol. The
two chief differences are for one, that for every lactic acid molecule converted to acetate, a CO2 molecule is
produced, resulting in a lower carbon efficiency. The other problem with using lactate as the source for
energy and reducing equivalents however, is the existing competing pathway, the acrylate pathway, through
which a shift from even-chained RBO to odd-chained RBO is caused [65, 66].
Figure 2: metabolic pathway for chain elongation of acetate to caproate through ethanol [65]
Substrate Product ΔGr0 (kJ/mol)
ATP generation Lactate + H2O Acetate + CO2 + 2H2 -8.79
Cycle 1 [65] Lactate + Acetate n-Butyrate + H2O + CO2 -37.52
9
Cycle 1 [36] 2 Lactate + H+ n-Butyrate + 2H2O -83.74
Cycle 2 Lactate + n-Butyrate n-Caproate + H2O + CO2 -57.65
Overall caproate formation 15 Lactate 5 n-caproate + 10H2 +
5H2O + 5CO2
-41.32
Table 5: thermodynamic balances for caproate formation through lactate [65]
From here, it can be seen that the chain elongation through ethanol is a thermodynamically more appropriate
pathway for the production of caproate when compared to the use of lactate, but this difference can be
attributed to the formation of a large amount of CO2, which implies a much lower carbon efficiency. It is also
not clear which conversions actually take place with lactate fermentation, seeing as how there is a higher
probability of odd-chained carboxylates being formed. As illustrated above, Cavalcante et al assume that
lactate and acetate are both required for butyrate production, whilst Zhu et al. [67] have shown that butyrate
production using only lactate is also feasible, and that the preferred substrate for chain elongation depends
on the inoculum.
As stated before, medium chain length carboxylic acids can only be produced by bacteria under strictly
anaerobic circumstances. This is due to the fact that, when oxygen is present, any present SCCAs and MCFAs
are consumed as an energy rich food source, and are oxidized to form CO2 and H2O. Not only does
fermentation with the intent of producing MCFAs require an anaerobic atmosphere, it is crucial for there to
be a minimal hydrogen partial pressure, PH2, of 10-2kPa. This minimal PH2 is required in order for the chain
elongation process to be thermodynamically feasible, more specifically, the bifurcation system which
regenerates NAD+ from NADH, which conserves energy and electrons[68, 69]. The PH2 also has an effect on
the metabolic pathways of homoacetogens and hydrogenotrophic methanogens. Homoacetogens use CO2 and
H2 present in the atmosphere to produce acetate, which can as a result be used by the chain elongating bacteria
to produce more MCFAs. To this end, the presence of homoacetogens is not a negative factor, providing that
they do not bring the hydrogen concentration to a level that is too low. The same goes for methanogens,
which also consume hydrogen, however, in contrast to homoacetogens, steps are taken in order to remove
methanogens from the fermentation culture. The reasoning behind the discrimination between the different
hydrogen consumers is the fact that amongst methanogens, there are acetoclastic methanogens which not
only consume hydrogen but also acetate. The consumption of acetate by acetoclastic methanogens would
cause the chain elongation to halt at the first step, which is why, in order to be able to produce MCFAs, a
method of inhibiting methanogens is needed. There are two widely used measures. The first is the use of 2-
bromoethanesulphonate (BES), an inhibitor with a greater selectivity for methanogenic bacteria when
compared to other inhibitors such as propionic acid or sodium nitrate which also inhibit the production of
MCFAs [70], and BES dosage is recommended to be used at concentrations of 0.5mM, for the inhibition of
methane producers [71]. However, to use BES at a large scale requires a large amount of BES, inflating the
cost of production, and although it is an inhibitor of methanogens, other bacteria are also influenced, which
would mean decreased production rates of MCFAs. The other option, which is cheaper and less intensive, is
to lower the pH of the reactor to a point where the growth of methanogens is inhibited. A pH of 6 is already
sufficient to completely limit methane production [72], but many studies that rely on pH to inhibit
methanogenic bacteria use lower pH values, ranging from 5 to 5.5 [73, 74].
10
Extracting MCFAs MCFAs have a low solubility. This particular quality means that evaporation, distillation, precipitation or
other energy-intensive extraction techniques normally reserved for organic molecules are not necessary,
unless the MCFAs have to be separated from other oily substances. The core of caproate extraction lies in
somehow increasing the concentration of undissociated caproic acid to the point at which phase separation
occurs, after which the oily layer can easily be harvested. The trouble lies in producing such a concentrated
stream when working with a biological production system, namely fermentation. Due to the long aliphatic
tail of MCFAs, these molecules can diffuse easily across the cytoplasmic membrane of cells. Once inside
they can interfere with cytoplasmic functioning [75]. The concentration of carboxylic acids needed to inhibit
growth and/or metabolism strongly depends on which carboxylic acid is taken into account, as well as the
organisms present in the fermentation culture. Microorganisms which produce MCFAs will naturally be able
to endure higher concentrations of the same MCFAs, but unless the fermentation is carried out in two stages
as opposed to single stage fermentation, presence of MCFAs will cause problems. In two stage fermentation,
with the first stage being a fermentor where sugars are oxidized to ethanol and acetate and the second being
the chain elongation fermentor, the non-MCFA producing bacteria are kept separate from the fermentor with
MCFA production. In a single-stage fermentation tank however, there is no separation of the primary
producers and the chain elongating bacteria. This means that there is a need to keep MCFA concentrations
as low as possible, so that production rates can be kept as high as possible. Undissociated butyric acid, for
example, can inhibit growth at levels of 47.9 mmol/l [76], whereas MCFAs such as caproate and caprylate
have a critical concentration of their undissociated forms at 10.5mmol/L and 0.62mmol/L [77]. At neutral or
high pH, this means that the concentration of caprylate in both dissociated and undissociated forms needs to
be higher than 78 mmol/L. However, if there is a need to work at pH values as low as 5, the maximum
allowable total concentration of caproate and caprylate is 25.3 and 1.4mmol/L (calculated using table 6)
respectively. To this end, an investigation of various extraction techniques will be discussed here.
pKa %Dissociated
form at pH 3
%Dissociated
form at pH 4
%Dissociated
form at pH 5
%Dissociated
form at pH 6
%Dissociated
form at pH 7
Acetate 4.756 1,72 14,92 63,69 94,61 99,43
Propionate 4.87 1,33 11,89 57,43 93,10 99,26
Butyrate 4.83 1,46 12,89 59,66 93,67 99,33
valerate 4.83 1,46 12,89 59,66 93,67 99,33
Caproate 4.85 1,39 12,38 58,55 93,39 99,30
Caprylate 4.89 1,27 11,41 56,30 92,80 99,23
Capric acid 4.9 1,24 11,18 55,73 92,64 99,21
Table 6: Theoretical dissociation concentrations of various VFAs at different pH values
Precipitation
Through the application of salts such as calcium carbonate or ammonium bicarbonate directly to the
fermentation tank, carboxylic acids in solution are converted to calcium- or ammonium carboxylate salts.
The reactor effluent can then be concentrated by means of dewatering processes to the point where crystals
of salt carboxylates are left behind [78]. However, this type of process is not an in-line extraction technique,
which means it would not serve to fulfill the goal of reducing MCFA concentrations inside the reactor in
order to limit product inhibition and toxicity.
11
Membrane distillation
Membrane distillation is a low-cost, low energy process in which fermenter liquid is heated up to 55C. On
the other side of the membrane, the permeate is kept cool, with the temperature gradient being the driving
force of MCFA diffusion. The end result of this membrane step is a mixture with up to 10% MCFA by
weight. This concentrated stream is then further processed using an extraction column, rectification and
water-stripping column. In the extraction column, solvents such as ethanol or methyl tert-butyl ether are
used, which can be recovered in a different down-stream process[78]. This type of process requires
multiple steps, which come at a cost and environmental impact. This process can be performed in-line with
a reactor, however, the recycle stream of the membrane separation step will be changed in some way, due
to having been heated up and cooled down again.
Liquid/liquid separation
This technique relies on the affinity that carboxylic acids have between two liquid media, the first being the
fermentation broth, and the other a solvent which has properties that would attract carboxylic acids across a
membrane, without the loss of water or other components. These liquids can include alcohols, ketones, ethers,
aliphatic hydrocarbons, organophosphates, and aliphatic amines [79].
A specific application of liquid/liquid separation is pertraction. A pertraction system consists of a
hydrophobic membrane, often in the form of a tube, through the inside of which which an extractant product
flows, whilst the feed consisting of fermentation broth flows around the membrane. The extractant product
used for extracting organic compounds is typically an oil based solvent, such as mineral oil [77, 80]. In these
studies, a second pertraction unit was applied to extract the MCFAs from the mineral oil to an alkaline
solution. Typically, once the alkaline solution has a high enough concentration of MCFAs, acid is added until
almost all of the dissolved caproate undergoes phase separation. The caproic acid can then be siphoned off.
Another method is by keeping the extractant alkaline, and allowing the MCFAs to precipitate as salts, which
can be recovered after filtration. In this type for dual pertraction system, the membrane which extracts the
desired component from the feed is called the forward module, and the membrane which extracts the
components from the first extractant product into another aqueous solution is called the backward module.
Whilst the first extraction phase relies on the hydrophobicity of MCFAs, the second is simply based on a
concentration gradient. In order to keep the gradient steep enough, the concentration in the alkaline solution
had to be kept low, which is achieved through continuous inline extraction using membrane electrolysis.
Electrochemical separation
There are two different kinds of electrochemical separation, with the first being membrane electrolysis, and
the other being electrodialysis.
Membrane electrolysis
Membrane electrolysis is an extraction where two liquids, namely the anolyte and catholyte, are separated by
a membrane, and depending on the type of membrane, only anions or cations can cross over. Anion exchange
membranes are used for the extraction of MCFAs, and the fermentation broth is recycled over the cathode
compartment. The dissociated MCFAs have a negative charge, and will thus cross over the membrane to the
anode compartment. The potential across the membrane is heavily affected by the concentration gradient
across the exchange membrane (among others, conductivity, distance between electrodes, resistance of the
membranes, pH gradient, etc.). When the concentration of dissolved molecules in the anolyte compartment
is too high, the power that is required to extract additional amounts from the fermentation broth/catholyte
increases rapidly. This explains why this technique is especially useful for extracting MCFAs such as
12
caproate. When the molecules reach the anode, they become protonated by the protons produced at the anode,
due to the protonation, are only present in their undissociated form. As seen before, the undissociated form
has a very low solubility, due to their long hydrophobic chains and their neutral charge, and from a certain
concentration (10g/L for caproate), they undergo phase separation. By recycling the anolyte over an external
anolyte chamber, oils phase droplets are removed from the inner workings of the electrochemical cell and
can accumulate in the external receptacle. Due to this, the concentration of MCFAs dissolved in the anolyte
liquid will never surpass the solubility limit, and cause an exponential increase in power usage/ potential
across the exchange membrane.
The use of membrane electrolysis has shown positive results in multiple studies, both in terms of extraction
efficiency, and in terms of increased productivity of MCFAs. This is achieved on one hand thanks to
increased hydrogen partial pressure[73, 81], and on the other by the removal of the RBO products which
indirectly inhibits RBO due to inhibiting the microorganisms responsible for the production of ethanol and
acetate from sugars and carbohydrates [81].
Another added advantage of using an electrochemical cell is that the hydroxide ions created by hydrolysis at
the cathode [82] also reduce the need for pH control in the fermentor through adding of caustic. In fact,
electrochemical cells are regularly used primarily for pH control, and can easily be set up to only become
active when the pH of the fermentation broth drops below a certain value, as determined by the operator.
This represents a decrease in operational costs, as well as removing an environmentally hazardous parameter,
increasing the sustainability of the project.
Electrodialysis
This is a setup composed of two anion and one cation exchange membranes arranged in parallel. The anode
is submerged in an acidic solution (or distilled water which becomes acidified due to hydrolysis at the anode
producing protons). Next is a cation exchange membrane, which allows protons from the acidic solution to
cross over into the next compartment, through which a distilled water stream, serving as an extractant, flows.
The extractant is separated from a third compartment by an anion exchange membrane. The third
compartment is the compartment through which a mixed stream containing the to-be-extracted carboxylic
acids flows, from which the carboxylic acids need to be extracted. Separating the third and fourth
compartments is another anion exchange membrane. In the fourth compartment, the cathode is submerged in
an alkaline solution (or distilled water which becomes alkaline due to hydrolysis at the cathode producing
hydroxide ions). The hydroxide ions formed at the cathode cross over the anion exchange membrane, and
force the dissociation (also deprotonation) of carboxylic acids in the mixed stream. Due to their dissociation,
the carboxylates gain a negative charge. Due to the subsequent electrostatic attraction between the negatively
charged carboxylates and the positively charged anode, they are pulled across the next anion exchange
membrane into the extractant, but cannot continue on to the anode due to the cation exchange membrane
limiting movement of carboxylates. However, the cation exchange membrane does allow for the protons
generated at the anode to pass over into the extractant. Once the protons come into contact with the
carboxylates, the carboxylates once more attain their undissociated carboxylic acid state. The carboxylic
acids at this point do not have a net charge anymore, and thereby remain unaffected by electrostatic
interactions so long as there are sufficient protons in the extractant stream [83].
