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Journal of Biotechnology 157 (2012) 524–546

Contents lists available at SciVerse ScienceDirect

Journal of Biotechnology

journa l homepage: www.e lsev ier .com/ locate / jb io tec

reatment of mezcal vinasses: A review

ania Robles-Gonzáleza, Juvencio Galíndez-Mayera, Noemí Rinderknecht-Seijasb,éctor M. Poggi-Varaldoc,∗

ENCB-IPN, México D.F., MexicoESIQIE-IPN, México D.F., MexicoEnvironmental Biotechnology and Renewable Energy Rand D Group, Department of Biotechnology and Bioengineering, Centro de Investigación y de Estudios Avanzados del IPN,pdo. Postal 14-740, México D.F. 07000, Mexico

r t i c l e i n f o

rticle history:eceived 14 February 2011eceived in revised form 5 August 2011ccepted 7 September 2011vailable online 16 September 2011

eywords:erobicnaerobiciohydrogen productioniological treatmenthemical treatmento-compostingolor removalungal treatmentezcal vinasse

hysical treatment: Recalcitrant effluents

a b s t r a c t

Mexican distilleries produce near eight million liters of mezcal per year, and generate about 90 millionliters of mezcal vinasses (MV). This acidic liquid waste is very aggressive to the environment becauseof its high content of toxic and recalcitrant organic matter. As a result, treatment is necessary beforedischarge to water bodies. It is interesting, yet disturbing; verify that there is a significant gap on thetreatment of MV. However, there is an abundant body of research on treatment of other recalcitrant toxiceffluents that bear some similarity to MV, for example, wine vinasse, vinasses from the sugar industry,olive oil, and industrial pulp and paper wastewaters. The objective of this review is to critically organizethe treatment alternatives of MV, assess their relative advantages and disadvantages, and finally detectthe trends for future research and development.

Experience with treatment of this set of residuals, indicates the following trends: (i) anaerobic digestion,complemented by oxidative chemical treatments (e.g. ozonation) are usually placed as pretreatments,(ii) aerobic treatment alone and combined with ozone which have been directed to remove phenoliccompounds and color have been successfully applied, (iii) physico-chemical treatments such as Fenton,electro-oxidation, oxidants and so on., which are now mostly at lab scale stage, have demonstrated asignificant removal of recalcitrant organic compounds, (iv) fungal pretreatment with chemical treatment

eview followed by oxidative (O3) or anaerobic digestion, this combination seems to give attractive results,(v) vinasses can be co-composted with solid organic wastes, particularly with those from agriculturalactivities and agro-industies; in addition to soil amenders with fertilizing value to improve soil qualityin typical arid lands where agave is cultivated, it seems to be a low cost technology very well suited forrural regions in underdeveloped countries where more sophisticated technologies are difficult to adopt,due to high costs and requirements of skilled personnel.

. Introduction

.1. Mezcal industry in Mexico

Mexican mezcal production in 2006 and 2007 was 8 and 6 mil-ions liters, respectively, according to the Consejo Mexicano Regu-ador de la Calidad del Mezcal A.C. (COMERCAM, Mexican Councilor Quality Regulation of Mezcal A.C.; http://www.comercam.org/).here are seven states in Mexico that can produce Mezcal, a Denom-nation of Origin distillate: Oaxaca, Durango, Guerrero, San Luis

otosi, Zacatecas, Guanajuato, and Tamaulipas (Fig. 1). The Statef Oaxaca produces ca. 65% of the total amount of mezcal.

∗ Corresponding author. Tel.: +5255 5747 3800x4324.E-mail address: hectorpoggi2001@gmail.com (H.M. Poggi-Varaldo).

168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.jbiotec.2011.09.006

© 2011 Elsevier B.V. All rights reserved.

The Mexican industrial standard NOM-070-SCFI-1994 (NOM-070-SCFI, 1994) (http://www.profeco.gob.mx/) defines mezcal asthe regional alcoholic beverage obtained by distillation and rectifi-cation of broths that are directly prepared by fermentation of sugarscontained in juices extracted from the mature hearts (or heads or“pinas”) of agaves. These mature “pinas” are previously hydrolyzed(usually by cooking in ovens) and the extracts are subjected toalcoholic fermentation with yeasts. In general, Mezcal process con-sists of four stages, i.e., cooking of agave hearts, milling the cookedhearts, juice or must fermentation, and distillation/rectification. Inthe distillation/rectification stage, between 8 and 15 L of distilleryslops (also called mezcal vinasses, MV) are generated for each literof mezcal (Robles-González et al., 2010; Jiménez et al., 2005; Preeti

and Aniruddha, 2006). In 2007, the volume of MV representedapproximately 90 million liters.

MV contains a variety of organic substances such as acetic andlactic acids, glycerol, phenols, polyphenols, melanoidins, as well

V. Robles-González et al. / Journal of Bio

Nomenclature

AC aromatic compoundsAD anaerobic digestionAnSBR anaerobic sequencing batch reactorAOP advanced oxidation processAPT anaerobically pre-treatedAFBR anaerobic fluidized bed reactoras specific surface area, area per unit volume of a pack-

ing or supportBM batch mode or batch operationBOD0 initial BODBv volumetric organic loading rateBx specific organic loading rate, i.e., per unit of mass of

biomassCF coagulation/flocculationCOD0 initial CODCV cotton gin compost with vinasseCW cotton wasteEF electro FentonEO electrochemical oxidationEGSB expanded granular sludge bed reactorGI germination indexGM grape marcHRT hydraulic retention timekO3 consumption ratio of ozoneMnP manganese peroxidaseMP melanoidin productsMV mezcal vinasseOM organic matterOT operation timeRO reverse osmosisTOC0 soluble initial soluble total organic carbonTPT-V thermally pretreated vinasseU units of enzyme activityUASB upflow anaerobic sludge (granular) blanket reactorUF ultrafiltrationUS ultrasoundV vinasseVBM vinasse from fermented beet molassesVFSM vinasse from fermented sugar-cane molassesVOA volatile organic acidsVWD vinasses from wine distillationWS wheat strawY yieldYı pseudoyield

Greek letters�arom removal efficiency of aromatic compounds�COD removal efficiency of organic matter on COD basis� removal efficiency of color

ahiu2thorct

color�phen removal efficiency of phenolic compounds

s inorganic species such as sulphates and phosphates salts. Itas been pointed out that vinasses composition and character-

stics may vary, depending upon the feedstock and the processsed for distillate production (Duarte et al., 1997; Robles-González,011; Robles-González et al., 2010). In spite of that, MV shareshe main following features: low pH in the range of 3–5, andigh contents of organic matter (35,000–50,000 mg O2/L as BOD

r 100,000–150,000 mg O2/L as COD) which is usually toxic andecalcitrant. Vinasses are of environmental concern because its dis-harge to water bodies or onto soils may have a negative impact onhe ecosystem. As a result, treatment is necessary before discharge

technology 157 (2012) 524–546 525

to water bodies. It is interesting, yet disturbing, to verify that thereis a significant gap on the treatment of MV. However, there is anabundant research on treatment of other recalcitrant toxic effluentsthat bear some similarity to MV, for example, wine vinasse, vinassesfrom the sugar industry, olive oil, and industrial pulp and paperwastewaters. The objective of this review is to critically organizethe treatment alternatives of MV, assess their relative advantagesand disadvantages, and finally detect the trends for future researchand development.

The scope of this review encompasses the following topics:(i) Mezcal vinasses characteristics and environmental impact; (ii)Biological treatment of vinasses, covering experiences with anaer-obic digestion which are the most used method in the treatmentof vinasse and aerobic treatment that has studied mainly forremoval of color (melanoids) and some toxic compounds (phe-nols and polyphenols) present in the vinasse; (iii) Co-compostingwith organic solid wastes, and fungal treatment, under the head-ing of Biological Treatment; (iv) Physico-chemical treatment, withfocus on ozonation and Fenton oxidation, this type of treatmentaddresses the removal of organic matter recalcitrant to biologicaldegradation. Whenever available, data from MV treatment will bepresented and discussed; otherwise, references from similar recal-citrant effluents will be included.

1.2. Characteristics of mezcal vinasses

Mezcal vinasses mainly consist of distillery slops that is themain contributor to volume and the organic load, although thereare minor components that usually contribute to the effluent vol-ume and load variability. Among those components, it can be foundthe effluents from fermenter cleaning (low in volume although theorganic load can be around 5,000 mg COD/L), condensates, cool-ing water, etc. (Robles-González, 2011; Duarte et al., 1997). Sincecooling water may represent a high flowrate but esentially a verylow pollutant load, it is recommended its segregation from MV.Tables 1–3 display typical characteristics of MV and vinasses fromsugar cane molasses fermentation/distillation and those originatedin the alcohol production from wine.