Adsorption
Carboxylic acids can be adsorbed onto ion exchange resins, and provide a clean and otherwise unchanged
stream that can be recycled to the reactor. The problem lies in the high capital and expenditure costs, as well
13
as the need for continuously cleaning the resins or exchanging them for fresh resins. No references to
industrial application of this extraction method were found.
Context of this thesis This thesis was meant to play a supporting role in a demonstration project ongoing at the Center for Microbial
Ecology and Technology. In this project a pilot scale fermentation and extraction system has been built that
produces a mixture of MCFA from undiluted thin stillage. The pilot scale installation is comprised of a
fermenter where MCFA are produced at a pH of 5-6, and an electrochemically assisted pertraction unit that
extracts MCFA from the filtrate of the fermentation broth into an alkaline solution. The MCFAs are then
subsequently removed from the alkaline solution through electrochemical membrane separation, and are
transferred into an anode compartment. Once the concentration of undissociated MCFAs reaches their
respective maximum solubility points, an oil layer will form in the anolyte. This oil layer can then be siphoned
off. Through the operation of the pilot plant, a number of challenges have surfaced, and an investigation of
possible answers or alternative solutions was needed, which resulted in this thesis.
Challenges and hypothesis’s:
In the pilot, fed with a mixture of fermentation beer (rich in ethanol) and thin stillage from wheat
fermentation, there was no net ethanol consumption. There were two hypothesis’s which could explain this
occurrence. The first hypothesis was that PH2 affected the rate at which ethanol was consumed, as well as the
production rate of chain elongation products. From literature, it has been seen that a minimum PH2 is required
in order for chain elongation to start, although it has also been shown that high PH2 cause buildups of butyrate
and propionate, leaning towards propionate which is not a substrate for even-chain elongation but for odd-
chain elongation. At PH2 pressures above 0.098atm, ethanol oxidation to acetate coupled with ATP formation
is thermodynamically unfavorable, as discussed earlier in the carboxylate platform. To see whether the PH2
truly is a cause of reduced ethanol consumption, a first experiment was performed to evaluate the effects of
high and low PH2 on reactors fed with an ethanol enriched stream. A second hypothesis for the decreased
ethanol consumption rates can be attributed to the presence of solids, or more specifically, the presence of
products that originate from the hydrolization of solids. Although the exact mechanisms are not known, it
has been shown that increasing the solids loading rate causes increased ethanol titers [84]. The increased
ethanol titers in the given example are desired, however reports have been made on the inhibitory result that
hydrolyzation of cellulosic material could potentially cause [15].
A second goal is to investigate the application of membrane electrolysis as means to extract MCFAs from
fermentation medium with high extraction rates, and lower costs without liquid-liquid extraction. At an early
stage, the pilot used a pertraction system to deliver stream of dissolved MCFAs to an electrochemical cell.
The major disadvantages are firstly, the fact that the flowrate of MCFAs from fermentation medium across
the pertraction system and into the catholyte of the electrochemical cell is heavily limited by the surface area
of the membrane. This means that, if it is desired to increase the speed at which MCFAs are extracted, it is
necessary to apply larger separation membranes. A second disadvantage is the fact that organic solvents are
used in the pertraction process. These organic solvents do not only represent an additional expenditure, but
are also not environmentally friendly. From a life cycle assessment[85], the three largest contributors to
pollution and global warming were the addition of ethanol to stimulate chain elongation, solid waste
management during an acidification step, and the use of organic solvents in the liquid-liquid extraction
system. The third major disadvantage is the fact that a pertraction system consists of numerous parts, these
being the membrane modules, but also the organic solvent and alkaline solution. Pumps are needed to pump
the organic solvent and alkaline solution. In short, the inclusion of a pertraction system not only limits the
14
rate at which MCFAs can be extracted from the reactor, but also increases operational expenditures, due to
the number of pumps necessary for recirculation of the various liquids
As for the third area of investigation, the hypothesis was that by using solid-free undiluted thin stillage, chain
elongation would still be possible. Not only because of the mechanical issue with clogging that was explained
before, but also because there is reason to believe that hydrolyzation of the solids present in the feed are
partially responsible for decreased ethanol consumption and MCFA production rates. When solids derived
from plant matter are hydrolyzed, compounds named tannins are released into the surrounding broth. Tannins
are polyphenols which can comprise up to 50% of dry matter in leaves and stems of floral organisms, and
inhibit the growth of a large spread of organisms, including yeasts, bacteria of the genus Clostridium and
fungi[86]. Tannins are concentrated in the husks of spent grains, which form the vast majority of the solids
present in thin stillage. The presence of tannins has led many studies in chain elongation on thin stillage or
similar streams to be performed with diluted thin stillage. In this way, the inhibitory effects of tannins and
other such compounds are lessened in impact. However, in this thesis and the pilot project, efforts are made
to keep the whole process as environmentally friendly as possible, and using large amounts of clean water to
dilute a dirty stream is the opposite. But there is another perspective, that there might still be a use for the
solids. At the pilot, the culture used for RBO is a mixed culture, meaning that there are many different species
of bacteria, each with their own metabolic part in the grand scheme of things, and each with their own
optimum survival strategy. There is a case to be made for the possibility that there are certain bacteria which
require carrier materials in order to thrive. In the case that bacteria that fit this description are the primary
producers, or in other words are responsible for primary conversion of dissolved carbohydrates and other
compounds to RBO substrates, then removing of solids from the process might prove detrimental, because
chain elongating bacteria are unable to process larger polymers. To test once and for all whether or not the
solids are of importance to the chain elongation process, a continuous experiment was set up where undiluted
solid-free stillage was fed to duplicate reactors.
15
Materials and methods
Influence of PH2 on ethanol consumption and MCFA production In this experiment, four batch reactors were implemented. Two had a volume of 300ml, and two had a volume
of 1200ml. the smaller reactors were used to simulate environments with a higher PH2, hence forth referred
to as HI reactors, and the two large reactors were used to simulate an environment with low PH2, referred to
as LO reactors from this point onwards. One of each type of reactors (one HI and one LO) were inoculated
with inoculum obtained from the pilot, given the prefix PI, whilst the other two reactors were inoculated with
an inoculum derived from a continuously running reactor where an enrichment culture (EC) performing chain
elongation was grown anaerobically on synthetic medium (see material and methods: experiment 3) enriched
with ethanol and acetate, at pH 7. The latter inoculum is positive for chain elongation through ethanol, and
is used in this experiment as the positive control. The reactors inoculated with the known chain elongator are
given the prefix EC. Both inocula were obtained by centrifuging medium from respectively the pilot and
from the EC, and washing the pellets three times with substrate-free synthetic medium (SM), the composition
of which is described in tables 10, 11 and 12. The starting conditions of the experiment were 20ml of
respective inocula, and 180ml of tap water. Every day, 20 ml was removed and 20ml of feed (reduced by
volume needed for pH balancing) was fed to each of the reactors. Stillage was obtained from Tereos Starch
and Sweeteners, Aalst (Belgium), where bio-ethanol is produced from the fermentation of wheat and
subsequent distillation. After distillation, the remaining biomass left after the removal of most of the ethanol
is separated into a light fraction (thin stillage) and a heavy fraction (DDGs). The DDGs are sold as an animal
feed additive, whereas the thin stillage, rather than being concentrated to stillage syrup, is simply send to an
A.D. facility, for energy recovery in the form of biogas. Beer was also used in this experiment. The
fermentation beer is, like the thin stillage, obtained from Tereos Starch and Sweeteners. Rather than being a
waste stream, this is the pre-distillation product, which would normally have the ethanol extracted from it.
The difference between the two waste streams is that fermentation beer still contains a high concentration of
ethanol, as well as the other components found in thin stillage. Because this product is not separated into a
heavy and light fraction, it has a higher amount of solids compared to the thin stillage. The reactors were
constantly stirred using magnetic stirring devices, and were kept at a constant temperature of 28˚C. The
headspaces of the LO reactors were flushed (20 cycles) with N2 gas every day after sampling and oxygen
free for anaerobic conditions, after which the headspace pressure was relieved in all reactors. The pH of all
reactors was adjusted to pH6 on a daily basis. Because the duration of this experiment was scheduled to be
longer than 30days (>3HRTs), no biological triplicates were implemented for this experiment. In the
beginning, a mix of beer and thin stillage was used as feed, but was later changed to thin stillage with ethanol
dosed. In both feeds, ethanol concentrations varied around 12g/L, with the compositions of the three different
feed batches shown in table 7.
From the daily discharge of effluent, 10ml was frozen for total soluble solids (TSS)/volatile soluble solids
(VSS) measurements. 5ml was centrifuged for 10 minutes ate 7800 rpm and filtered using 0.45µm filters
(Chromafil® Xtra PA-45/25, Macherey-Nagel, Germany). 4ml was kept for pH measurement, and for
calculating (equation 1) the amount of 1M NaOH needed to bring each of the reactors back to pH 6. The last
1ml was centrifuged for 10 minutes at 14000rpm, and the pellet was kept in frozen state for potential DNA
analysis of the culture.
𝑌 = ((
𝑋0.1𝑀4 ) ∙ 180
10) + 1.3
With Y being the volume (ml) of 1M NaOH needed to bring the reactor back to pH 6, X0.1M being the amount
of 0.1M NaOH needed to bring 4ml of sample to pH6, and 1.3 being the volume of 1M NaOH needed to
16
bring 20 ml of feed to pH6. Every day, prior to sampling and feeding, pressure in the headspace was measured
using a tensimeter (INFIELD 7, UMS, Germany) and a sample of the headspace was analyzed for each
reactor.
Inhibition of ethanol by the feed matrix This experiment is separated in two runs, a primary run and a rerun. In the primary run, 5 series of small
batch reactors (penicillin bottles) in triplicates with a working volume of 15ml were used. For fermentation
medium, each triplicate group had a different ratio of SM:Reactor effluent without solids, as shown in table
10, with SM compositions shown in tables 12,13 and 14. The reactor effluent without solids was obtained by
siphoning off permeate from the solid-liquid separation membrane installed at the pilot. Prior to mixing the
two media in the corresponding ratios, the ethanol concentration in both media was adjusted to 12g/L, and
acetate to 6g/L, while the pH was adjusted to pH6. After preparation of the batch reactors, each reactor was
inoculated with EC inoculum. The reactors were kept on a shaker in a 34˚C room, and were flushed 20 times
with N2 gas at the start of the experiment. 0.6ml samples were taken on days 0,1,3,5,7,9 and 12. Samples
were centrifuged for 10 minutes at 14000rpm and the supernatant collected whilst the pellets were discarded.
pH was measured again at the end of the experiment.
Percentage broth (series #) 0% (1) 10% (2) 25% (3) 50% (4) 100% (5)
Volume Broth (mL) 0 1.35 3.375 6.75 13.5
Volume SM (mL) 13.5 12.15 10.125 6.75 0
Volume inoculation (mL) 1.5 1.5 1.5 1.5 1.5
Total (mL) 15 15 15 15 15
Table 8: Composition of fermentation media of the various series used in: Inhibition of ethanol oxidation
by the feed matrix: primary run
In the rerun, 6 series of triplicates were run, also in penicillin bottles, but this time with working volumes of
50ml. The sixth series was added so as to observe the effects of solids on ethanol consumption and MCFA
production. Solids from 45 ml of pilot effluent were separated from the broth by centrifugation. The resulting
pellet was then washed three times in SM without substrates. The solids were then resuspended in 45ml of
SM with substrates, and added to the series 6 batch reactors. Each batch was also inoculated with 5ml EC
inoculum, which has been prepared according to what was seen in experiment one and in the primary run of
this experiment. Other changes were also implemented. The beginning acetate concentration was reduced
from 6g/L to 4g/L, sampling was done every four hours as opposed to daily sampling, and headspace
composition was analyzed on a daily basis as well.