Vinasses generaly contain high concentrations of dissolvedsolids; up to 50% of this parameter can be reducing sugars (Sangaveet al., 2007a), non volatile compounds coming from the fermen-tation broth, phenolic and polyphenolic compounds (Sales et al.,1987; Capasso et al., 1992; Robles-González et al., 2010), rela-tively high concentrations of mineral salts that reflect on a highelectrolytic conductivity (250–300 dS m−1), and ash. Vinasses areacid with a pH that usually ranges from 3.5 to 5, dark colored(brownish, ascribed to the presence of melanoids) (García et al.,1997; Jiménez et al., 2003; Coca et al., 2005). The organic pollutantload is very high with extremely elevated values of biochemicaloxygen demand (35,000–50,000 mg O2/L) and chemical oxygendemand (70,000–150,000 mg/L). Biodegradability indices in therange 0.2–0.5 mg BOD/mg COD are very common (Robles-Gonzálezet al., 2010; Nandy et al., 2002; Sangave et al., 2007a; Madejón et al.,2001a,b). Given this profile, vinasses are very aggressive and recal-citrant effluents, whose direct discharge to water bodies and soilmay cause severe environmental impact.

1.3. Pollution and environmental impact of vinasses

Uncontrolled discharge of vinasses onto soils can negativelyimpact soil quality. For instance, the high content of soluble saltsin MV can lead to soil salinity and sodicity (Tejada et al., 2009;

Shojaosadati et al., 1999). This, in turn, can seriously deterioratesoil structure, porosity, and fertility (Tejanda and González, 2005).Low pH of vinasses can be associated to heavy metal remobiliza-tion in soils (García et al., 1997). High suspended solids loads can

526 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546

Fig. 1. Regions of Mexico with significant mezcal, tequila and others spirits beverage production. Keys: 1. Jalisco – Tequila and Racilla; 2. Nayarit – Tequila; 3. Guanajuato –M TequilL – Sotm

ctbhevs(ie(a

5cg

TC

R

ezcal and Mezcal; 4. Michoacan – Tequila and Sikua; 5. Tamaulipas – Mezcal anduis Potosi – Mezcal; 10. Zacatecas – Mezcal; 11. Sonora – Bacanora; 12. Chihuahuaexico-wahaka-tequila-sotol-and-more).

log pores in soils, thus leading to development of anaerobic condi-ions that not only are noticeable for their bad aesthetic symptoms,ut can also contribute to lower the soil pH and remobilization ofeavy metals mentioned previously (García et al., 1997; Jiménezt al., 2005) and phytotoxicity to crops due to accumulation of aariety of substances generated in the fermentation of vinassesuch as acetic acid, lactic acid, glycerol, and ammonia nitrogenYavuz, 2007). Also, phenolic and polyphenolic compounds presentn vinasses (Table 3) can inhibit seed germination and damage sev-ral crops, as well as to negatively impact soil microbial activityDíaz et al., 2002a,b; Kannabiran and Pragasam, 1993; Mattiazzond de Glorie, 1987).

Since vinasses leave the factory at temperatures around0–80 ◦C, if not cooled before discharge to water bodies, theyan increase water temperature and to diminish dissolved oxy-en below its critical level for fish survival (Jiménez et al., 2005;

able 1haracteristics of mezcal vinasses from different manufacturing facilities in the State of O

Parameter IMF-1a

pH 3.7Alkalinity (mg de CaCO3/L) NDConductivity (mS/cm) 2.6 ± 0.02Color (475 nm) 4.6 ± 0.3COD (mg O2/L) 56,230 ± 162BOD5 (mg O2/L) 26,500 ± 710Phenol (mg gallic acid/L) 478 ± 24Fructose (mg/L) 14.8 ± 2.3Nitrogen Kjeldahl (mgNH3-N/L) 660 ± 37Total solid (mg/L) 26,830 ± 1120Total suspended solids (mg/L) 3130 ± 168Volatile suspended solids (mg/L) 1130 ± 88Fixed suspended solids (mg/L) 2000 ± 80Phosphate (mg/L) 290 ± 5Sulphate (mg/L) 308 ± 14

obles-González (2011).a Notes: IMF-1: Industrial mezcal factory 1.b IMF-2: Industrial mezcal factory 2.c TMF: Traditional or handcraft mezcal factory.

a; 6. Oaxaca – Mezcal; 7. Durango – Mezcal and Sotol; 8. Guererro – Mezcal; 9. Sanol; 13. Coahuila–Sotol (http://www.elmezcal.org/general/map-of-spirits-made-in-

Mane et al., 2006). On the other hand, turbidity and color asso-ciated to vinasses suspended solids and melanoidins respectively,may impair light penetration and associated photosynthetic pro-cesses and severely impact aquatic life (Fitzgibbon et al., 1995).The relatively high concentrations of nutrients P and N may causeeutrophication in water bodies, reservoirs, and channels (Vlyssideset al., 1997.)

Furthermore, presence of putrescible organic compounds suchas indol, 3-methyl indol as well as other sulphur-containing sub-stances are associated to serious aesthetic problems as well aspossible toxicity (Pant and Adholeya, 2007a.)

Currently, several countries have enacted more stringent stan-

dards for discharge of effluents from alcohol distilleries. Forexample, in 2005, the Indian environmental authorities made thedecision to convert the distillery industry in a zero-discharge indus-try in a few years (Pant and Adholeya, 2007a). This approach should

axaca, México.

IMF-2b TMFc

3.6 3.8ND ND3.9 ± 0.03 4.2 ± 0.056.0 ± 0.2 10.6 ± 0.560,560 ± 1004 122,860 ± 227022,000 ± 2830 33,600 ± 2260521 ± 16 542 ± 4825.4 ± 4.2 50.0 ± 6.4843 ± 97 5,650 ± 50343,450 ± 1490 94,7130 ± 40553905 ± 156 8400 ± 5042500 ± 100 6850 ± 4111405 ± 56 1550 ± 93850 ± 14 1705 ± 30947 ± 12 842 ± 14

V. Robles-González et al. / Journal of Bio

Table 2Typical characteristics of vinasses obtained from different substrates.

Parameters FSMa DGAb

pH 3.8–4.71,2,3,7 3.7–4.15,6

Alkalinity (mgCaCO3/L)

5800–60001,2 4501

Turbidity (NTU) 30,000Total solid (mg/L) 63,000–79,0001,2 21,410–100,0004,5

Total volatile solids(mg/L)

50,0004

Total suspended solids(mg/L)

3000–11,0002,3,7 39006

Total volatile supendedsolids (mg/L)

2500–90002,3 –

BOD5 (mgO2/L) 31,500–75,0001,3 11,150–42,2285,6

COD (mgO2/L 59,000–80,5001,2,7 24,500–120,0004,5

Kjeldahl N (mg N/L) 1600–18001,2 30,0004

Ammonia N (mg N/L 150–70001,7

Total N (mg/L) 9753 16004

NO3-N (mg/L) 9978 –Sulphate (mg/L) 31001 46004

Phosphate (mg/L) 20–3333,8 614

Total organic volatileacids (acetic acidbasis) (mg/L)

85002 13305

Sodium (mg/L) – 0.0324

Calcium (mg/L) – 0.64

Potassium (mg/L) 30,0008 1.924

Iron (mg/L) 203,0008 0.0354

Zinc (mg/L) 15,0008 0.00184

Total phenol 450 mg gallic acid/L 477 mg caffeic acid/L469 Total phenol/L2,3 735 mg gallic acid/L6,9

Total O-diphenol(mg/L)

343

TOC (mg/L) 26,000–32,0003,7 36,2756

Fixed suspended solids(mg/L)

– 10006

Total dissolved solids(mg/L)

49,000–510007 –

Reducing sugars (mg/L) 4000–50007 –

References: 1. Durán-de-Bazúa et al., 1991; 2. Jiménez et al., 2006; 3. García et al.,1997; 4. Harada et al., 1996; 5. Benitez et al., 2003; 6. Martín et al., 2002; 7. Sangaveet al., 2007a; 8. Madejón et al., 2001a,b; 9. Beltrán et al., 1999a.

a FSM: vinasse from distillation of fermented sugar molasses.b DGA: vinasse from distillation of grape alcohol; the number after A: means ‘Ref-

erence for vinasse type in column A’; the number after B: means ‘Reference forvinasse type in column B’.

Table 3Concentration of phenolic compounds in vinasses.

Phenolic compoundsconcentration (mg/L)

Basis ofexpression

Type of vinasses Ref.

4300 Total phenols From distillation ofgrape alcohol

1

244 Caffeic acid From distillation ofgrape alcohol

2

450 Gallic acid From distillation offermented sugarmolasses

3

477 Caffeic acid From ethyl alcoholproduction line

4

735 Gallic acid From distillation ofgrape alcohol

5

843 Total phenols From distillation ofgrape alcohol

6

469 Total phenols From the fermentationof sugar molasses

7

210 Gallic acid From distillation ofgrape alcohol

8

480–540 Gallic acid Mezcal vinasses 9

References: 1. Beltran-de-Heredia et al. (2005a); 2. Benitez et al. (2003); 3. Jiménezet al. (2003); 4. Martín et al. (2002); 5. Beltrán et al. (1999a); 6. Beltrán et al. (1999b);7. García et al. (1997); 8. Jiménez et al. (2005); Robles-González (2011).

technology 157 (2012) 524–546 527

rely in a sort of “kidney” scheme, including in-plant treatment ofeffluents, reuse, and recycling similar to zero-discharge pulp millsthat are in operation in Europe (Asghar et al., 2008; Gavrilescu andPuite, 2007; Ritchlin and Johnston, 1998.)

In Mexico there is still no standard that specifically regulatesthe discharge of mezcal vinasses or alcohol industry wastewaterin general. So far, the applicable environmental regulation is theMexican Official Standard NOM-001-ECOL-1997 (NOM-ECOL-001,1997) (Secretaría de Medio Ambiente y Recursos Naturales, SEMAR-NAT), which sets the maximum limits of pollutants in wastewaterdischarges waste in water bodies, soil, wetlands, etc., and possi-bly additional requirements (particular discharge conditions) thatcan be imposed by the environmental agency in Mexico SEMAR-NAT. Despite the lack of specificity, maximum permissible limitsfor BOD5, total suspended solids, and other pollutants of the Mex-ican regulation are much lower than the corresponding parametervalues in raw MV. Therefore, a significant extent of MV treatmentis required.

2. Biological treatment

As previously discussed, treatment of vinasses prior to theirdischarge or recycling is mandatory from regulatory and envi-ronmental standpoints in Mexico (environmental regulationsNOM-003-SEMARNAT-1997 (NOM-003-SEMARNAT, 1997) “Estab-lishing the permissible maximum levels of contaminants fortreated wastewater is reused in the public services” and NOM-001-ECOL-1996 “Establishing the maximum permissible levels ofcontaminants in wastewater discharges into national waters”).Among the most commonly treatment methods used we can findbiological and physico-chemical techniques (Maiorella et al., 1983).Their main goal is to reduce vinasse’s pollution impact by removingthe degradable organic matter, removing or degrading or at leasttransforming major toxic organic substances to compounds thatmay be more susceptible to biodegradation, and desirably convert-ing pollutants to resources such as bioenergy or higher added-valuemetabolites. In this way, a large menu of techniques have beenexplored and applied, such as lagoons, anaerobic bioreactors ofthe fluidized bed type, UASB (abbreviation of upflow anaerobicsludge blanket reactor), packed ones, etc. in the biological treat-ment category, as well as evaporation, combustion, and controlleddischarge onto soils to reclaim vinasses fertilizer value (Durán-de-Bazúa et al., 1991; Gemtos et al., 1999; Harada et al., 1996; Madejónet al., 2001a,b; Rao, 1972; Sheehan and Greenfield, 1980).

2.1. Anaerobic digestion

Anaerobic digestion has been one of the most employed systemfor vinasses treatment because of low operational costs, aerationsavings, low sludge production and the obtaining of by-productsas methane gas (Durán-de-Bazúa et al., 1991; Harada et al., 1996;Jiménez et al., 2006; Lalov et al., 2001). In this regard, a varietyof bioreactor configurations has been tested. For example Moletta(2005) reported work with bioreactors with suspended biomassgrowth or flocs (suspended biomass as used in anaerobic con-tact digesters, sequential batch anaerobic reactors and anaerobiclagoons) and immobilized biomass (i.e., granular sludge anaerobicreactors); he also mentioned the use of a combination of the twoapproaches systems: granular sludge bed reactors with anaerobicfilters, known as hybrid digesters. A summary of selected works onanaerobic treatment of vinasses is shown in Table 4.

Anaerobic digestion is successful in dealing with the degradablefraction of organic matter in vinasses; however, there is alwaysan important recalcitrant compounds fraction (i.e., brown poly-mers melanoidins and other compounds) that still remain after

528 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546

Table 4Selected results on anaerobic treatment of vinasses.

Process and experimental design Results Remarks Ref.

Vinasses from Winery distillation (VWD) (i) �COD = 76% at organic load rate(Bv) = 6.29 kg COD/m3 d

(i)Adequate for wastewater treatmentscontaining organic compounds easilyassimilated or that do not require high rates ofCOD removal

1

Anaerobic fluidized bed (AFBR); Supports: (i)corrugated plastic and (ii) open poresintered-glass media; Laboratory scale (LS)

(ii) �COD = 96% at Bv = 5.88 kgCOD/m3 d

(ii)Promotes cell transport and ensures contactbetween the microorganisms and thesubstrate, suitable for effluent toxic andrecalcitrant compounds

VWD �COD = 80% at Bv = 30 kg COD/m3 d Excellent support for use in a high rateanaerobic fixed bed

2

Anaerobic packed bed reactor; inert media:small floating support of polyethylene(Bioflow 30: � = 0.93 g/cm3; as = 320 m2/m3);LS

Bioflow 30 showed high retention capacity ofbiomass (4.6 g dry solids per support) at theend of the experiment, biomass accounted for57 g solids/L reactor

VWD �COD = 81–89% at Bv = 29.6 kgCOD/m3 d

At the end of the experiment 83.4% of the totalbiomass was attached to the support

3

Anaerobic moving bed biofilm reactor (ABBR);filled with cylinders of polyethylene(� = 0.84 g/cm3; LS

66% of the bioreactor was filled withcylindrical polyethylene support

VFSM vinasse from fermented sugar-canemolasses

�COD = 90% at Bv = 20 kg COD/m3 d Biomass concentration attached to zeolite inreactor was 40–45 g volatile solids/L

4

AFBR; carrier support was zeolite particles of0.25–0.50 and 0.50–0.80 mm diameter; LS

Experiments were carried out 30 ± 2 ◦C

VWD �COD = 96.5% at Bv = 5.88 kgCOD/m3 d and HRT = 2.55 days

Experiments were carried out at 55 ◦C 5

AFBR filled with porous support media; LS �COD = 81.5% at Bv = 32 kg COD/m3 d Previously colonization of the support used inAFBR in semicontinuous anaerobic fixed-bedreactors treating VWDBioreactor acidogenic crash at Bv = 40.5 kgCOD/m3 d, corresponding HRT to 0.37 days,dramatic decrease of COD removal and noproduction of biogas

VFSM �COD = 70% at Bv = 10 kg COD/m3 d The activated carbon + natural zeolite showedgood qualities as AFBR support

6

AFBR; carrier support zeolite and activatedcarbon; LS

VFSM anaerobic column reactor (ACR);Support material studied: coconut coir; LS

�COD = 64% at Bv = 23.25 kgCOD/m3 d; HRT = 8 d

Coconut coir as the support material appearsto be a cost effective and promising technologyfor treatment distillery effluent, howeverdisadvantages are the operation at longHRTand moderate efficiencies moderate�COD = 64%

7

Mezcal vinasses �COD = 85% at Bv = 1.96 COD/m3 d Methanogenesis deteriorated at Bv > 10.7 kgCOD/m3 d as revealed by decreases in theremoval efficiency (60–70%) as well as biogasproduction, along with a jump in the alfa factorup to values 0.51–0.64.

8

AFBR, LS, 35 ◦C [CH4] = 83%, v/vBv = 1.96, 2.73, 5.70, 10.70 y 30.40 kg COD/m3 d, �COD = 61% at Bv = 30.4 COD/m3 d

[CH4] = 49% v/v

Notes: AFBR, anaerobic fluidized bed reactor; VWD, vinasses from wine destilation; VFSM, vinasse from fermented sugar molasses; Bv, volumetric organic loading rate; HRT:hR tta (2A

ao(Cac

2

wibaas

ydraulic retention time; LS: laboratory scale; �COD, removal efficiency of COD.eferences: 1. Pérez-García et al. (2005); 2. Thanikal et al. (2007); 3. Sheli and Molecharya et al. (2008); 8. Robles-González (2011).

naerobic treatment (Durán-de-Bazúa et al., 1991). In this regard,ther treatment techniques such as advanced oxidation processesAOP) (Lucas et al., 2010; Martín et al., 2002; Sreethawong andhavadej, 2008; Zeng et al., 2009) can be used in combination withnaerobic digestion to enhance removal of biologically-recalcitrantomponents.