Percentage broth (series #) 0% 10% 25% 50% 100% 0%S
Volume Broth (mL) 0 4.5 9 22.5 45 0
Volume SM (mL) 45 40.5 36 22.5 0 45
Volume inoculation (mL) 5 5 5 5 5 5
Total (mL) 50 50 50 50 50 50
Feed batch 1 (mix of beer and
thin stillage) (1:5)
Feed batch 2 Feed batch 3
Ethanol 20.9 ±2.24 12.7 ± 0.77 14.3 ± 1.84
Lactate 0.32 0.40 0
Acetate 0 0.67 3.0
Propionate 0.02 0.10 0.8
Butyrate 0 0 1.4
Table 7: Compositions of the various feed batches used in: Influence of PH2 on MCFA production and
ethanol consumption
17
Table 9: Composition of fermentation media of the various series used in: Inhibition of ethanol oxidation
by the feed matrix
Component Concentration (g/L)
K2HPO4 0.31
KH2PO4 0.23
NH4Cl 0.25
MgCl2 0.06
MgSO4•7H2O 0.05
NaHCO3 2.5
Yeast extract 1
Se-W 1mL
SL-10 1mL
7Vit(10x) 0.1mL
Table 10: Composition of SM used for inoculum preparation and fermentation medium in: Inhibition of
ethanol oxidation by the feed matrix
HCl (25%; 7.7 M) 10.00 ml
FeCl2 x 4 H2O 1.50 g
ZnCl2 70.00 mg
MnCl2 x 4 H2O 100.00 mg
H3BO3 6.00 mg
CaCl2 x 6 H2O 190.00 mg
CuCl2 x 2 H2O 2.00 mg
NiCl2 x 6 H2O 24.00 mg
Na2MgO4 x 2 H2O 36.00 mg
Distilled water 990.00 ml
Table 11: SL-10 composition (1L) used for SM medium
Vitamin B12 100.00 mg
p-Aminobenzoic acid 80.00 mg
D(+)-Biotin 20.00 mg
Nicotinic acid 200.00 mg
Calcium pantothenate 100.00 mg
Pyridoxine hydrochloride 300.00 mg
Thiamine-HCl x 2 H2O 200.00 mg
Distilled water 1000.00 ml
Table 12: 7vit (10x) composition (1L) used for SM medium
Continuous fermentation of solid-free undiluted thin stillage Two 5L continually stirred tank reactors (CSTR) (FLC-6, BelachBioteknik) were fed with solid free thin
stillage for an HRT of 10days, with one serving as a control (R2) and the other being an experimental reactor
(R1) during phase 1 of this experiment. The CSTRs had a built in pH probe (EasyFerm Plus, Hamilton,
Bonaduz, Switzerland), 2 pumps with pumpheads (Watson-Marlow, USA), 2 small pumps for acid and base
dosage, a level probe, a gas production column, a temperature probe and heating blanket which kept the
18
temperature of both CSTRs at 30ºC, and a stirrer with a helix blade with stirring kept at 100rpm, with reversal
of stirring direction. The solid-free thin stillage was obtained by allowing the thin stillage which was kept in
a 1000L cubic container to settle over the course of a few days, and collecting the supernatant, which had a
TSS of less than 0.1g/L. Feeding was done once an hour, and effluent was automatically discharged when
the level in the reactor exceeded 5L, as detected by the level probe. Both reactors were kept at a pH of 5,
with 5M NaOH dosage for pH control. The pH set point was later increased to 5.5, and then later again, to 6.
After 30 days, in phase 2, a ceramic membrane was attached to the R1, where a sludge retention time (SRT)
of 20 days was achieved, although later the SRT was changed to complete retention (C.R.). A pump capable
of high flowrates (600 series mid-flow process pump, Watson-Marlow, USA) was used to run the reactor
broth through the ceramic membrane with an rpm of 65. On day 60, at the commencement of phase 3, a direct
electrochemical extraction was performed on R1 using a three-chamber electrochemical cell, using a current
of 0.710 A. The electrochemical cell was built locally, using plexiglass (PMMA) frames (Vlaeminck A. NV,
Belgium). The internal chamber area was 0.0002m2 for each compartment (5*2*20cm). The outer plates
(14*2*24) were held together with bolts and wingnuts. Between each compartment, a rubber sheet was used
to prevent leaks. For the parts of the middle compartment, thicker versions of the same rubber sheets were
used, due to the corrosive nature of the middle compartment. The end plate of the anode compartment
contained an extra hole, through which the anode could be linked to a power bank. For the anode, a 5*20cm
titanium electrode coated with iridium oxide was used, whilst the cathode was a 5*15cm stainless steel gauze
with 0.5cm mesh. The anode was connected to an external anolyte reservoir, filled with 10% sulphuric acid,
which was continuously recirculated across the anode compartment. The middle compartment was also
connected to an external reservoir, where the solution in the middle compartment was recirculated from the
bottom of the reservoir to the bottom of the middle compartment, so as to avoid any oil-phase layer formed
in the middle compartment from flowing through and damaging various parts. The cathode compartment was
fed directly from R1, and returned to the feed port of the reactor.
Sampling during phases one and two consisted of taking 16ml samples from both CSTRs, 8ml kept separate
in a frozen state for TSS/VSS analysis, and 8ml was centrifuged for 10minutes at 7800rpm, and filtered using
a 0.45µm filter, before storage in a frozen state. From phase three onwards, samples from R1 were taken
from a sampling port placed after the cathode compartment of the electrochemical cell. Increased sample
volume from R2 was used, 25ml per sampling, in order to perform TSS/VSS from this reactor in triplicate.
8ml samples was also taken from the middle compartment by means of a sampling port.
day -8 0 31 47 56 60 67
Feed (R1, R2) TS SF-TS SF-TS SF-TS SF-TS SF-TS SF-TS
pH (R1, R2) 5 5 5 5.5 5.5 5.5 6
Sludge retention (R1) HRT HRT 2 X HRT 2 X HRT C.R. C.R. C.R.
Extraction (R1) X X X X X ✓ ✓
Table 13: overview of the timeline of experiment 4. Phase 1: days 1-30, Phase 2: days 30-60, Phase 3: days
60-end
Analytical methods
VFA analysis
C2-C8 fatty acids (including isoforms C4-C6) were measured by gas chromatography (GC-2014,
Shimadzu®, The Netherlands) with DB-FFAP 123-3232 column (30m x 0.32 mm x 0.25 µm; Agilent,
Belgium) and a flame ionization detector (FID). Liquid samples were conditioned with sulfuric acid and
sodium chloride and 2-methyl hexanoic acid as internal standard for quantification of further extraction
19
with diethyl ether. Prepared sample (1 µL) was injected at 200ºC with a split ratio of 60 and a purge
flow of 3 mL min-1. The oven temperature increased by 6ºC min-1 from 110ºC to 165ºC where it was
kept for 2 min. FID had a temperature of 220ºC. The carrier gas was nitrogen at a flow rate of 2.49 mL
min-1.
TSS/VSS analysis
TSS and VSS were performed according Standard Methods 2540D and E (APHA, 1997).
Organic Ion Analysis
Volatile fatty acids were analysed using a Dionex DX 500 ion chromatography system using an IonPac
ICE-AS1 column with 0.4 mM HCl as eluent and an ED50 conductivity detector. The lower limit of
detection was XX (here you fill in your calculated limit of detection, also taking care of any dilution
factors).
Inorganic Ion Analysis
Cloride, nitrite, nitrate, sulphate and phosphate were determined on a 761 Compact Ion Chromatograph
(Metrohm, Switzerland) equipped with a conductivity detector.
Headspace analysis
The gas phase composition was analyzed with a Compact GC (Global Analyser Solutions, Breda, The
Netherlands), equipped with a Molsieve 5A pre-column and Porabond column (CH4, O2, H2 and N2) and a
Rt-Q-bond pre-column and column (CO2, N2O and H2S). Concentrations of gases were determined by
means of a thermal conductivity detector.
COD analysis
COD was analysed with Nanocolor® kits (CODE; Macherey-Nagel)
Calculations
(1) Conversion g/L to COD/L
𝑋 𝑔(𝐶𝑎𝐻𝑏𝑂𝑐𝑁𝑑) 𝐿⁄ = 𝑋 ∙ (𝑎 + 𝑏
4−
𝑐
2−
3𝑑
4) 𝑔𝐶𝑂𝐷 𝐿⁄
(2) Organic loading rate
𝑂𝐿𝑅 = 𝑔𝐶𝑂𝐷 𝐿⁄ (𝑓𝑒𝑒𝑑)
𝐻𝑅𝑇
(3) Calculation of production rates
𝑔𝐶𝑂𝐷 𝐿⁄ 𝑑⁄ = 𝑔𝐶𝑂𝐷 𝐿⁄
𝐻𝑅𝑇
(4) Partial Pressure
𝑃𝑥(𝑎𝑡𝑚) = [1 + 𝑃𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 ∙ (
𝑉𝐻𝑆 + 𝑉𝑆𝑉𝐻𝑆
)
1013,25] ∙ 𝐶𝑥
With
▪ Px = The partial pressure of component x in atmospheric units (1atm = 1013.25 hPa)
▪ Pmeasured = The gas pressure measured in the reactor after sampling in hPa
▪ VHS = the volume of headspace in the reactors = Lfull-L0.2L
20
▪ Vs = the volume of sample taken + the volume removed from reactor prior to
measuring
▪ Cx = the concentration of component x in the headspace
Results Influence of PH2 on MCFA production and ethanol consumption In figure 3, a general overview of the PH2 in all reactors is shown for the duration of the experiment. On day
5 and day 6, PH2 in PiHI and ECHI reached local maxima respectively 0.50 and 0.77atm, after which they
respectively dropped down to 0.01atm and 0.12atm on day 10 for PiHI and ECHI respectively. From day 10
onwards, PH2 in both reactors increased gradually until day 21 in PiHI and day 29 in ECHI. Following this
gradual increase is a jump in PH2, where PH2 in PiHI remained relatively stable at 0.90 ±0.05atm between
days 25 and 35. In ECHI, a similar peak was observed, with a PH2 of 0.76 ±0.07 between days 30 and 35.
From day 35 onwards, in both of the HI reactors, PH2 then starts decreasing, at which point the experiment
ended. The PH2 in PiLO and ECLO remained at a low value, with maximum daily PH2 levels of 0.10±0.01
and 0.10±0.03atm, respectively.
Figure 3: Daily maximum hydrogen partial pressure in reactors
Figure 4 shows the evolution of component concentrations of the various VFAs and. Both of the EC reactors
show a lag phase for caproate production, which started to accumulate from day 7 onwards. Caproate
production in PiHI started immediately, whilst PiLO showed a 7-day lag-phase. In both of the LO reactors,
a slight buildup of lactate accumulated in the beginning of the experiment until day 8 for PiLO and day 10
for ECLO. In contrast, in treatments with high PH2 lactate buildup only occurred from day 23 in PiHI and
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 5 10 15 20 25 30 35 40 45
Pre
ssu
re (
atm
)
Time (days)
PH2
PiHI PiLO ECHI ECLO
21
from day 28 in ECHI, with a larger amount in PiHI than in ECHI. Higher acetate concentrations accumulated
at low PH2, averaging 5.64±0.24gCOD/L from day 28 onwards in PiLO and 5.15±0.15gCOD/L in ECLO,
whilst in PiHI and ECHI, acetate concentrations averaged 3.40±0.12g/L from day 32 onwards and 3.67±0.18
from day 28 onwards respectively. Propionate concentrations remained low in all reactors throughout the
experiment, although concentrations rose slowly over time, most notable in the LO reactors. Looking at
butyrate, PiLO maintained a concentration 2g/L higher than PiHI from day 9 onwards. This trend is not seen
in the EC reactors, although towards the end of the experiment, from day 30 onwards, ECLO sees a slow
accumulation of butyrate, which is not seen in ECHI, with a difference of 1.3g/L on the last day. For valerate,
a similar trend is seen as with propionate. Low concentrations were maintained throughout the experiment,
although concentrations did increase slowly over time in both of the LO reactors. Looking at caproate, stable
concentrations were reached on days 28-38, 30-35, 25-34 and 27-38 for PiHI, PiLO, ECHI and ECLO
respectively. After the stable phase seen in PiLO and ECHI, caproate concentrations dropped to 8.90±0.36
and 9.91±1.10gCOD/L respectively. In both the HI reactors, octanoate concentrations reached up to 0.66 and
0.59gCOD/L in PiHI and ECHI, whilst octanoate concentrations in PiLO and ECLO barely reached
concentrations of 0.33 and 0.14gCOD/L.
Figure 4: Concentrations of even-chain RBO products and lactate
22
Figure 5: Product distribution (%-COD) for all reactors
In figure 5, the relative proportions of VFA COD production are shown. This figure allows for a clearer view
of how the difference in PH2 affected VFA production performance in the four reactors. Apart from the
difference in the lag time which is required for production of MCFAs, the distribution of even and odd length
VFAs is affected, as well as the total amount of MCFA production. In both of the HI reactors, from day 28
onwards, 83.82%±1.86 and 84.01%±2.71 of the VFA-COD is in the form of even chained VFA products for
PiHI and ECHI respectively. On the other hand, during the same period, even chained VFA products took up
only 79.96%±1.18 and 77.93%±1.27 of the total VFA COD in PiLO and ECLO respectively. When observing
the total amount of MCFAs produced in each of the reactors from day 28 onwards, 64.16%±2.28 of VFA-
COD was in the form of MCFAs in PiHI, compared to 52.88%±3.89 in PiLO. Similarly, 63.71%±3.73 of
VFA-COD in ECHI was in the form of MCFAs compared to 53.08%±3.47 in ECLO.
In the four reactors, the applied levels of PH2 did not have a significant effect on ethanol consumption (Fig.
6). Ethanol concentrations remained similar in all four reactors throughout the duration of the experiment,
with a few solitary outliers spread throughout the data. The data does show, however, that the ethanol
concentrations in the LO reactors had the tendency of being slightly lower than in the HI reactors.
23
Figure 6: Ethanol production rates (gCOD/L.d) for the four reactors
24
Inhibition of ethanol oxidation by the feed matrix An experiment was designed to assess if chain elongation, with ethanol as electron donor, was negatively
affected by the composition of the fermented thin stillage. An enriched culture, adapted to utilize ethanol for
chain elongation, was exposed to the liquid fraction of the pilot effluent diluted to different levels with
modified M52 medium. The impact of the broth on the microbial process was evaluated by comparing ethanol
oxidation rates under the different conditions tested. Figure 7 shows the average concentrations of ethanol
measured in each of the batch series for the duration of the experiment. All treatments showed a lag phase in
ethanol oxidation that lasted 0.9d. Despite this initial lag-phase, all the ethanol was oxidized in the control
treatment after 2.9 d, and the same occurred in the rest of the treatments after 5 days.