.1.1. Anaerobic fluidized-bed reactorThis process has been used in the treatment of high strength

astewaters and can effectively operate at very high organic load-ng rates and high feed rates. Biomass does not wash-out from the

ioreactor because of its immobilization on support particles; withn adequate start-up it is relatively easy to the develop a bed of veryctive bioparticles that is “fluidized” inside the bioreactor, that is,uspended by drag forces of upcoming effluent.

007); 4. Fernández et al. (2007); 5. Pérez et al. (1999); 6. Fernández et al. (2001); 7.

Anaerobic fluidized bed bioreactors display all the typical ben-efits of anaerobic digestion (Chen et al., 1988; Iza, 1991; Pérezet al., 1997–1999). In addition to those, more specific advantagesof this bioreactor configuration can be quoted (Collivignarelli et al.,1991; Durán-de-Bazúa et al., 1991; Holst et al., 1997; Meunier andWilliamson, 1981; Tay and Zhang, 2000): it can carry a high con-centration of biomass attached to a dense carrier, which cannotbe easily washed out from the bioreactor and increases overallpollutant depuration rate; a very large surface area for biomassattachment and wastewater/biocatalyst contact is provided; typi-cally high mass transfer rates are achieved for substrate from bulkliquid to bioparticles and for products from bioparticles to the

bulk liquid phase enhanced by liquid recirculation and by eitherin situ generation or recirculation of biogas (Dos Reis and Silva,2011; Fuentes et al., 2008; Godia and Sola, 1996; Holst et al., 1997;Wei et al., 2011); it allows for the treatment of either high or low

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trength wastewaters; it has the ability to control and optimize theiofilm thickness; the biomass carrier can be chosen for a specificpplication to enhance removal; recirculation of treated effluenteans that the reactor shows an excellent hydraulic pattern that

voids plugging, shortcircuiting and dead zones; for the same rea-on an excellent dilution of influent with the effluent is achieved,hich provides alkalinity (and consequently, some neutralization)

nd reduces the concentrations of pollutants and toxic substancesimportant for high organic wastewaters and/or toxic wastewatersuch as vinasses). Last, but not least, anaerobic fluidized bed biore-ctors exhibit a fast start-up (even when non anaerobic inocula aresed), good stand-by features, and an outstanding process stabil-

ty (Durán-de-Bazúa et al., 1991; Garibay-Orijel et al., 2005; Iza,991; Pérez et al., 1997; Poggi-Varaldo and Rinderknecht-Seijas,996; Switzenbaum, 1983). These are the reasons why the anaer-bic reactors have been scaled up at pilot and industrial levels.

Robles-González (2011) treated mezcal vinasses in a lab scale,esophilic anaerobic fluidized bed reactor with organic loading

ates of 2.0, 2.7, 5.7, 10.7, and 30.4 kg COD/m3.d. The carrier mediumas 6/20 mesh granular activated carbon. Removal efficiency of

rganic matter was in the range 61.5–84.8% as COD. Methanogen-sis started to deteriorate at the loading rate 30.4 kg COD/(m3.d)s revealed by decreases in the removal efficiency (60–70%) asell as biogas production, along with a jump in the alfa factor up

o values 0.51–0.64. Methane content in biogas was in the range8.9–82.9%; higher values corresponded to lower organic loadingates of operation. In summary, these experiments confirmed theechnical feasibility of anaerobic treatment of mezcal vinasses inuidized bed bioreactors.

Pérez et al. (1999) performed studies in distillation winesinasses degradation with a termophilic fluidized bed reac-or (55 ◦C) at lab scale, they used SIRAN carbon as support

edium (previously colonized in a semicontinuous fixed bed).hey reported 96.5% COD removal operating at organic volumetricoads BV = 5.88 kg COD/m3.d. When the bioreactor was challengedt BV = 32 kg COD/m3.d, a substrate to biomass ratio Bx = 0.55 kgOD/kg VS.d, and 2.55 h hydraulic retention time (HRT), a volumet-ic methane production of 1.08 m3/(m3.d) was obtained during anperation period of 94 days. The increase of organic load provokedhe decrease in the contaminants removal (COD) and in biogas pro-uctivity. Durán-de-Bazúa et al. (1991) reported the operation of300 L capacity, mesophilic pilot anaerobic fluidized bed reactor

oaded with 700 �m diameter spent ion exchange resin as supportedium. They studied three HRT (4, 3 and 2 d, based on the biore-

ctor volume) and they reported up to 70% COD removal efficiencyat BV = 34 kg COD/m3.d and HRT of 2 days), and 7 m3 (STP)/m3.diogas volumetric productivity with 70–80% methane. In a anothertudy, Sheli and Moletta (2007) treated wine distillation vinassesn a bioreactor with a 66% poliethylene support material (density.84 g/mL); it was mixed during 1.25 min periods with a submergedump in the bottom, and they obtained 81.3–89.2% COD removalfficiency at BV = 29.6 kg COD/m3.d. At the end of the experimentn increase in the biomass attached to the carrier was observed83.4%).

.1.2. Anaerobic filtersAnaerobic filters are packed columns with a type of static

edium support colonized by an anaerobic microbial consortium.hese bioreactors can work in upflow and downflow mode; theatter achieves in general better, sustained and more reliable oper-tion. The downflow anaerobic filter has the capability to minimizelogging of the packed bed when operated with effluents with high

oncentrations of suspended solids (Nicolella et al., 2000). Braunnd Huss (1982) treated vinasses from molasses fermentation in ailot scale anaerobic filter reactor during a year operation, at load-

ng rates Bv up to 50 g VS/m3.d, with HRT approximately 1 day.

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They reported a biogas yield between 430 and 460 L/kgVS with a13–20.5 m3/(m3 d) biogas volumetric productivity. In experimentson treatment of winery wastewater in a lab scale upflow anaerobicfilter at ambient temperature (19–27 ◦C), BV = 37.68 kg COD/(m3.d),and HRT of 8 h, Yu et al. (2006) observed 82% COD removal effi-ciency (initial concentrations 8.34–37.68 kg COD/m3), and a yield0.30–0.35 m3-CH4/kg COD, the best results were obtained workingwith multiple feed points. In another study, Thanikal et al. (2007)treated vinasses in a lab scale anaerobic reactor packed with smallpieces of low density poliethylene (0.93 g/cm3, Bioflow 30) as sup-port medium. They found COD removal efficiencies over 80% evenat a challenging BV of 30 kg COD/m3.d; the retention biomass capac-ity obtained was 4–6 g dry solids per g support, representing afixed biomass of 57 g solids/L reactor. Bories et al. (1988) treatedvinasses from sugar molasses fermentation vinasses in a down-flow fixed bed anaerobic digestor using plastic as support medium;they obtained 85–97% BOD removal efficiencies and 60–73% COD,at BV = 14.2–20.4 kg COD/m3.d, HRT of 2.5–3.3 d, with a biogas pro-ductivity of 6.5–8.4 m3/(m3.d).

When treating effluents from beet sugar industry (range ofconcentration from 5000 to 15,000 mg COD/L) in a mesophilic, con-tinuous lab scale packed bed anaerobic reactor using polyestercloth as support medium, Hamoda and Kennedy (1986) obtaineda 87% COD removal and 4 m3/(m3 (reactor volume).d) methaneproduction rate at BV = 24 kg COD/m3.d and HRT of 5 h. Farhadianet al. (2007) used an upflow anaerobic fixed bed reactor filledwith different support materials to treat beet sugar effluents;they observed that COD removal efficiency depended on the typesupport employed. Best results were obtained using a standardindustrial corrugated packing (75–93%) whereas lower results cor-responded to a PVC packing (65–77%).

2.1.3. Upflow anaerobic sludge blanket and related reactorsSince the late 1980s this bioreactor configuration has been

applied with success in the treatment of a great variety of wastew-aters (Lettinga and Hulshoff-Pol, 1991). The UASB operates incontinuous and upflow regime, it can accept high feed rates, itpossesses an internal biogas collection system, and it can workunder mesophilic and termophilic conditions (Wiegant and DeMan, 1986). A few drawbacks of the UASB are: slow start-up,since granulation of the sludge is a long time process not com-pletely understood, or at least, reliable protocols are not availablein the open literature; poor mixing and existence of dead zoneswhen operated at low flowrates; active biomass wash-out whenthe reactor is subjected to hydraulic loading upsets or when granu-lar sludge entraps gases; granular sludge destruction due to toxicityepisodes; extreme susceptibility to suspended solids in the influ-ent, which accumulate in the reactors and pose a major problemfor the operation of the reactor and reduce the reactor capacity(Nicolella et al., 2000). To some extent, some disadvantages such asthat of poor mixing have been overcome with the modification ofthe so called expanded granular sludge bed reactor (EGSB) that is anUASB with effluent recycling. In this way, several advantages of theanaerobic fluidized bed bioreactor were incorporated to the EGSB(Garibay-Orijel et al., 2005; Nicolella et al., 2000; Poggi-Varaldo andRinderknecht-Seijas, 1996).