Figure 7: average ethanol concentrations (gCOD/L) over time for all series
Looking at the average maximum ethanol consumption rates, a clear progression in average rates at which
ethanol is consumed can be seen from figure 8, where with increasing broth concentrations, ethanol
consumption rate decreases. The exception to this trend is the series with 100% broth, which has an almost
as high ethanol consumption rate as the series with 0% broth, which has the highest.
25
Figure 8: maximum ethanol consumption rates (gCOD/L.d) for all series
A clear progression of VFA concentrations (Fig. 9) was seen in function of medium composition. VFA
production ends its lag phase at 0.9 days, with the exception of the 50% broth series, which has a lag phase
duration of 2.9 days. As seen in Figs. 8 and 9, trends can be seen in the series with 0% broth through to the
series with 50% broth, with the 100% broth series breaking the trends. The first trend is the production of
butyrate, where the concentration of butyrate present in each series increases in relation to the amount of
pilot fermentation broth present in the fermentation medium. Another major trend is the production of
caproate, where the attained concentrations at the end of the batch experiment decrease from the 0% broth
series to the 50% broth series. In order of increasing broth concentration, butyrate concentrations at the end
of the batch experiment were 5.99±0.75g/L, 7.26±0.66g/L, 8.63±0.77g/L and 11.08±2.48g/L. Caproate
concentrations, in the same order, reached final concentrations of 6.37±0.25g/L, 4.90±0.22g/L, 4.49±0.44g/L
and 1.53±0.21g/L. In both of these trends, butyrate and caproate concentrations in both of these series differed
from the trend in the 100% broth series, with final concentrations of 8.31±0.86g/L and 7.80±0.39g/L
respectively.
26
Figure 9: VFA concentrations (gCOD/L) shown for all series
In each of the batch series, during the initial stages of the experiment, acetate and propionate production
occurred. With the exception of the 50% broth series, this initial production lasted for approximately one
day, whilst in the 50% broth series the initial production lasted 3 days, as seen in fig. 10. The rates at which
acetate and propionate were initially produced in the batches show some trending nature as well, shown in
fig. 11, with decreasing acetate production rates from the 0% to 25% broth series, but increasing propionate
production rates. In these two trends, both the 50% and 100% broth series do not follow the same pattern,
where the 50% broth series shows a higher initial acetate production rate and a lower initial propionate
production rate than expected. On the other hand, the 100% broth series shows minimal initial production
27
rates for both acetate and propionate, and is also the only series in which propionate is not consumed, but
produced throughout the duration of the experiment, as seen in fig. 10.
Figure 10: initial acetate and propionate production rates for all series
The composition of the fermentation medium also affects the speed at which the substrates are consumed,
and the speed at which the RBO products are produced. The maximum consumption and production rates
then (fig. 12) are also impacted. Maximum acetate and ethanol consumption rates are highest in the 0%
broth series, and the consumption rates of the two substrates decreases with increasing Pilot fermentation
broth in the fermentation medium of the batches, to the point where in the 50% broth series, the maximum
caproate production attained during this experiment was only 0.36±0.07gCOD/L.d. On the other hand, in
the 50% broth series, a maximum butyrate production rate of 9.00±1.21gCOD/L.d.
28
Figure 11: Maximum production and consumption rates after lag phase (gCOD/L.d) for all series
When looking at the total change of concentrations of dissolved VFAs and ethanol in each of the series (fig.
12), in each series, more COD was produced than there was being consumed. However, the 50% broth series
shows the lowest net production of VFA-COD when compared to the other series, even though the same
amount of COD in the form of acetate and ethanol was consumed, suggesting a greater CO2 production. In
the 100% broth series, a far larger net production of total VFA-COD was observed compared to the other
series. This suggests that there were additional sources of carbon, energy and reducing equivalents present
in the pilot fermentation broth.
Figure 12: net consumption/production of substrates and products in gCOD/L
29
When looking back at the results obtained from this experiment (Fig 10.), it can be seen that the major
changes in composition of the fermentation broth in terms of production and consumption occurred within
the first three days. Due to the fact that samples were not consecutively taken during this transitionary
period, the resolution of the obtained data was not great enough for a more in-depth look into the manner in
which the fermentation matrix influenced ethanol oxidation. Furthermore, when looking at the difference in
ethanol consumption in the 100% broth series (Fig. 8) and the ethanol consumption in the PH2 testing (Fig.
7), a hypothesis was formed that the presence of solids could possibly be responsible for the inhibition of
ethanol oxidation. In order to perform a more close-up investigation, a second batch test was designed and
implemented. In this second batch test, the goals were to strengthen the findings from the first batch test, as
well as to investigate the possible influence of solids derived from thin stillage on ethanol oxidation using
an additional batch series, the 0% broth with solids (0%S) series.
Figure 13: Average ethanol concentrations in each series (gCOD/L)
The results of the second batch experiment were markedly different in certain aspects from the first batch
experiment, such as the ethanol consumption. From figure 13, it can be seen that the ethanol concentration
in the various series decreased a lot less over time when compared to figure 8 of the first batch experiment.
However, a trend can still be seen, where less ethanol was consumed in the batches with higher pilot
fermentation broth than in the series with less. In contrast to the first batch experiment, there was no batch
series in which all of the available ethanol was consumed.
0
5
10
15
20
25
30
35
40
0.0 0.8 1.2 1.8 2.2 2.8 3.2 3.9 4.2 5.8
Co
nce
ntr
atio
n (
gCO
D/L
)
Time (days)
0% 10% 25% 50% 100% 10%S
30
Figure 14: Maximum consumption rates of ethanol (gCOD/L.d) in each of the series
When looking at the maximum consumption rates observed in each of the batch series (Fig 14), a similar
trend can be seen as was seen in figure 9 of the first batch experiment. The difference lies in the fact that,
where in the first experiment the 100% broth series did not follow the trend of decreasing maximum ethanol
consumption rates with increasing pilot fermentation broth, here the 100% broth series does follow the trend,
with only 0.22±0.17gCOD/L as the maximum consumption rate. Lastly, the 0%S broth series also showed a
much lower maximum ethanol consumption rate, with 0.97±0.34 gCOD/L.d compared to
5.28±0.37gCOD/L.d in the 0% broth series, even though neither of these series have any pilot fermentation
broth.
Figure 15: Average VFA concentrations (gCOD/L) in each of the batch series for the duration of the
experiment
31
Apart from the trend in ethanol consumption, trends can also be seen in the production of the various VFAs
(Fig. 15). Looking at the progressions of acetate concentration, in the 0%, 0%S, 10% and 25% broth series,
acetate concentrations start decreasing from day 2.2 onwards, whilst the acetate concentration in the 50%
and 100% broth series does not seem to be consumed in function of time. In the series with 50% broth, a bare
minimum of change in each of the VFAs can be seen, whilst in the 100% broth series, based on the data
shown in figure 14, changes in VFA concentrations might as well be statistical errors, rather than a
quantitative shifting of medium composition over time. From the graphs in figure 16, it is also possible to
see that there was a considerable amount of caproate and octanoate present in the filtered reactor fermentation
broth, with initial caproate concentrations of 6.11±0.46gCOD/L at the start of the experiment in the 100%
broth series. Broth concentration is also reflected in the Initial octanoate concentrations, with the 100% broth
series containing 1.06±0.15gCOD/L, and the 50% broth series containing 064±0.17gCOD/L. by looking at
the graphs in figure 16, estimates can be made of the respective lag phases for some of the series. The 0%,
10%, 25% and 0%S broth series both end their lag phases between 2.8 and 3.2 days, and no end to the lag
phase can be identified for the 50% and 100% broth series. In each of the series, butyrate and caproate
production begins from the start of the experiment, but production rates do not increase drastically until the
lag phase is over.
As in the primary run, a brief period of acetate and propionate production was seen in every series, although
the total amount produced and the production rate changed from series to series. In figure 16, the average
initial production rates for acetate and propionate are given. From the figure, a general trend can be seen,
where the series with higher broth concentrations have greater acetate production rates than the batches with
more SM. The 0%S series shows itself to be the exception to this rule, with almost exactly the same average
initial acetate production rate as the series with 100% broth.
Figure 16: Average initial production rates (gCOD/L.d) for acetate and propionate in each of the series
0
0.2
0.4
0.6
0.8
1
1.2
0% 10% 25% 50% 100% 0%SPro
du
ctio
n r
ate
s (g
CO
D/L
.d)
acetate propionate
32
Figure 17: Average maximum consumption and production rates of the various VFAs and ethanol
Some trends similar to figure 13 can also be seen in figure 16, where the maximum consumption and
production rates of each batch series are shown for the various VFAs and ethanol. After the series with 0%
broth, the series with 25 and 10% broth have the next greatest ethanol consumption rates, with 3.8±1.84 and
3.3±0.76 gCOD/L.d each. The series with 50% broth comes in fourth place, with a maximum ethanol
consumption rate of 1.5±1.19 gCOD/L.d, followed by the 0%S series, with 1.26 gCOD/L.d and rounding of
is the series with 100% broth, where almost no ethanol consumption took place, with 0.22±0.17 gCOD/L.d
being consumed. Butyrate production rates are in the same order, with the exception of series with 0%S
broth, which takes the number 2 place, with 6.2±0.98 gCOD/L.d being produced as opposed to the series
with 0% broth, where a maximum butyrate production rate of 8.3±1.64gCOD/L.d being observed. Worth
noting is the fact that, whilst acetate was consumed in the series with 0, 10, 25 and 0%S broth, a continuous
production of acetate was observed occurring in the series with 50% and 100% broth.
-10
-5
0
5
10
15
0% 10% 25% 50% 100% 0%S
Pro
du
ctio
n a
nd
C
on
sum
pti
on
Rat
es
(gC
OD
/L.d
)
acetate propionate butyrate
valerate caproate heptanoate
octanoate ethanol
33
Figure 18: Net consumption/production of the various VFAs and ethanol (gCOD/L)
In figure 19, net changes in the COD present in the form of the various VFAs and ethanol is shown. The 0%
broth series experienced a net decrease of 1.01±1.14gCOD/L, the 10%broth series a decrease of
0.82±0.69gCOD/L, the 25% broth series an increase of 4.64±9.23gCOD/L (large variation due to highly
elevated butyrate concentration being measured in one of the triplicates of this series on the last day), the
50%broth series an increase of 2.25±4.28gCOD/L (large variation due to one triplicate in the series
effectively consuming more COD), an increase of 1.85±1.11gCOD/L in the 100% broth series, and finally
an increase of 2.77±1.40gCOD/L was observed in the 0%S broth series.
Looking at the changes in COD present in acetate, the 0% broth series consumed the most acetate,
2.70±0.03gCOD/L, followed by the 10% and 25% broth series, which consumed 2.28±0.74 and
1.63±1.20gCOD/L respectively. In the 0%S series, a net decrease of acetate COD of 1.52±0.45gCOD/L was
seen. In contrast, both the 50% and 100% broth series rendered a net increase in acetate COD, with a net
production of 0.96±1.25gCOD/L in the 50% broth series, and 0.99±0.64gCOD/L in the 100% broth series.
What can clearly be seen from figure 19 is that the net production of butyrate increases from the 0% to 25%
broth series, with 7.17±1.32, 9.55±0.55 and 18.69±9.25gCOD/L net production in the 0%, 10% and 25%
broth series respectively. As for the 50 and 100% broth series, only 2.42±0.73 and 0.75±0.44gCOD/L of
butyrate was produced. In the 0%S broth series, a net production of 7.90±0.69gCOD/L was seen.
In terms of net caproate production, highest production was observed in the 0% broth series, with
7.10±0.85gCOD/L. as the concentration of broth in each series increases, the concentration of caproate COD
decreases. In the 0%S broth series, 1.56±1.15gCOD/L was produced.
-20
-15
-10
-5
0
5
10
15
20
25
30
35
0% 10% 25% 50% 100% 0%S
Ne
t ch
ange
in c
on
cen
trat
ion
s (g
CO
D/L
)
acetate propionate butyrate valerate
caproate heptanoate octanoate ethanol
34
Continuous fermentation of solid free undiluted thin stillage As this experiment was a complex one, there are many different streams of data which have to be analyzed
and compared.
Phase 1: Feeding solid-free undiluted thin stillage
In phase one, solid-free thin stillage was fed to both the experimental reactor (R1) and the control reactor
(R2), for 3HRTs (30 days) in order to get a baseline for both reactors, and to have an acclimatized culture
for the following phases.
During phase 1, the feed, which is a complex real feed subject to changes in composition as can be seen
from figure 20 was the only experimental condition. Figure 19 shows an overview of the composition of
the feed during phase 1. Apart from lactate and the components which are not present in large amounts, the
remainder of substrates such as ethanol, glycerol and 1,3-propandil (1,3 PDO), which contain the remainder
of observable COD in the feed continuously vary in concentrations over time. Lactate had an average
concentration of 2±0.7gCOD/L during phase 1. Maximum acetate concentrations in the beginning (days 0-
5) were 0.277±0.039gCOD/L and towards the end of phase 1 (day 25,26) was 0.74±0.07gCOD/L. Glycerol
concentrations were 2.72±0.031gCOD/L during the first 5 days, and from day 10 onwards,
5.21±1.32gCOD/L. 1,3PDO varied as well, averaging 10.32±0.51 on days 11 till 17, an isolated peak on
day 21, with 14.10 gCOD/L., and overlapping with phase 2, 12.74±0.54 gCOD/L on days 28-32.