Harada et al. (1996) treated alcohol distillation spentwash ina UASB reactor under termophilic conditions (55 ◦C) at BV = 28 kgCOD/m3.d, COD influent concentration of 10 kg COD/m3. Theyobserved up to 45 and 80% of COD and BOD removal efficiency,respectively. In studies performed by Espinosa et al. (1995) in alab scale UASB reactor treating vinasses from sugar cane molasses

distillation at BV = 17.4 kg COD/(m3.d), a poor 44% COD removal effi-ciency was achieved. Yet, supplementation of the influent witha solution containing Fe (100 mg/L), Ni (15 mg/L), Co (10 mg/L)and Mo (0.2 mg/L) was associated to significant increases in the

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OD removal efficiency(58%) and biogas production (from 10.7o 14.8 NL/d). Shivayogimath and Ramanujam (1999) tested a labcale hybrid UASB reactor (UASB at the bottom, and packed witholypropilene rings in the upper zone as a separation gas–liquidnd biomass retention system) for the treatment of a distillerypentwash. At BV = 36 kg COD/m3.d and HRT of 6 h a 80% CODemoval was achieved; the produced biogas had 80% methane andhe methane pseudoyield was up to 0.40 m3 (NPT) CH4/kg CODfed,here the pseudoyield is defined as the amount of methane gen-

rated per unit mass of substrate fed or applied (as g or kg COD orOD fed) and the amount of methane is expressed in m3 at 273 Knd 101.325 kPa absolute pressure.

The variation and low COD removal efficiencies obtained inome of the reported investigations could be a consequence of theermented material origin, the distillation process (Harada et al.,996) as well as due to the presence of some organic compoundsphenolic compounds) that have been reported as being toxic andecalcitrants for the methanogenic systems (Borja et al., 1993a;enitez et al., 2003; Field and Lettinga, 1989).

.1.4. Biohydrogen productionIn recent years a wide group of investigators have proposed

nd chosen hydrogen as an alternative renewable energy source.onsequently, scientists have tried to look for new alternativeso generate hydrogen from organic compounds present in liquidr solid agroindustrial wastes using microorganisms (Fountoulakisnd Manios, 2009; Kapdan and Kargi, 2006; Valdez-Vazquez andoggi-Varaldo, 2009; Wang and Zhao, 2009). It has been shown thatrganic compounds present in vinasses could be used as substrateor hydrogen-producing microorganisms, particularly in dark fer-

entation approach. In this regard, Lay et al. (2010) used vinassesrom distillation of fermented molasses fermentation as growth

edium for the hydrogen production in a lab scale, complete mixeactor, operating at HRT of 3–24 h. Their bioreactor was seededith activated municipal wastewaters treatment plant sludgeshere the presence of Clostridium acetobutylicum and Clostridium

asteurianum bacterial genuses were identified. A production of upo 390 mmol H2/(L.d = was observed using Bv = 320 g COD/(L.d) at

HRT of 3 h. In another study Yu et al. (2002) treated vinassesrom rice fermentation in a lab scale upflow reactor at a HRT of–24 h, COD 14–36 gCOD/L, pH 4.5–6.0 and temperature 20–55 ◦C.he obtained biogas composition showed 53–61% hydrogen con-entration and 37–45% carbon dioxide in the biogas. The optimumydrogen production rate was 9.33 LH2/(gVSS.d) with 2 h HRT andhe corresponding yield ranged 1.37–2.14 mol H2/mol-hexose at4 g/L COD, pH 5.5 and 55 ◦C.

.1.5. Ozonation combined with anaerobic digestionVinasse from bioethanol manufacturing (COD0 = 68.56 g/L) was

ubjected to a very short ozonation pre-treatment (15 min) in batchaboratory-scale reactors at 35 ◦C (Siles et al., 2011). Under thisondition 50% of reduction of phenols was obtained. The stable con-entration of organic carbon concentration showed that phenolsere transformed into other simple forms. Anaerobic biodegrad-

bility of raw and pre-treated vinasses were similar (values closeo 80% as COD). However the pre-ozonation process enhanced the

ethane yield coefficient (14% increase) and methane productionate (by 41.16%). The integrated chem-biological process proved toe a viable option for the treatment of vinasses.

Martín et al. (2002) tested the anaerobic treatment ofinasse from ethyl alcohol production line (COD0 = 97.5 g/L;OD0 soluble = 42.228 g/L; TOC0 soluble = 36.275 g/L) pretreated with

zone; ozone/UV light and Ozone/UV light/TiO2. The last pretreat-ent decreased COD by 32%, although TOC removal was very low

7%). In the anaerobic digestion stage, pretreated vinasses gave anncreased yield coefficient (1.12 mL CH4/mg TOC) and also a higher

technology 157 (2012) 524–546

specific rate of methane production (20%) compared to those fromraw vinasse.

Sangave et al. (2007b) examined the effect of the ozonationand ultrasound (US) on aerobic biodegradability of two vinassesfrom the fermentation of sugar-cane molasses. Thermally (TPT-V)and anaerobically pre-treated (APT) were tested. COD was diluteddown to 10–12 g/L; COD reductions of 45.6% and 13% were reportedfor ozonation and US, respectively. In both cases, the subsequentaerobic oxidation rate was enhanced. The aerobic biodegradabilityincreased 25 times with the ozonation treatment of TPT-V, how-ever the use of US and ozone did not improve the biodegradabilityof the APT. Overall, ozonation peformed better than US regardingthe increase of both COD removal and aerobic biodegradability.

In another work, Alvarez et al. (2005) treated cherry stillage inan anaerobic sequencing batch reactor (AnSBR); influent COD wasvaried between 5 AND 50 g/L. Different cycle times were selectedto test specific organic loading rates (Bx), from 0.3 to 1.2 g COD/(gVSS.d). They observed COD and TOC removal efficiencies higherthan 80% for influent COD up to 28.5 g/L and low Bx. Yet, volatileorganic acids (VOA) were accumulated and methane productiondeteriorated at influent COD higher than 10 g/L; the AnSBR showedsigns of instability and could not operate efficiently at Bx > 0.3 gCOD/(g VSS.d) possibly due to toxic polyphenols in cherry stillage.A pre-ozonation step was useful, since more than 75% of polyphe-nols could be removed by ozone. The integrated process was shownto be a suitable treatment technology as the following advantagescompared to the single AnSBR treatment.

2.2. Aerobic treatment

There are several studies on aerobic treatment of vinassesmainly for removal of color (melanoids) and, particularly, toxicsubstances such as phenols and polyphenols. In general, aerobictreatment is not recommended for wastewaters with high contentsof organic matter such as vinasses, because of the high energy costs,limitation of oxygen transfer in the aeration, and the high amountof biomass generated (waste sludge, ca. 50% of BOD is transformedin biomass or sludge) that may represent huge additional costs ofsludge treatment and disposal (Jiménez et al., 2005; Metcalf andEddy, 2004.)

Aerobic treatment of vinasses has used microbial consor-tia (mixed cultures with bacterial predominance, sampled fromwastewater treatment plants) (Beltrán et al., 1999a; Benitez et al.,2000) as well as bacterial pure cultures (Dahiya et al., 2001a;Sangave et al., 2007a; Sirianuntapiboon et al., 2004), ligninolytic(Fahy et al., 1997; Jiménez et al., 2005; Raghukumar and Rivonkar,2001; Raghukumar et al., 2004) and non-ligninolytic fungi (Fadilet al., 2003; García et al., 1997).

Either single aerobic process or aerobic treatment combinedwith phys-chem processes such as ozonation (Beltrán et al., 1999a;Rehman et al., 2006; Robles-González et al., 2010; Sangave et al.,2007a), Fenton-like reaction, and advanced oxidation with UV lightand H2O2 have been explored and reported (Beltran-de-Herediaet al., 2005a; Benitez et al., 2003). Most of these studies have beencarried out at lab scale. Also, there have been efforts for reclaimingbacterial biomass as a source of alternative protein, i.e., unicellu-lar protein for animal feed (Barrocal et al., 2010; Díaz et al., 2003c;Durán-de-Bazúa et al., 1991.)

2.2.1. Aerobic treatment combined with other methodsRobles-González et al. (2010) reported results on the series

treatment of MV by ozonation followed by aerobic biological. In the

ozonation stage, they found organic matter removals in the range4.5–11% COD (contact times up to 1.5 h), whereas the removal ofaromatic compounds and phenols (expressed as gallic acid) werewithin the ranges 16–32% and 48–83%, respectively. In the aerobic

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ost-treatment, the maximum removal of COD was 84% that cor-esponded to ozonated vinasses with previous 1.5 h of ozonation.he overall removal efficiency (COD) of the combined treatmentzonation + aerobic degradation reached a maximum value of 87%,here the major contribution corresponded to the biological stage.eltran-de-Heredia et al. (2005a) carried out batch, lab scale exper-

ments with aerobic pre-treatment and chemical oxidation withenton reagent and hydrogen peroxide for the removal of organicatter as COD and phenols present in vinasses from wine distil-

ation (inlet COD0 = 18.5 g/L). An aerobic seed sampled from a fullcale wastewater treatment plant was used, the inoculum was pre-iously acclimatized to vinasses (in the acclimatization step CODas 5–18 g/L). Removal efficiencies were 75% and 54% in the aero-

ic pre-treatment for COD and phenols, respectively, whereas thealues observed for the chemical oxidation with Fenton-H2O2 were4% and 79%.

Benitez et al. (2003) studied the oxidation of the organicubstrate present in wastewaters generated in wine vinassesCOD0 = 24.5 g/L and BOD0 = 11.15 g/L) by both an ozonation pro-ess and an aerobic activated sludge system. The ozonation wasarried out in a subsequent first discontinuous and a second con-inuous periods. Organic matter removals ranged from 5% to 25.2%s COD; total aromatic compounds removal varied between 16.8%nd 51.4%. The effects of the inlet ozone partial pressure, theydraulic retention time in the continuous reactor (inlet COD0round 21.7 g/L and 0.454 Abs at 254 nm for aromatic compoundsAC)) and the presence of UV radiation and H2O2 in addition tozone (COD0 = 3.8 g/L and 0.05 Abs for AC) were evaluated. It wasound that AC removals increased with the increase of ozone partialressure (16.8–31.2%) and the COD removal was low (5.0–7.5%).he increase of ozone contact time from 3 to 9 h also favoredollutant removals with up to 25.2% COD and 51.4% AC. The usef ozone + UV + H2O2 was much more efficient than ozone aloneCOD0 = 3.8 g/L); the first approach lead to removals of 58.4% and6.9% of COD and AC, respectively. Kinetic fittings for batch andontinuous periods lead to the evaluation of the apparent rate con-tants for pollutant degradation as 216 L/(mol O3 h) and 232 L/(gOD h), respectively. In the aerobic degradation (COD0 = 19.56 g/L)y the activated sludge system COD removals from 31% to 85% werechieved at hydraulic retention times between 24 and 72 h. The aer-bic treatment satisfactorily fitted the Contois model for organicatter degradation.Sangave et al. (2007a,b) conducted lab-scale experiments in

rder to assess the effect of ozone as pre-aerobic treatment andost-aerobic treatment for the treatment of vinasses from sugarane. The treatment was carried out by ozonation, aerobic biolog-cal degradation processes alone and by using the combinationsf these two processes. Seed for the aerobic treatment step wasampled from a full scale wastewater treatment plant and furthercclimatized to vinasses before tests. Ozone removed up 27% ofrganic matter as COD during the pretreatment step itself. In theombined process, pretreatment of the influent led to enhancedates of subsequent biological oxidation step. Nearly a 2.5 timesncrease in the initial oxidation rate was reported. Post-treatment

ith ozone led to further removal of COD along with the com-lete discoloration of the effluent. The 3-step integrated processozone–aerobic oxidation–ozone) achieved 79% COD reductionlong with discoloration of the effluent sample as compared to4.9% COD reduction for non-ozonated vinasses over a similar treat-ent period.In another research, wine vinasses were treated separately first

y means of a chemical ozonation (COD0 = 34–36 g/L) and a biologi-

al aerobic degradation in an activated sludge system, and secondlyy a combined process which consisted of an aerobic pretreatmentollowed by an post-ozonation treatment (Benítez et al., 2000).rocesses were continuous in all cases. Ozonation effected low

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COD removals in the range 4.4-16%, with increased when boththe hydraulic retention time and the ozone partial pressure wereincreased. An ozonation model based on a mixed flow reactor forthe liquid phase and plug flow reactor model for the gas phase,yielded a rate constant for the ozone reaction and the consump-tion ratio kO3 = 3.6 L/(g COD h) and b = 22.5 g COD degraded/mol O3consumed, respectively. The activated sludge stage results fitted amodified Contois model with kinetic parameters K−1 = 5.43 L/g VSSand qmax = 6.29 g COD/(g VSS.h). Finally, the authors reported thatthe combined process significantly improved the efficiency of theozonation stage compared to the ozonation alone; this was alsoreflected by a 100% increase of kO3 = 5.6 L (g COD h) of the post-ozonation stage of the combined process.

Beltran et al. (2001) reported the performance of the inte-grated aerobic digestion and post-ozonation for the treatmentof high strength distillery wastewater (i.e., cherry stillage,COD0 = 145–180 g/L and BOD0 = 100–140 g/L). In the batch acti-vated sludge step, BOD and COD overall conversions of 95% and82% were registered, respectively. However, removals of polyphe-nol content and absorbance at 254 nm (A254) were much lower, i.e.,35% and 15%, respectively. The aerobic digestion process was fittedto a Contois’ model kinetics.The post-ozonation stage was effectivefor the removal of polyphenols and A254.

2.2.2. Treatment using bacterial consortia and bacterial purecultures

Vinasses discoloration has been frequently performed usingfungi as these latter have the capacity to degrade organic poly-mers and to adsorb them onto the mycelium (Fahy et al., 1997;González et al., 2000; Kumar et al., 1998; Strong and Burgess,2008). Nevertheless in recent years it has been reported the fre-quent use of bacterial consortia as well as pure microbial cultureshaving the capacity to reduce color and remove the COD from thevinasses. Mohanaa et al. (2007) isolated and characterized a bac-terial consortium that consisted of Pseudomonas aeruginosa PAO1,Stenotrophomonas matophila and Proteus mirabilis employing 16srDNA analysis; this consortium was able to decolorize vinasses (67%color removal in 24 h) and to reduce 51% COD in 72 h at 37 ◦C usingan effluent supplemented with 0.5% glucose, 0.1% KH2PO4, 0.05%KCl and 0.05% MgSO4·7H2O.

Sirianuntapiboon et al. (2004) tested an acetogenic bacterialstrain BP103 and achieved 32.3% and 73.5% color removal fromraw vinasses from sugar molasses distillation and anaerobically-treated vinasses, respectively. Both effluents were supplementedwith 3.0% glucose, 0.5% malt extract, 0.1% KH2PO4, 0.05% KCl and0.05% MgSO4·7H2O. Without the nutrient supplementation the dis-coloration decreased. The same effect was observed when the strainwas evaluated in a sequencing batch reactor at 7 days HRT using 10times diluted anaerobically-treated vinasses supplemented with30 g/L glucose (Tondee and Sirianuntapiboon, 2008). COD removalefficiency was significant (65.2%) as well as removal of otherpollutant parameters (BOD5, 82.8%; TKN, 32.1%; and melanoidinpigment, 50.2%).

Chavan et al. (2006) isolated a Pseudomonas sp. strain from soilnear a sugar-cane alcohol distillation factory (COD0 = 146.38 g/Land BOD0 = 70.84 g/L, 10% vinasse dilution was necessary). Colorbefore treatment was dark brown. The strain was capable of remov-ing 56% color and 63% COD in 72 h, at pH 6.8–7.2, temperaturein the range 30–35 ◦C and with the optimum medium compo-sition 4 g/L glucose, 0.2 g/L KH2PO4 and 0.009 g/L MgSO4·7H2O.Removals of COD and color by Pseudomonas sp. were presum-ably associated with degradation stimulation via supplementation

with another source of carbon (Dahiya et al., 2001a,b; Tondeeand Sirianuntapiboon, 2008), as well as by the production ofenzymes, such as sorbose oxidase, manganese peroxidase depen-dant, and glucose oxidase. The latter, for instance, is activated in the

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resence of glucose, and yields H2O2 as a by-product (Scheleissnert al., 1997). H2O2, in turn, can oxidize organic matter thus yieldingimpler compounds like volatile acids (Kumar and Viswanathan,991), facilitating their further degradation to carbon dioxide ando biomass production. This, in turn, results in removal of CODGhosh et al., 2002; Watanabe et al., 1982) and reduction of colorOhomomo et al., 1988).