Figure 19: Feed composition during Phase 1
During phase 1, even chained RBO substrates and products showed similar trends in R1 and R2, as seen in
the comparative figure 19. In R1, there was a lag phase of 7days in caproate production and lactate
consumption, which was not seen in R2. In R1, ethanol was present throughout phase one, with an average
of 0.86±0.64gCOD/l, whereas in R2, ethanol concentrations from day 1-12 were around 0.23gCOD/l, after
which a period of 10 days of higher ethanol concentrations emerged with an average concentration of
1.85±0.39gCOD/L. However, as stated, the intention of phase 1 was to reach a stable baseline in both
0
2
4
6
8
10
12
14
16
0 5 10 15 20 25 30
Co
ncn
etr
atio
n
(gC
OD
/L)
Time (days)
lactate acetate glycerol 1,3 PDO ethanol
35
reactors. This was achieved towards the end of phase 1, for the last 4 days (days 20-28). In figure 20, the
average concentrations of even-chained RBO substrates and products are shown. From figure 20, it can be
seen that the two reactors show similar trends, though in R1, higher butyrate and caproate concentrations
and production rates are seen. On the other hand, R2 shows a higher octanoate concentration and
production rate, and a lower ethanol concentration and production rate.
Figure 20: Even-chained RBO substrates and products concentrations in both reactors
Figure 21: Average Concentrations (gCOD/L) and production rates (gCOD/L.d) for both reactors during days 20-28
The results from figure 20 are re-represented in numerical form in table 14. From these results, it can be
seen that there is a higher odd-chain RBO production in R2, whilst at the same time, total MCFA
production in R2 is higher with 47.32% as opposed to 44.83% in R1.
acetate propionate butyrate valerate caproate heptanoate octanoate
R1 2.91±0.66 0.13±0.17 47.18±6.10 4.95±1.11 36.87±4.57 1.02±1.19 6.94±2.16
R2 6.97±4.50 0.33±0.08 41.53±3.33 3.85±0.70 34.02±4.65 2.40±0.25 10.90±2.60 Table 14: Average concentrations (gCOD/L) for both reactors during days 20-28
A last important parameter which will be discussed during phase one is the progression of TSS in both of
the reactors, as shown in figure 21. In the beginning of phase 1, there were still solids in the reactors left
over from before the reactors were fed with solid-free thin stillage. As a result it takes until day 5
0
1
2
3
4
5
6
7
8
0 5 10 15 20 25 30
Co
ne
ntr
atio
n
(gC
OD
/L)
Time (days)
R1
Lactate Acetate Butyrate Caproate Octanoate Ethanol
0
1
2
3
4
5
6
7
8
0 5 10 15 20 25 30
R2
36
approximately before the remaining solids in the reactors are washed out. Even after the solids remaining
from the reactor startup were washed out, TSS continuously decreased, even during the period from day
20-28, although the concentrations were more stable compared to earlier in the experiment, with 3.7±0.89
and 3.0±0.16 gTSS/L in R1 and R2 respectively.
Figure 22: Concentration of suspended solids (gTSS/L) in both reactors
Phase 2: sludge retention
In phase 2, a ceramic membrane was first attached to R2 (originally the experimental reactor) and later to
R1 (originally the control reactor). SRT was set up to be twice the HRT, and was changed towards the end
of phase 2 to C.R.
In figure 22, the composition of the major constituents in the feed is shown for days 31-59. In the
beginning, until day 32, some 1,3 PDO is still present from phase 1, which was discussed in the previous
section. In phase 2, lactate, acetate and glycerol values remained relatively constant, with
3.85±0.52gCOD/L in the form of lactate, 0.64±0.20gCOD/L in the form of acetate and 7.47±1.03gCOD/L
as glycerol. On day 42, a sudden shift occurred in the composition of the feed, concerning ethanol and 1,3
PDO concentrations. From day 42 until day 56, average ethanol concentrations were 18.6±0.51gCOD/L,
and average 1,3PDO concentrations were 10.73±1.41gCOD/L.
37
Figure 23: Composition of the feed during phase 2
Due to the changes in the feed, the composition and production rates of both R1, shown in figure 23 and
R2, shown in figure 24 changed drastically. Until day 33, the stable phase which was accomplished in R1
maintained its composition. From day 34 onwards, an increase in ethanol concentrations was seen in R1, as
well as an increase in butyrate concentrations during the same time period. In the period of 34-40, ethanol
concentrations increased from 0.02gCOD/L to 3.47gCOD/L. In the same period, butyrate concentrations
increased from 3.56gCOD/L to a maximum of 7.23gCOD/L. After this period, a sudden increase in ethanol
concentration is apparent, jumping from 3.47 to an average of 11.60±0.56gCOD/L until day 54, after which
concentrations dropped down to 3.58±0.16gCOD/L for the remainder of phase 2. Looking at the
productivity values for the products of even-chained RBO, shown on the right in figure 23, caproate
production levels averaged around 0.37±0.16gCOD/L.d until day 52, after which a short period of
heightened caproate production appeared, with a maximum of 2.5gCOD/L.d. During this period, octanoate
productions also increased in production rates, from an average of 0.07±0.017gCOD/L.d before day 52 to
0.25±0.04gCOD/L.d.
38
Figure 24: Even-chained RBO substrates and products concentrations (gCOD/L) and production rates (gCOD/L.d) in R1
In figure 24, the concentrations of substrates and products of even-chained RBO in R2 are shown on the
left, and the production rates on the right. On day 32, R2 was reinoculated after leakage with a mix of R1
effluent and water. During the following days, the system was still in the process of stabilizing, with a
momentary lapse due to unknown causes on day 40. After this lapse, a sudden increase in ethanol
concentration can be seen, with an average of 11.39±0.44gCOD/L during days 40-54, after which
concentrations decrease to 3.42±0.33gCOD/L. Looking at production rates, the average butyrate production
rate from day 39 until day 54 was 1.11±0.37gCOD/L.d, after which production rates decreased. Caproate
production restarted on day 42, with continuously increasing production rates until day 54, at which point a
peak in production rates occurred, with a maximum production rate reached of 3.7gCOD/L.d. After this
peak in caproate production, a decrease in production rates are observed, although the caproate
concentration in the reactor remained above 18gCOD/L.
Figure 25: Even-chained RBO substrates and products concentrations (gCOD/L) and production rates
(gCOD/L.d) in R2
39
Figure 26: Concentraction of soluble solids (gTSS/L) in both reactors during phase 2
From figure 25, which portrays the TSS in both reactors, it can be seen that the TSS in both reactors
remained low, averaging 3.22±0.95 and 2.78±1.60 in R1 and R2 respectively, with only a small amount of
fluctuation in R2, and almost none in R1.
In order to observe whether the elevated ethanol and 1,3PDO concentrations in the feed between days 42
and 56 had a different effect on R1 and R2, a comparative analysis is shown in figure 26. For lactate, the
concentrations in R1 remained low, with an average of 0.95±0.43gCOD/L from day 30 until day 47.
Between day 47 and day 56, no lactate was observed in R1, although afterwards, towards the end of phase
2, lactate concentrations increase. After the restarting of R2, a period of elevated lactate concentrations was
observed, with a maximum concentration of 7.46gCOD/L. This period lasted 5days, after which lactate
concentrations in R2 remained under the limit of detection. On day 40, a significant increase in
concentration in ethanol was observed in both reactors, going from 2.70gCOD/L in R1 and 0.96gCOD/L in
R2 to 11.61±0.56 and 11.39±0.44gCOD/L for R1 and R2 respectively. Between day 42 and day 45, ethanol
in the feed increased from 0 to 18.60±0.51gCOD/L. During the same period that increased ethanol
concentrations were observed in the feed, increased 1,3PDO concentrations were likewise observed, with
an average of 10.73±1.41gCOD/L. During this same period, elevated 1,3PDO concentrations were likewise
observed in R2, with an average of 3.30±0.50gCOD/L, but no concentration increase was observed in R1.
40
Figure 27: Concentrations (gCOD/L) of lactate, glyerol, 1,3 PDO and ethanol in the feed, R1 and R2
during phase 2.
41
Phase 3: Direct electrochemical extraction
In phase 3, a three-chamber electrochemical cell was added to R1. The current applied over the membranes
started at 0.511A, and was later increased to 0.711A. The application of electrochemical extraction caused
the pH in R1 to rise to a pH of around 6. The pH setpoint of R2 was then similarly increased from 5.5 to 6.
Figure 28: Composition of feed during phase 3 (gCOD/L)
After phase 2, at the end of which the ethanol and 1,3 PDO concentrations dropped down to 0gCOD/L as
seen in figure 27, there was a recurring situation as seen in phase 2, where elevated concentrations were
measured in the feed for both of these components. On day 63, 1,3PDO has a peak in concentration of
13.11gCOD/L, and is present for 5 days before not being present anymore until day 82, from which point
on, concentrations average at 0.92±0.65gCOD/L. As for ethanol, similar to 1,3 PDO, concentrations rose
again on day 63, and increased over time to a maximum of 8.88gCOD/L on day 77. After day 77, ethanol
concentrations decreased again to 3.39±0.44gCOD/L until day 96, where it dropped down to 0gCOD/L
once more. Lactate and acetate concentrations in the fee show a similar trend, with a slow increase in
concentration from 4.07 and 0.75gCOD/L to 6.12 and 2.96gCOD/L respectively on day 77. After day 77, a
constant in concentration of 5.57±0.28gCOD/L for lactate and 2.38±0.31gCOD/L was reached until day 98.
After day 98, lactate decreased over time to an end of 3.86gCOD/L, and acetate remained at
0.61±0.07gCOD/L from day 98 onwards. Looking at glycerol, there was a continuous increase in
concentration over time, 6.72 to 12.15gCOD/L.
42
Figure 29: Even-chained RBO substrates and products concentrations (gCOD/L) and production rates (gCOD/L.d) in R1
In order to observe the effects of the direct electrochemical extraction, the data from R1 has to be split into
multiple sections. In this first section, in figure 28, an overview is given of the complete concentration of
even-chained RBO substrates and products, compiled from the fermentation liquid, the middle
compartment of the electrochemical cell and the oil harvested from the electrochemical cell. A very
noticeable occurrence is the fluctuations of caproate concentrations and production rates during phase 3,
with local maximums of 10.3gCOD/L and 1.27gCOD/L.d on day 73, 14.70gCOD/L and 1.61gCOD/L.d on
day 89 and 1.44gCOD/L.d on day 98. During phase 3, lactate concentrations remained below 2gCOD/L,
except for a solitary peak on day 80, where the concentration was 2.53gCOD/L. Acetate showed a clear
trend, with increasing concentrations over time until a maximum concentration of 5.82gCOD/L is observed
at the end. Similar to the caproate concentrations and production rates, butyrate also has a fluctuating
characteristic, though not to the same extent. When looking at octanoate, for the first time during this entire
experiment, concentrations surpassed 2gCOD/L, with a maximum of 5.02gCOD/L and a maximum
production rate of 0.62gCOD/L.d on day 73. After this maximum, octanoate concentrations and production
rates steadily decreased over the course of the experiment until the end of phase 3, where a stable
concentration of 0.51±0.02gCOD/L was measured for the last three days. Ethanol concentrations were
relatively stable throughout phase 3, slightly decreasing over the course of phase 3 from 5.11gCOD/L until
a concentration of 3.71±0.16gCOD/L.
43
Figure 30:Concentration of undissociated and dissociated even-chain VFAs and lactate in the middle
compartment
The electrochemical cell experienced multiple mishaps, the frequency of which can be deduced from figure
29. These mishaps occurred on days 67, 76, 90, 98 and 101. The cause of these mishaps was due to
pressure buildup causing the middle compartment to be fouled as the result of a tear in the anion exchange
membrane, an error during attempted oil harvesting or spontaneous fouling of the middle compartment,
most likely due to leaks. Oil harvesting succeeded on two different occasions, once on day 101 and on day
105. In the first attempt at extracting, total caproate levels (both the dissociated form and the undissociated
form) reached a maximum of 11.22gCOD/L, or 5.09g/L. In the second attempt, total caproate
concentrations reached a maximum of 15.61gCOD/L, or 7.08g/L. In both of these cases, total caproate
concentrations stabilized before phase separation occurred. In the third attempt, total caproate
concentrations exceeded 22.87gCOD/L, or 10.38g/L and phase separation took place. Regrettably, in the
process of trying to harvest the formed oil layer, the middle compartment was fouled, and the extraction
had to recommence. In the fourth attempt, total caproate concentrations reached 27.7gCOD/L, or 12.58g/L,
and in the fifth attempt, 30.76gCOD/L or 13.96g/L. During the last week of phase 3, a more thorough
extraction was planned, with a daily follow up instead of sampling once every two days, but during this
period, most likely to the last fouling, R1 experienced heavy decreases in productivity of VFAs, and the
maximum total caproate concentration achieved during the last trial was only 15.23gCOD/L or 6.91g/L.
44
Figure 31: Even-chained RBO substrates and products concentrations (gCOD/L) and production rates
(gCOD/L.d) in R2
In order to determine the effect of the direct electrochemical extraction on production rates, a comparison
can be drawn between R1 and R2, using figures 28 and 30. By comparing the two figures, it can be seen
that for every analyzed component except for caproate, concentrations and production rates were a lot more
stable in the R2 reactor. Lactate concentrations remained low throughout phase 3 in R2, with a solitary
maximum of 2.45gCOD/L on day 84. For the remainder of phase 3, lactate levels remained below
1gCOD/L. Acetate concentrations were similarly low throughout phase 3 in R2, averaging
1.76±0.30gCOD/L. Butyrate concentrations showed a little more fluctuation when compared to acetate and
lactate, but even so the concentration remained relatively stable at 6.08±1.27gCOD/L during phase 3, with
a maximum of 8.59gCOD/L on day 88. As for caproate, the concentration at the start of phase 3 was
16.19gCOD/L. This high concentration was a remnant of the period during phase 2 where caproate
production was inflated due to the feed gaining a large amount of ethanol. From 16.19gCOD/L the
concentration decreased in a logarithmic manner until day 84, where a minimum caproate concentration of
3.53gCOD/L was measured. From this point onwards, the caproate concentration and production rate once
more increased to 16.87gCOD/L and 1.75gCOD/L.d on day 94. This was followed by a slight decrease
until day 103, after which, during the final week of the experiment, caproate concentrations increased once
more. Octanoate concentrations increased throughout phase 3, starting from day 49 during phase 2. At the
end of phase 3, a maximum octanoate concentration of 3.63gCOD/L was measured, with a production rate
of 0.37gCOD/L.d. finally, ethanol concentrations also remained stable throughout phase 3, with an average
concentration of 4.82±0.97gCOD/L.
In order to compare the whether the application of direct electrochemical extraction had an influence on the
consumption of the various components in the feed, the comparative study shown in figure 31 was
analyzed. With the exception of glycerol, the consumption behavior of R1 and R2 was similar for lactate,
1,3 PDO and ethanol. In the case of glycerol, however, it can be seen that more glycerol was consumed in
R1 when compared to R2.
45
Figure 32: Concentrations (gCOD/L) of lactate, glyerol, 1,3 PDO and ethanol in the feed, R1 and R2
during phase 3
46
Discussion
Influence of PH2 on MCFA production and ethanol consumption This experiment resulted in some interesting and novel observations. For one, the PH2 applied to the
reactors seems to have influenced the lag duration before lactate consumption began. As seen from figures
3 and 4, The reactors which were maintained at a lower PH2 showed some lactate accumulation during the
first HRT, whilst the HI reactors experience lactate accumulation during the latter part of the experiment. It
has been found that by decreasing the PH2 in a reactor would facilitate the consumption of lactic acid [87,
88], which the findings from the later part of the experiment support. However, the reason as to why the
LO reactors experienced a lag phase is not clear yet, however, it could be due to the fact that an elevated
PH2 allows for greater ethanol production from sugars [89]. Given that in the beginning of the experiment
the measured ethanol concentrations in all reactors was low, this endogenous ethanol production stimulated
by high PH2 could have caused a greater biomass production rate at the beginning of the experiment, which
translated into a faster start to lactate consumption.
Another finding which did not add up to what was found in literature, was the necessary PH2 required for
chain elongation, and therefore MCFA production, to occur. It is commonly accepted that, when the PH2 is
below 0.098atm, chain elongation is thermodynamically unfeasible [68, 69]. However, production of
caproate in the LO reactors started on day 6 and on day 8 for PiLO and ECLO respectively, whilst the
threshold of 0.098atm was not reached until day 13 and day 19 for PiLO and ECLO, respectively. Seeing as
how the headspaces of the reactors were sampled before feeding and flushing, this means that in 24 hours’
time, there was at no point an accumulation of H2 to the point that chain elongation was possible, according
to theory.
In literature, it was found that by maintaining a high PH2, ethanol oxidation is diminished, due to
unfavorable thermodynamics, but that this was not the case for MCFA producing bacteria, due to their
ability to get rid of excess reducing equivalents [90]. So according to this study, low increases in PH2 should
not have an effect on the ability of a chain elongating mixed culture to oxidize ethanol. This is in line with
what was seen in this experiment, where elevated PH2 up to 1atm did not influence the oxidation of ethanol,
but on the other hand, lowering the PH2 did not increase ethanol consumption rates either, which the same
source suggests it should. This finding lowered the expectation that the high PH2 achieved in the pilot
reactor was responsible for the decreased ethanol consumption.
Another difference which was observed between the HI and Lo reactors was the manner in which the VFA-
COD was distributed between SCCAs and MCFAs, but also in the distribution between odd- and even-
chain VFAs. In the HI reactors, more VFA COD was in the form of MCFAs compared to in the form of
SCCAs. In PiHI, 44.72±2.25% of the VFAs produced from day 24 onwards were in the form of SCCAs,
compared to PiLO, where 58.02±3.15% of VFA COD was in the form of SCCAs (p=0.005, paired t-test).
This is recurrent in the EC reactors, with ECHI possessing 47.73±5.76% of its VFA COD in the form of
SCCAs, and ECLO 56.74%±1.77. As for the odd-even distribution, 84.05±1.65% of COD in PiHI was in
the form of even-chained products, whilst in PiLO even-chained VFAs took up 79.91±1.01% of total VFA
COD. In ECHI and ECLO, a similar result is apparent, with 83.40±2.60 and 78.64±1.93% respectively of
VFA-COD in the form of even chained VFAs.
When looking at the effect of PH2 on chain length of produced VFA-COD, it has been established that at
higher hydrogen partial pressures, acetate is more readily converted to butyrate [91, 92], and butyrate in
turn to caproate, although in the case of the latter, it is only up to the point where caproate starts inhibiting
micro-organisms [93], after which hydrogen accumulation can be observed. In another study, it was
hypothesized that in order to maintain high caproate production rates, lower hydrogen partial pressures
47
would be advisable, but that an increase in PH2 would push the chain elongation process to longer chained
molecules [54].
When regarding the distribution between odd-and even chain VFAs, it is suggested that higher PH2 would
induce a greater propionate production, which would lead to more odd-chain VFAs [65, 66]. However, in
this experiment the opposite was found, where higher PH2 seemed to push the chain elongation more
towards even-chain elongation. After extensive research, a possible cause for this phenomenon was found.
As stated before, odd-chain VFAs are produced by chain elongation bacteria when propionate instead of
acetate is used as the starting molecule, which is then further elongated to larger odd-chained VFAs.
Propionate is a common hydrolization product of proteins, which are a major constituent in thin stillage,
where up to 19% of dry matter in thin stillage derived from corn consists of crude protein[94]. It has been
observed before, that at high hydrogen partial pressures, hydrolization of substrates including proteins is
inhibited [95]. This could be a plausible explanation for why more odd-chain VFAs were observed in the
LO reactors when compared to the HI reactors.
Conclusions:
• Lower or higher hydrogen partial pressure did not affect ethanol consumption. Under the
conditions, no ethanol consumption occurred even when the inoculum was derived from en
enrichment culture known to be positive for chain elongation through ethanol.
• Higher hydrogen partial pressures were beneficial for steering chain elongation towards the
production of MCFAs.
• Higher hydrogen partial pressures caused a greater amount of VFA-COD to be produced in the
form of even-chain products.
Inhibition of ethanol oxidation by the feed matrix After the first experiment, where the possibility of hydrogen pressure being responsible for the lack of
ethanol consumption was investigated, a second experiment was designed to investigate the second
hypothesis, namely whether there were components in the matrix of the feed which would inhibit ethanol
consumption such as phenolics, melanoidins and furfurals [15].
In the primary run of this experiment, reliable results were gained from the 0% broth through 4, which
showed a clear progression in parameters such as initial acetate and propionate production rates, subsequent
acetate and ethanol consumption rates as well as butyrate and caproate production rates. Because the series
with 100% broth broke the trend in every single one of these cases, and because a rerun of this experiment
was performed, for the discussion on the results of the primary run, the series with 100% broth will be left
out of consideration. The reason as to why the 100% broth series behaved in a different trend as opposed to
the other series is unknown, but could be worth investigating further, but in this case, the experiment was
simply done over.
Starting with the progression of ethanol consumption in the 0-50% broth series, figures 8 and 9 show
without a doubt that the higher the concentration of Pilot fermentation broth in fermentation medium is, the
lower the maximum ethanol consumption rate is. However, contrary to what was expected to be observed,
there was an (almost) complete consumption of ethanol in every batch series, whilst there was an
expectation of there being a much more inhibitory effect of the pilot fermentation broth on ethanol
consumption, along the lines of what was seen in the Influence of PH2 on MCFA production and ethanol
consumption experiment where no discernable amount of ethanol was consumed. Therefore, in the primary
run of this experiment, although we can say with relative assurance that the pilot fermentation broth does
inhibit ethanol consumption to some extent, the matrix of the pilot fermentation broth used was not the key
48
inhibitor. In the end, we came upon a single hypothesis, namely that it could be the presence of the solids in
the Influence of PH2 on MCFA production and ethanol consumption experiment which were not present in
the primary run of this experiment, which are the ones responsible for the inhibited ethanol consumption.
And thus the secondary run of this experiment was carried out, with an extra series where solids from the
pilot fermentation liquid were added to a SM fermentation medium. In the end, by comparing the ethanol
consumption rates and net consumption, it was seen that the presence of solids reduced net consumption of
ethanol by more than half, and reduced the maximum ethanol consumption rate by over fivefold. However,
this inhibitory effect caused by the solids was a lot less when compared to the inhibitory effect of the feed
matric in the secondary run. In the secondary run, the series with 50% broth had a maximum ethanol
consumption rate of 0.57gCOD/L.d and the series with 100% broth only 0.22gCOD/L.d. In the secondary
run of this experiment, net ethanol consumption and maximum consumption rates were a lot lower when
compared to the primary run. Seeing as how this phenomenon also took place in the 0%broth series, the
most likely cause is that the continuous reactor from whence the inoculum was derived was in a period of
stress, and therefore the mixed culture from there was less metabolically active.
As for the effects which the Pilot fermentation broth and solids derived from the Pilot had on the other
fermentation medium composites, figures 10 and 16 portray very different stories, and support the claim
that the inoculum for both runs, despite being obtained from the same source, had very different
characteristics. In the primary run, initial acetate and propionate production rates decreased as the
concentration of Pilot fermentation broth in the fermentation liquid increased, whilst the opposite was
observed in the secondary run, although in both runs the 50% broth series did not follow the trend, the other
series did. Combined with figures 9 and 15, it can be assumed that this difference in trends is due to the
lagphase during which the inoculum was forced to adapt to the new environment. In the first run, where
there was a healthy inoculum, the initial acetate and propionate production rates likely remained low in the
series with low Pilot fermentation broth due to the inoculum having the ability of producing MCFAs after
only 1 day. This is in contrast to the secondary run, where the lagphase had a duration of 2-3 days, which
means that during the lagphase, the only way to procure energy was by oxidizing ethanol to acetate. In both
cases, the evidence shown by figures 10 and 16 show that the 0%broth series had a significant advantage
over the 100%broth series.
Looking back once more at figures 9 and 15, it can be seen that the net production of caproate was highest
in the 0% broth series, followed by the series with less Pilot fermentation broth (leaving the 100%broth
series from the primary run aside). In contrast, butyrate accumulation occurred in the series with higher
Pilot fermentation broth concentrations, except for the 50% and 100% broth series in the secondary run,
which did not experience any major change in composition of the fermentation medium. The results from
the primary run show that although chain elongation is possible in the Pilot fermentation broth, this
primarily extends to the elongation of acetate to butyrate, and only a little bit of elongation from butyrate to
caproate, and in the secondary run, that chain elongation in Pilot fermentation broth requires either a strong
inoculum, or a modification of the matrix.
Conclusions:
• The presence of Pilot fermentation broth has a negative effect on ethanol consumption, confirming
the hypothesis that there are inhibitory components present in the thin stillage matrix.
• Production of MCFAs through chain elongation is also inhibited by components present in the Pilot
fermentation broth matrix, with accumulation of butyrate occurring in the series with higher broth
concentrations compared to caproate production in the series with lower broth concentrations.
49
Continuous fermentation of solid-free thin stillage
Phase 1: feeding solid-free undiluted thin stillage
During the first phase of this experiment, a heterogeneous and constantly changing feed was fed to the two
reactors. This meant that throughout the experiment, no truly stable state was achieved, however, from
phase 1, from day 21-28, an average butyrate and caproate production rate of 0.40±0.03 and
0.32±0.08gCOD/L.d was achieved in R1, whilst for R2, the values were 0.31±0.03 and
0.26±0.05gCOD/L.d were obtained for butyrate and caproate respectively. Maximum production rates of
caproate were 0.68 and 0.85gCOD/L.d for R1 and R2 respectively during the same period. When looking at
octanoate, a maximum production rate of 0.09gCOD/L.d was observed in R1, compared to 0.11gCOD/L.d
in R2. When compared to other studies where MCFAs were produced through chain elongation in a
fermenter fed with a complex “real” feed, we can see that this base-line which was obtained during phase 1
did not lack when compared to certain other studies. In an experiment as performed by Steinbusch et al
[49], a maximum caproate production rate of 1.09gCOD/L.d was achieved. Towards the end of phase 1,
octanoate concentrations increased to 0.89 and 1.17gCOD/L from days 10 and 7 onwards in R1 and R2
respectively, suggesting that the bacterial communities were acclimatizing to the environment and the feed.