Adsorption could be another mechanism of color removal,lthough it has been reported that previous acclimatization of cellso a medium containing melanoidins products (MP) may decreasehe adsorption activity (Sirianuntapiboon and Prasertsong, 2008).ontribution of volatilization to color and COD removal is unlikely,ince concentration of volatile compounds in vinasse is negligi-le. Dahiya et al. (2001a) achieved 76% discoloration employingPseudomonas fluorescens strain immobilized in a porous cellu-

ose carrier under non sterile conditions; color reduction was 90%nder sterilized medium for the wastewaters supplemented withlucose 0.5% (w/v) provenient from molasses fermentation and dis-illation process. They discussed that this difference might be alsoue to the role of the high temperatures reached during the ster-

lization process that could have caused the pigment degradationnto low molecular weight compounds. Kumar and Viswanathan1991) isolated and acclimatized an unidentified bacterial strainy continuous increasing the distillery wastewater concentration.his strain was capable of removing 80% COD of the influent in 4–5ays.

In the precedent cases the vinasses had to be supplemented withlucose (0.4–30 g/L) and other essential nutrients, the discolorationas possible after 3–10 days. This could mean an economicalrawback for these treatments since the mezcal vinasse quan-ity generated each year is approximately 90,000,000 L, whereashe annual amount of vinasses in India is nearly 40 billions litersRaghukumar et al., 2004).

.2.3. Alternative sources of proteinDue to the costs of wastewater treatment the obtaining of by-

roducts as part of the process is becoming, without doubt, anmportant factor that could enhance the economic feasibility ofhe treatment. For instance, microbial biomass is a by-product thatould be used as unicellular protein source for animal feed. On thene hand, this is of particular interest in countries where high qual-ty fodder and proteins for animal feed are scarce, such as Mexico,ndia, and others. On the other hand, even in developed countries

here conventional, quality animal feeds are available, microbialiomass protein could play a significant role in abating animal hus-andry costs.

Several researchers have carried out studies to solve the envi-onmental problems provoked by the vinasses high organic loadsooking for producing unicellular biomass as alternative sourcef protein. Research included bacterial, fungal, and algal biomass.urán-de-Bazúa et al. (1991) treated vinasses from sugar molasses

ermentation in lab and pilot tests in aerobic rotating disk bioreac-or system. At lab scale they used diluted vinasses and obtained

icrobial biomass with a raw protein content between 18 and7%, and 1.8 kg wet biomass/kg COD removed system yield; CODemoval efficiency obtained was 65% and 70% and BOD removalfficiency was ca. 95%. At pilot plant scale they employed non-iluted vinasses with a concentration 60 to70 kg COD/m3; a similariomass yield to that the lab scale tests was obtained, with amall increase in the protein content (20% and 30%). However, itas observed that organic matter removal efficiency significantlyecreased compared to lab tests (40–50% and 60–70% for COD and

OD5 removal efficiency, respectively). The raw protein percent-ges were similar to those found by Díaz et al. (2003c). In effect,he latter reported 30.1% when using Candida utilis in a lagoonystem. Shojaosadati et al. (1999) isolated a fungus species of the

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genus Hansenula from an alcohol plant effluent. In batch tests withthis strain and vinasses they observed a concentration of 5.7 g/Lbiomass, with 39.6% raw protein and 31% COD removal efficiencywith no addition of external nutrients source. When supplementingnutrients they found a considerable increase in the biomass con-centration (8.5 g/L biomass), raw protein (50.6% and COD (35.7%).

Nitayavardhana and Khanal (2010) studied the potential useof Rhizopus microsporus (var. oligosporus) as protein ingredientin feed for aquaculture. They cultivated the microorganisms invinasses from sugar-cane alcohol distillation; vinasses were sup-plemented with nitrogen and phosphorus. It was found a highfungal growth at pH 5.0 and 30 ◦C, the obtained biomass containedca. 46% raw protein and the COD removal was 42%. Barrocal et al.(2010) tested a batch system to produce Spirulina maxima capa-ble of growing in a Schlösser medium with 5 g/L de sugar beetvinasse; they reported biomass concentrations of 3.5 and 4.8 g/Land productivities of 0.15–0.24 g/(L.d). The biomass concentrationand productivity increased to 8 g/L and 0.7 g/(L.d), respectively,when they used a photobioreactor with Schlösser medium with2 g/L sugar beet vinasse.

2.3. Vinasse co-composting with organic solid wastes

A summary of several works that used vinasses for co-composting a variety of agroindustrial and other wastes ispresented in Table 5. Land disposal of raw vinasse may impairthe physical, chemical and biological properties of soil, which isreflected in the increase of soil loss, reduced vegetation coverand increased values of exchangeable sodium (Tejada et al.,2008). These problems can be overcome by co-composting thevinasse with other solid wastes. Because no information aboutco-composting of mezcal-vinasse was found in literature, severalexamples of this procedure that used other kinds of vinasse arediscussed.

Using a mixture of vinasse and cotton waste, Díaz et al. (2003a)investigated the influence of vinasse addition and incubation timeon the properties of the co-composted products (pH, electrical con-ductivity, organic matter content, Total Kjeldahl Nitrogen (TKN)and C/N ratio). The products obtained with 20–30% of addedvinasse, at moderate operating times (20–35 days), showed highbiodegradability and minimum losses of nitrogen.

Three mixtures of a concentrated vinasse and solid wastes(grape husks, squeezed olive paste and cotton wastes) wereco-composted in static windrows (Madejón et al., 2001a). The com-posts obtained were used in field experiments to study the effectof their application as deep fertilizer on three crops: corn, sugar-beet and sunflower. At the end of the experimental period, soiloxidizable-C, total humic extract-C and humic acids-C contents sig-nificantly increased in soils treated with composts when comparedwith an unamended control and inorganic fertilizer treatments.Organic fertilization also increased the Kjeldahl-N content of thesoil. A slight increase of soil salinity was observed both in thecomposts and the inorganic fertilizer treatments. Nevertheless, thisincrease did not cause sodium hazard to the soil.

In another study, Díaz et al. (2003b) composted mixtures ofvinasse and grape husks in a lab scale reactor at 55 ◦C. They foundthat the best properties of the co-compost were obtained withadditions of vinasse in proportions between 10% and 20%. Highervinasse proportions increased the NKT losses, the acidity, and thesalinity of compost extracts.

Tejada et al. (2008) showed that the soil application of a prod-

uct obtained from co-composting beet vinasse and green manure(Trifolium pratense L.), combined with vermicompost, had a posi-tive effect on soil physical and biological properties with respectto a control soil. The use of this product would contribute to soil

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Table 5Use of vinasses in co-composting.

Substrate, process and operationalconditions

Features of the process; compostcharacteristics; effects of application

Remarks Ref.

Vinasse from wine; Lab scale (LS);aerobic conditions at 55 ◦C for 43 days;determination of ratio grapemarc/vinasse for an optimumcomposting

pH did not show great differences among themixtures (7.9–8.31).

Higher vinasse ratio the higher thesalinity of the product

1

Organic matter (OM) content decreased in allmixtures

Recommended 10–20% vinasse in themix might as the best for theoptimization of the process and highquality compost

At lower vinasse ratio higher OM losses andhigher values of biodegradabilityIncreasing the ratio of vinasse in the mix wasan increase in the losses of Kjeldahl-N; Pcontent decreased. Possible limit to vinasseaddition because of the optimum range of C/Pratio is 75–150

Mixture of depotassified beet vinasse(45%, dry weight) and cotton gin waste(55%) using two different aerationsystems: static aerated pile (process A)and windrows (process B); Pilot scale

A faster increase of temperature in the processB (54 ◦C at 7 days) than in process A (45 ◦C at21 days) was observed

The decrease of NH4+–N content could

be used as a criteria of compostingmaturity

2

In both systems high NO3−–N content at the

beginning of composting was observed; itdecreased during and slightly increased duringmaturation phase

Decreased phytotoxicity

Increase of the NH4+–N in both processes

during the thermophilic phase. At the end ofthe process a decrease in NH4

+–N content wasobserved

High salinity; compost should be usedat moderate doses due to the relativehigh values of electrolytic conductivity(21.7 and 12.9 dS/cm for A and Bprocesses respectively)

Mass porosity did not change in process A,whereas it increased in process B

Windrow process recommended

High amounts of macronutrients, particularlyN and KLow concentrations of heavy metals

Mixtures of depotassified beet vinassewith three different residues wereco-composted in static windrows,under cover, with aeration andcontrolled conditions

Composts presented low concentrations ofmicronutrients

Compost had a moderte positive effecton plant nutrition and yield, and onsoil chemical fertility

3

Mixtures: A, 82% grape-marc and 18%vinasse; B, 76% olive pressed cake, 17%vinasse and 6% leonardite; C, 47%cotton gin trash, 49% vinasse and 3%leonardite

Risk of use for its high content of salts presentin vinasse is decreased with the combinationof urea with compost Vinasse and P2O5

No serious risks of salinization orsodification for coarse textured or welldrained soils under irrigation