However, as seen in the course of the TSS in the two reactors during phase 1, no increase in TSS was
observed during phase 1. These results combined indicate that at pH 5, where 58.55% of caproate is
theoretically present in its dissociated form, no significant metabolic inhibition takes place, however the
cellular growth might be inhibited to the point where an HRT of 10 days did not allow for a net increase in
biomass in the reactors.
Phase 2: Sludge retention
During this phase, a ceramic membrane was attached to R1, in the hope of seeing an increase in biomass
accumulation in R1 by increasing the SRT. When applying an SRT of 20 days (2xHRT), still no increase in
biomass was seen in R1. It was hypothesized that the pH would be the determining factor for increasing
biomass growth, both by reducing the ratio of caproate present in its undissociated form, as well as
improving the acidity of the fermentation broth to a more suitable level for chain elongating bacteria. On
day 47, the pH in both reactors was increased to 5.5, which still did not produce any result in increasing the
TSS in either reactor.
As illustrated in figure 22, on day 42, a large spike of ethanol concentration was registered in the feed.
After this time period, where 18.6±0.51gCOD/L of ethanol was fed into both reactors, spikes in caproate
production were achieved, with maximum caproate production rates of 2.50 and 3.70gCOD/L.d were
observed in R1 and R2 respectively. These production rates showed that, in contrast to the pilot reactor, the
mixed cultures in the two reactors were capable of consuming roughly a third of the ethanol introduced into
the reactors. This finding fits with what was seen in the secondary run of inhibition of ethanol oxidation by
the feed matrix, where the batch series with 0% broth with solids (0%S) showed a decreased ethanol
consumption, drawing a comparison between the 0%S broth series and the pilot, which was fed with
undiluted thin stillage with solids. When the caproate reached its maximum concentration in both reactors,
these were 13.46gCOD/L (6.11g/L) in R1 and 19.53gCOD/L (8.86g/L)in R2, with 77.6% being in its
dissociated form. This means that in R1, 1.37g/L and in R2, 1.98g/L of undissociated caproic acid were
present at these times. These values were already well above the theoretical inhibition/toxicity values, and
as such, direct electrochemical extraction needed to be implemented so as to avoid continuous inhibition.
Indeed, after these peaks were attained, caproate production in R2 dropped for more than 20 consecutive
days, hinting at a decreasing chain-elongating biomass presence in the reactor. In an effort to save the
experimental reactor R1 at least, from the excessive MCFA concentrations, Direct electrochemical
extraction was implemented.
50
Phase 3: Direct electrochemical extraction
In phase 3, the direct electrochemical cell suffered from multiple breakdowns, which affected not only the
extraction rates, but also the stability of the reactor R1 whenever the middle compartment mixed with the
cathode compartment, resulting in a steep increase in acidic, concentrated VFA solution entering the
reactor. The electrochemical cell had to be restarted 6 times in total. In the first trial(d60-67),
concentrations of VFAs stabilized at relatively low concentrations, for an unknown reason. In the second
trial(d67-76), VFA concentrations quickly reached higher levels when compared to the first trial, but
caproate concentration stabilized at around 5.5g/L, which, assuming everything was present in its
undissociated form, would still be well below the concentration needed to achieve phase separation (10g/L
undissociated caproate). In the third trial (d79-88), even higher concentrations were achieved, with
maximum caproate concentration of 8.49g/L. During this third trial, the formation of an oil layer was
observed, however, the middle compartment was fouled before harvesting, quantification and analysis of
the oil layer was possible. The fourth trial (d88-95) was when 15.1g of oil was able to be harvested for the
first time, at the end, after the oil was harvested, however, the middle compartment fouled once more due
to unknown reasons. In the fifth trial (d96-101), an oil phase layer developed at a more rapid pace than in
previous instances, and 6.7g of oil was harvested. After harvesting, the anion exchange membrane ripped,
causing a complete mixing of the middle compartment and the cathode compartment, which caused an
influx of organic acids and other anions into the reactor. This caused severe problems in the reactor itself,
where caproate concentrations in R1 dropped from above 4g/L to 2.24±0.10g/L, octanoate concentrations
dropped from above 0.3g/L to 0.20±0.01g/L.
During the course of phase 3, other observations were made. For instance, acetate concentrations in the
reactor (not accounting for the middle compartment) rose dramatically from day 60, the start of phase 3,
dramatically. Acetate concentrations during phase 2 remained below 1g/l, except for a solitary peak on day
52. After applying direct electrochemical extraction, acetate concentrations increased. During the first trial
of the electrochemical cell, acetate concentrations did not increase, but from day 68 onwards,
concentrations remained continuously above 1.5g/L, and went as high as 2.99g/L, and from day 94
onwards, concentration increased in an exponential manner from 1.88g/L to 4.63g/L on the final day.
Neither of these observations were seen in R2, where acetate concentrations remained below 2.26g/L
during phase 3. When taking into account the acetate extracted from R1 into the middle compartment of the
electrochemical cell, then it can be seen that the concentration of acetate in R1+MC is significantly higher
than in R2 throughout the duration of phase 3, with the opposite being the case throughout phase 2. This
indicates that the functionality of the electrochemical cell was an impediment to the consumption of
acetate, and, by extension, chain elongation.
Butyrate concentrations in R1 were between 2 and 2.5g/L during phase 2, and remained almost constantly
below 1.7g/L during phase 3, whilst butyrate concentrations in R2 increased (after a period of product
inhibition after the ethanol spike in phase 2) from above 2g/L to almost 4g/l, and even 4.7 on day 87. This
means that during phase 3, most likely due to the repeated failing of the electrochemical cell, butyrate
concentrations decreased in R1, supporting the earlier finding that the extraction limited chain elongation.
Caproate concentrations inside R1 fluctuated vigorously during phase 3, and no clear correlation between
this fluctuation and the direct electrochemical extraction can be seen. As with butyrate, caproate
concentrations and production rates were higher in R2 than in R1
Octanoate concentrations in R1 increased after the ethanol spike occurred during phase 2 from around
0.5g/L to just below 2.2g/L on day 73. Afterwards, during the remainder of phase 3, octanoate
51
concentrations continuously dropped until a final concentration of around 0.2g/L. This is in contrast to what
was seen in R2, where we see the concentration of octanoate increase for the duration of phase 3, from
1.55gCOD/L on day 60 to 3.63gCOD/L on day 105.
Conclusions:
• Caproate or MCFA production in a mixed-culture fermentation process fed with solid-free thin
stillage is possible. At a pH of 6, with an HRT of 10 days, no sludge retention and no in-line
extraction, production rates of 1.48±0.17gCOD/L.d caproate and 0.33±0.04gCOD/L.d of octanoate
were achieved.
• No net-growth of biomass was observed when the reactor was kept at 5 or5.5, regardless of
whether sludge retention was applied or not. This means that even if MCFA production is
stimulated under more acidic conditions, biomass growth will be stifled [97], and in danger of
washing out in continuous fermentation. Net biomass growth was only observed when the pH was
increased to 6.
• Ethanol consumption was observed during phase 2, where 37-38% of ethanol present in the feed
was consumed, increasing caproate production rates from 0.27 and 0.04gCOD/L.d to 2.50 and
3.7gCOD/L.d in R1 and R2 respectively. However, little no ethanol consumption was observed
during phase 3, even though there was 3.5-6.5gCOD/L present in the feed. This suggests that there
is a minimum ethanol concentration needed before it is used as a substrate for chain elongation.
• The fact that there was at most 37-38% consumption of ethanol means that there are inhibitory
components present in the solid-free thin stillage, reinforcing the findings from inhibition of
ethanol oxidation by the feed matrix.
• In the event that direct electrochemical extraction cannot be maintained well, and function without
failure for a long period of time, there can be severe impacts on the fermentative process,
decreasing production rates.
52
Bibliography 1. Agency, U.S.E.I., International Energy Outlook 2016 2016. 2. Eurostat. Water Statistics. 2015 [cited 2017 8/4]; Available from:
http://ec.europa.eu/eurostat/statistics-explained/index.php/Water_statistics. 3. Dodson, J.R., et al., Elemental sustainability: Towards the total recovery of scarce metals.
Chemical Engineering and Processing, 2012. 51: p. 69-78. 4. Das, A., A. Vidyadhar, and S.P. Mehrotra, A novel flowsheet for the recovery of metal values from
waste printed circuit boards. Resources, Conservation and Recycling, 2009. 53(8): p. 464-469. 5. Lastella, G., et al., Anaerobic digestion of semi-solid organic waste: biogas production and its
purification. Energy Conversion and Management, 2002. 43(1): p. 63-75. 6. Fabiani, C., et al., Treatment of waste water from silk degumming processes for protein recovery
and water reuse. Desalination, 1996. 105(1): p. 1-9. 7. Visakh, P.M. and S. Thomas, Preparation of Bionanomaterials and their Polymer Nanocomposites
from Waste and Biomass. Waste and Biomass Valorization, 2010. 1(1): p. 121-134. 8. YourDictionary, biomass, in Your Dictionary. 2017: Web. 9. Mor, S., et al., Municipal solid waste characterization and its assessment for potential methane
generation: A case study. Science of The Total Environment, 2006. 371(1): p. 1-10. 10. Staley, B.F. and M.A. Barlaz, Composition of Municipal Solid Waste in the United States and
Implications for Carbon Sequestration and Methane Yield. Journal of Environmental Engineering, 2009. 135(10): p. 901-909.
11. Noor, Z.Z., et al., An overview for energy recovery from municipal solid wastes (MSW) in Malaysia scenario. Renewable & Sustainable Energy Reviews, 2013. 20: p. 378-384.
12. Faaij, A., et al., Characteristics and availability of biomass waste and residues in the Netherlands for gasification. Biomass & Bioenergy, 1997. 12(4): p. 225-240.
13. Eurostat. Afvalstatistieken. 2017 [cited 2017 17-08]. 14. Healy, M.G.C., et al., Resource recovery from sewage sludge. Sewage treatment plants: economic
evaluation of innovative technologies for energy efficiency, ed. K.P.T. K. Konstantinos. 2015: IWA. 15. Wilkie, A.C., K.J. Riedesel, and J.M. Owens, Stillage characterization and anaerobic treatment of
ethanol stillage from conventional and cellulosic feedstocks. Biomass and Bioenergy, 2000. 19(2): p. 63-102.
16. Noukeu, N.A., et al., Characterization of effluent from food processing industries and stillage treatment trial with Eichhornia crassipes (Mart.) and Panicum maximum (Jacq.). Water Resources and Industry, 2016. 16: p. 1-18.
17. Kim, Y., et al., Composition of corn dry-grind ethanol by-products: DDGS, wet cake, and thin stillage. Bioresource Technology, 2008. 99(12): p. 5165-5176.
18. Djukić-Vuković, A.P., et al., Effective valorisation of distillery stillage by integrated production of lactic acid and high quality feed. Food Research International, 2015. 73: p. 75-80.
19. Markovic, M., et al., The Possibility of Lactic Acid Fermentation in the Triticale Stillage. Chemical Industry & Chemical Engineering Quarterly, 2011. 17(2): p. 153-162.
20. Eskicioglu, C., et al., Anaerobic digestion of whole stillage from dry-grind corn ethanol plant under mesophilic and thermophilic conditions. Bioresource Technology, 2011. 102(2): p. 1079-1086.
21. Anderson, J.L., et al., Evaluation of Dried and Wet Distillers Grains Included at Two Concentrations in the Diets of Lactating Dairy Cows1. Journal of Dairy Science, 2006. 89(8): p. 3133-3142.
22. McKinnon, J.J. and A.M. Walker, Comparison of wheat-based dried distillers’ grain with solubles to barley as an energy source for backgrounding cattle. Canadian Journal of Animal Science, 2008. 88(4): p. 721-724.
23. Cromwell, G.L., K.L. Herkelman, and T.S. Stahly, Physical, chemical, and nutritional characteristics of distillers dried grains with solubles for chicks and pigs. Journal of animal science, 1993. 71(3): p. 679-686.
24. Ezeji, T. and H.P. Blaschek, Fermentation of dried distillers’ grains and solubles (DDGS) hydrolysates to solvents and value-added products by solventogenic clostridia. Bioresource Technology, 2008. 99(12): p. 5232-5242.
25. Lau, M.W., B.E. Dale, and V. Balan, Ethanolic fermentation of hydrolysates from ammonia fiber expansion (AFEX) treated corn stover and distillers grain without detoxification and external nutrient supplementation. Biotechnology and Bioengineering, 2008. 99(3): p. 529-539.
53
26. Nasr, N., et al., Comparative assessment of single-stage and two-stage anaerobic digestion for the treatment of thin stillage. Bioresource Technology, 2012. 111: p. 122-126.
27. Moestedt, J., et al., Biogas Production from Thin Stillage on an Industrial Scale—Experience and Optimisation. Energies, 2013. 6(11): p. 5642.
28. Liu, K. and K.A. Rosentrater, Distillers Grains: Production, Properties, and Utilization, ed. C. Press. 2016: CRC Press. 564.
29. Djukić-Vuković, A.P., et al., Lactic acid production on liquid distillery stillage by Lactobacillus rhamnosus immobilized onto zeolite. Bioresource Technology, 2013. 135: p. 454-458.
30. Bento, J.M.A. and H.L. Fleming, Membrane-based process for the recovery of lactic acid and glycerol from a "corn thin stillage" stream. 1993, Google Patents.
31. Yen, H.-W., Y.-C. Yang, and Y.-H. Yu, Using crude glycerol and thin stillage for the production of microbial lipids through the cultivation of Rhodotorula glutinis. Journal of Bioscience and Bioengineering, 2012. 114(4): p. 453-456.
32. Ahn, J.-H., B.-I. Sang, and Y. Um, Butanol production from thin stillage using Clostridium pasteurianum. Bioresource Technology, 2011. 102(7): p. 4934-4937.
33. Biebl, H., Fermentation of glycerol by Clostridium pasteurianum — batch and continuous culture studies. Journal of Industrial Microbiology and Biotechnology, 2001. 27(1): p. 18-26.
34. Gonzalez, R., P. Campbell, and M. Wong, Production of ethanol from thin stillage by metabolically engineered Escherichia coli. Biotechnology Letters, 2010. 32(3): p. 405-411.
35. Hsieh, C., T.-H. Hsu, and F.-C. Yang, Production of polysaccharides of Ganoderma lucidum (CCRC36021) by reusing thin stillage. Process Biochemistry, 2005. 40(2): p. 909-916.
36. Agler, M.T., et al., Waste to bioproduct conversion with undefined mixed cultures: the carboxylate platform. Trends in Biotechnology, 2011. 29(2): p. 70-78.
37. Cope, J.L., et al., Evaluating the performance of carboxylate platform fermentations across diverse inocula originating as sediments from extreme environments. Bioresource Technology, 2014. 155: p. 388-394.
38. Wu, G.H., et al., Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants. Nature Biotechnology, 2005. 23(8): p. 1013-1017.
39. Holtzapple, M.T. and C.B. Granda, Carboxylate Platform: The MixAlco Process Part 1: Comparison of Three Biomass Conversion Platforms. Applied Biochemistry and Biotechnology, 2009. 156(1-3): p. 525-536.
40. Varrone, C., et al., Comparison of Different Strategies for Selection/Adaptation of Mixed Microbial Cultures Able to Ferment Crude Glycerol Derived from Second-Generation Biodiesel. BioMed Research International, 2017. 2017: p. 1.
41. Dengate, S. and A. Ruben, Controlled trial of cumulative behavioural effects of a common bread preservative*. Journal of Paediatrics and Child Health, 2002. 38(4): p. 373-376.
42. Finch, C.A., Polyvinyl alcohol; properties and applications. 1973: John Wiley & Sons. 43. Serop, M., Cellulose acetate butyrate semipermeable membranes and their production. 1971,
Google Patents. 44. Weiss, S.C., et al., A randomized controlled clinical trial assessing the effect of betamethasone
valerate 0.12% foam on the short-term treatment of stasis dermatitis. Journal of drugs in dermatology : JDD, 2005. 4(3): p. 339-345.
45. Wang, J., et al., Anaerobic Fermentation for Production of Carboxylic Acids as Bulk Chemicals from Renewable Biomass, in Anaerobes in Biotechnology, R. Hatti-Kaul, G. Mamo, and B. Mattiasson, Editors. 2016, Springer International Publishing: Cham. p. 323-361.
46. Schäfer, T. and P. Schönheit, Maltose fermentation to acetate, CO2 and H2 in the anaerobic hyperthermophilic archaeon Pyrococcus furiosus: evidence for the operation of a novel sugar fermentation pathway. Archives of Microbiology, 1992. 158(3): p. 188-202.
47. Chen, Y., et al., High-purity propionate production from glycerol in mixed culture fermentation. Bioresource Technology, 2016. 219: p. 659-667.
48. Berlett, B.S. and E.R. Stadtman, Protein Oxidation in Aging, Disease, and Oxidative Stress. Journal of Biological Chemistry, 1997. 272(33): p. 20313-20316.
49. Liu, K., et al., Continuous syngas fermentation for the production of ethanol, n-propanol and n-butanol. Bioresource Technology, 2014. 151: p. 69-77.
50. Knifton, J.F., Syngas reactions. Journal of Catalysis, 1985. 96(2): p. 439-453.
54
51. Ding, Y.-H., W. Huang, and Y.-G. Wang, Direct synthesis of acetic acid from CH4 and CO2 by a step-wise route over Pd/SiO2 and Rh/SiO2 catalysts. Fuel Processing Technology, 2007. 88(4): p. 319-324.
52. Ganigue, R., et al., Microbial electrosynthesis of butyrate from carbon dioxide. Chemical Communications, 2015. 51(15): p. 3235-3238.
53. Buasri, A., et al., Biodiesel production from crude palm oil with a high content of free fatty acids and fuel properties. Vol. 8. 2009. 115-124.
54. Angenent, L.T., et al., Chain Elongation with Reactor Microbiomes: Open-Culture Biotechnology To Produce Biochemicals. Environmental Science & Technology, 2016. 50(6): p. 2796-2810.
55. Mancini, A., et al., Biological and Nutritional Properties of Palm Oil and Palmitic Acid: Effects on Health. Molecules, 2015. 20(9): p. 17339-17361.
56. Wasewar, K.L. and D.Z. Shende, Extraction of Caproic Acid Using Tri-n-butyl Phosphate in Benzene and Toluene at 301 K. Journal of Chemical and Engineering Data, 2010. 55(9): p. 4121-4125.
57. Adam, G.A. and S.A. Needham, Antimicrobial polymers and methods for their production. 2015, Google Patents.
58. Sun, C.Q., C.J. O'Connor, and A.M. Roberton, The antimicrobial properties of milkfat after partial hydrolysis by calf pregastric lipase. Chemico-Biological Interactions, 2002. 140(2): p. 185-198.
59. Gabbe, S., Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate (vol 348, pg 2379, 2003). New England Journal of Medicine, 2003. 349(13): p. 1299-1299.
60. Yalkowsky, S.H., Y. He, and P. Jain, Handbook of: Aqueous Solubility Data. Handbook of, ed. C. Press. 2010.
61. Marounek, M., K. Fliegrova, and S. Bartos, Metabolism and Some Characteristics of Ruminal Strains of Megasphaera-Elsdenii. Applied and Environmental Microbiology, 1989. 55(6): p. 1570-1573.
62. Lundquist, F., et al., Ethanol metabolism and production of free acetate in the human liver. J Clin Invest, 1962. 41: p. 955-61.
63. Steinbusch, K.J.J., et al., Biological formation of caproate and caprylate from acetate: fuel and chemical production from low grade biomass. Energy & Environmental Science, 2011. 4(1): p. 216-224.
64. Van Eerten-Jansen, M.C.A.A., et al., Bioelectrochemical Production of Caproate and Caprylate from Acetate by Mixed Cultures (vol 1, pg 513, 2013). Acs Sustainable Chemistry & Engineering, 2013. 1(8): p. 1069-1069.
65. Cavalcante, W.d.A., et al., Anaerobic fermentation for n-caproic acid production: A review. Process Biochemistry, 2017. 54: p. 106-119.
66. Kucek, L.A., M. Nguyen, and L.T. Angenent, Conversion of L-lactate into n-caproate by a continuously fed reactor microbiome. Water Research, 2016. 93: p. 163-171.
67. Zhu, X.Y., et al., The synthesis of n-caproate from lactate: a new efficient process for medium-chain carboxylates production. Scientific Reports, 2015. 5.
68. Gonzalez-Cabaleiro, R., et al., Linking thermodynamics and kinetics to assess pathway reversibility in anaerobic bioprocesses. Energy & Environmental Science, 2013. 6(12): p. 3780-3789.
69. Spirito, C.M., et al., Chain elongation in anaerobic reactor microbiomes to recover resources from waste. Current Opinion in Biotechnology, 2014. 27: p. 115-122.
70. Zhou, Z., Q. Meng, and Z. Yu, Effects of Methanogenic Inhibitors on Methane Production and Abundances of Methanogens and Cellulolytic Bacteria in In Vitro Ruminal Cultures. Applied and Environmental Microbiology, 2011. 77(8): p. 2634-2639.
71. Zhuang, L., et al., Methanogenesis Control using 2-Bromoethanesulfonate for Enhanced Power Recovery from Sewage Sludge in Air-cathode Microbial Fuel Cells. International Journal of Electrochemical Science, 2012. 7.
72. Van Kessel, J.A.S. and J.B. Russell, The effect of pH on ruminal methanogenesis. FEMS Microbiology Ecology, 1996. 20(4): p. 205-210.
73. Ge, S., et al., Long-Term n-Caproic Acid Production from Yeast-Fermentation Beer in an Anaerobic Bioreactor with Continuous Product Extraction. Environ Sci Technol, 2015. 49(13): p. 8012-21.
74. Vasudevan, D., H. Richter, and L.T. Angenent, Upgrading dilute ethanol from syngas fermentation to n-caproate with reactor microbiomes. Bioresource Technology, 2014. 151: p. 378-382.
55
75. Huang, C.B. and J.L. Ebersole, A novel bioactivity of omega-3 polyunsaturated fatty acids and their ester derivatives. Molecular Oral Microbiology, 2010. 25(1): p. 75-80.
76. van den Heuvel, J.C., et al., Determination of the Critical Concentration of Inhibitory Products in a Repeated Fed-Batch Culture. Biotechnology Techniques, 1992. 6(1): p. 33-38.
77. Kucek, L.A., et al., Waste Conversion into n-Caprylate and n-Caproate: Resource Recovery from Wine Lees Using Anaerobic Reactor Microbiomes and In-line Extraction. Frontiers in Microbiology, 2016. 7.
78. Fasahati, P. and J. Liu, Techno-economic analysis of production and recovery of volatile fatty acids from brown algae using membrane distillation. Proceedings of the 8th International Conference on Foundations of Computer-Aided Process Design, 2014. 34: p. 303-308.
79. Reyhanitash, E., et al., Extraction of volatile fatty acids from fermented wastewater. Separation and Purification Technology, 2016. 161: p. 61-68.
80. Xu, J., et al., In-line and selective phase separation of medium-chain carboxylic acids using membrane electrolysis. Chemical Communications, 2015. 51(31): p. 6847-6850.
81. Andersen, S.J., et al., Electrolytic extraction drives volatile fatty acid chain elongation through lactic acid and replaces chemical pH control in thin stillage fermentation. Biotechnology for Biofuels, 2015. 8.
82. Andersen, S.J., et al., Electrolytic Membrane Extraction Enables Production of Fine Chemicals from Biorefinery Sidestreams. Environmental Science & Technology, 2014. 48(12): p. 7135-7142.
83. Bermeo, M.A., et al., Use of electrodialysis as a vfa recovery process From acidogenic of msw synthetic leachates. 2014.
84. Zhang, J., et al., Simultaneous Saccharification and Ethanol Fermentation at High Corn Stover Solids Loading in a Helical Stirring Bioreactor. Biotechnology and Bioengineering, 2010. 105(4): p. 718-728.
85. Chen, W.S., et al., Production of Caproic Acid from Mixed Organic Waste: An Environmental Life Cycle Perspective. Environmental Science & Technology, 2017. 51(12): p. 7159-7168.
86. Scalbert, A., Antimicrobial properties of tannins. Phytochemistry, 1991. 30(12): p. 3875-3883. 87. Junicke, H., et al., Impact of the hydrogen partial pressure on lactate degradation in a coculture of
Desulfovibrio sp. G11 and Methanobrevibacter arboriphilus DH1. Applied Microbiology and Biotechnology, 2015. 99(8): p. 3599-3608.
88. Collet, C., et al., Acetate production from lactose by Clostridium thermolacticum and hydrogen-scavenging microorganisms in continuous culture—Effect of hydrogen partial pressure. Journal of Biotechnology, 2005. 118(3): p. 328-338.
89. Yerushalmi, L., B. Volesky, and T. Szczesny, Effect of increased hydrogen partial pressure on the acetone-butanol fermentation by Clostridium acetobutylicum. Applied Microbiology and Biotechnology, 1985. 22(2): p. 103-107.
90. Grootscholten, T.I.M., et al., Two-stage medium chain fatty acid (MCFA) production from municipal solid waste and ethanol. Applied Energy, 2014. 116: p. 223-229.
91. van Andel, J.G., et al., Glucose fermentation byClostridium butyricum grown under a self generated gas atmosphere in chemostat culture. Applied Microbiology and Biotechnology, 1985. 23(1): p. 21-26.
92. de Kok, S., et al., Impact of dissolved hydrogen partial pressure on mixed culture fermentations. Applied Microbiology and Biotechnology, 2013. 97(6): p. 2617-2625.
93. Zhang, F., et al., Fatty acids production from hydrogen and carbon dioxide by mixed culture in the membrane biofilm reactor. Water Research, 2013. 47(16): p. 6122-6129.
94. Mustafa, A.F., J.J. McKinnon, and D.A. Christensen, The nutritive value of thin stillage and wet distillers' grains for ruminants - Review. Asian-Australasian Journal of Animal Sciences, 2000. 13(11): p. 1609-1618.
95. Cazier, E.A., et al., Biomass hydrolysis inhibition at high hydrogen partial pressure in solid-state anaerobic digestion. Bioresource Technology, 2015. 190: p. 106-113.
96. Sun, Y. and J. Cheng, Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technology, 2002. 83(1): p. 1-11.
97. Ganigue, R., et al., Low Fermentation pH Is a Trigger to Alcohol Production, but a Killer to Chain Elongation. Frontiers in Microbiology, 2016. 7.