Field experiments to study composteffects on three crops

No phytotoxicity detected

Composts increased crops yields with respectto the control without vinasses

Mixtures of vinasse (V) with solidresidue cotton waste (CW) at: 0%, 11%,40%, 69% and 80% (V/CW, w/w wetweight); times 1, 7, 23, 38 and 45 days

pH and Kjeldahl-N were influenced by both OTand V ratio; more sensitive to OT. Kjeldahl-Nincreased with OT and V ratio

Conditions recommended: 4

LS at 55 ◦C; Observed the effect ofoperation time (OT) and vinasse addedover pH; OM; Kjeldahl-N; C/N ratio,biodegradability and germinationindex (GI)

OM and GI were similarly affected by OT and V.GI significantly increased with OT butdrastically decreased with V ratio

For OM, V: 0–11%; OT: 38–45 d

C/N decreased with V ratio For GI, V: 11–40% OT: 38–45 daysThe addition of V increased the concentrationsof K, Ca and Mg decrease in the concentrationof P

For C/N, V: 0–40%; OT: 7–23 d

Minimal Kjedahl-N losses with 11–40%of V and OT 7–23 dDecrease in P could limit the additionof vinasse since optimal C/P (75–150)would not be met

Pile composting, 12 weeks Sucession of microbial populations wasobserved and inferred by physicochemicalchanges

Temperature > 55 ◦C was enough tosanitisize the produced composts

5

Mixtures of rice straw, soybean residueand enriched with rock phosphatesupplemented with vinasse. Buffalo’smanure was used as inoculum,

Mineralization of organic matter waspromoted by microbial activities led to organicmatte

534 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546

Table 5 (Continued)

Substrate, process and operationalconditions

Features of the process; compostcharacteristics; effects of application

Remarks Ref.

External sources of microorganisms enhancedthe biodegradation of recalcitrant substancesThe compost maturity was satisfactory after 84days

Beet vinasse was mixing withvermicompost (constituted by a greenforages)

The adittion of vinasse plus vermicompost hada positive effect on the soil’s parametersevaluated (physical, chemical and biologicalproperties)

Mixing of beet vinasse withvermicomposts resulted a feasiblestrategy for protecting soil’s propertiesand recovering semiarid areas

6

Application tests, evaluation ofchanges on physical, chemical andbiological properties of soils

The loss of amended soil decreased by 31.2%;plant cover increased 68.7% compared withunamended soil

Beet vinasse alone (V) and mixtures ofcrushed cotton gin compost withvinasse (CV)

The porcentaje of plant cover increased 86% inCV-amended soils and decreased 58.3% withonly-V, compared to the control soil

The negative effect of only-V on thesoilıs properties was ascribed to highconcentrations cations such as Na+ andfulvic acid present in the fresh vinasses

7

Tests for evaluation of effects on plantcover, soil’s physical, chemical andbiological properties

Only-V addition on the soil had negative effecton the soil’s physical, chemical and biologicalproperties

Application of CV to soil helps in itsrestoration

CV had a positive effect on the soil’s physical(structural stability and bulk density), andbiological properties (microbial biomass, soilrespiration, and enzymatic activities) withrespect to the control soil

Mixtures of sugarbeet molassesdistillery slops V and olive mill husks

OT of 30–40 d and the addition of 0–10% Vassociated to best results in terms of OMbiodegradation, compost maturity andlimitation of Kjeldahl-N losses

Mixtures of 0–40% of V and up to 40days of OT were evaluated

8

In-vessel composting process.Evaluation of operating conditions andextent of the stabilization timeMixtures of sugar beet vinasses (V)with cotton gin (CW) and grape marc(GM)

CW and GM were adequate for co-compostingof V

9

Supplemented with phosphate (P) andLeonardite (L) in order to prevent greatlosses of Nitrogen

Lower losses of organic matter was observedwith V co-composted with grape marc

Pile composting The composts tested had high fertilizer values,high levels of stability and absence ofphytotoxicity

Pile A: GM (76%) + V (20%) + L (1%) + P(3%)

No lignin degradation; cellulose degradationonly observed in the pile A

Pile B: CW (76%) + V (20%) + L (1%) + P(3%)Mixtures of vinasses from alcoholdistillery and GM, wheat straw (WS).Wet pre-treatments with 7–15% NaOHon total dry matter at 70 ◦C

NaOH concentration 7–15% increaseddigestibility from 37% to higher than 50%

Increase in the digestibility of the twosolid components and utilization of thevaluable compounds contained in V

10

Mixture A: 100 g wet GM, 16 g WS, and300 ml V

Contact times of 3 h adequate Mixture A was superior to mixture B.

Mixture B: only changed 16 g for 30 gof WS

Quality of the mixture was significantlyaffected when increased the WS from 20.5% to32.6%

NaOH conc. < 5–6% did not improvedigestibility

Pretreated mixtures had acceptable values ofdigestibility, crude fibre, and crude protein;better property profiles than untreated GMaloine and WS aloneHigh Na, K, Fe, Cu and Zn content of bothmixtures as well as of WS

Notes: CV: cotton gin compost with vinasse; CW: cotton waste; GI: germination index; GM: grape marc; LS: lab scale; OM: organic matter; OT: operation time; V: vinasse,WS: wheat straw.References: 1. Díaz et al. (2002a); 2. Díaz et al. (2002b); 3. Madejón et al. (2001a); 4. Díaz et al. (2003a); 5. Rashad et al. (2010); 6. Tejada et al. (2009); 7. Tejada et al. (2007);8

pe

p(emp

. Díaz et al. (2003b); 9. Madejón et al. (2001b); 10. Vaccarino et al. (1993).

rotection, and to the eventual restoration of arid lands (Tejadat al., 2009).

An inspection of works in Table 5 suggests that the best com-osts are those with low ratios of vinasses in the mixtures (10–20%)

Díaz et al., 2002a,b, 2003b; Madejón et al., 2001b). In gen-ral, for those mixtures, high decreases of biodegradable organicatter, adequate nutrient profiles, and positive effects on soil

roperties and crop yields are reported (Madejón et al., 2001a).

Furthermore, the best composts formulated with vinasses havepresented a high fertilizing value as well as an adequate stabilityand absence of phytotoxicty (Díaz et al., 2002a,b; Madejón et al.,2001b).

For higher ratios of vinasses in the mixtures, undesirable highsalinity is observed, associated to high concentrations of cationssuch as Na,K,P, Mg y Ca; this issue certainly could limit compostusefulness and application (Díaz et al., 2002a,b, 2003a).

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Overall, application of composts from co-composting of vinassesnd other wastes to soils has helped in minimizing soil losses asell as increasing the vegetal cover (Tejada et al., 2007, 2009).

he co-composting of vinasse with agricultural and agro-industrialolid wastes is worth investigating because it can provide a low-ost treatment technology suitable for rural regions where moreophisticated technologies are difficult to implement, due to highosts and requirements of skilled personnel.

.4. Fungal treatment

The decomposition of organic matter is usually carried outy fungi, generally Basidiomycetes found in forests and meadows.hese molds colonize the surface soil, layers of humus, dead plants,nd grass wastes. The protective layer of lignin is converted in ligno-ellulose by nonspecific extracellular oxide-reductases producedy fungi (Hatakka, 2001; Heinzkill and Messner, 1997; Steffen et al.,000). Once that lignin was removed, the plant polysaccharides arevailable as a carbon and energy source. Four ligninolytic enzymesave been described; lignin peroxidase (LiP), manganese peroxi-ase (MnP), versatile peroxidase (VP), and laccase. The first threenzymes contain a hemo group as cofactor. All of them use H2O2s the first electron acceptor.

Laccase uses O2 as the final electron acceptor (Papinutti et al.,003; Thurston, 1994), and plays an important role in remediationecause it acts over many organic pollutants such as: polycyclic aro-atic hydrocarbons, phenols, pesticides, endocrine disrupters, and

yes (Dahiya et al., 2001b; López et al., 2004; Majeau et al., 2010;uzhu and Viraraghavan, 2001). Laccase enzyme has also been used

or bleaching pulp and vinasse, and for the discoloration of effluentsrom the textile industry (Couto and Toca-Herrera, 2007; Rodríguezt al., 1999; Strong and Burgess, 2007; Wesenberg et al., 2003).able 6 shows a summary of several vinasse treatments using fungi,ostly ligninolytic. It can be seen that most studies have relied on

upplementation with glucose or another easily degradable carbonource. This is a drawback for fungal treatment application at fullcale.

As it was mentioned above, one of the main features of vinasses its color as well as the content of phenolic compounds; the lat-er are known to be toxic and their concentration can be relativelyigh (Table 3, ca. 500 mg gallic acid/L). So, efforts for degradinghese substances by using aerobic treatment with fungi have been

ade. Unlike the characteristic brown color of stillage generatedy the fermentation of molasses, which is attributed to recalcitrantelanoidines generated by the condensation of reducing sugars

nd amines or amino acids (Cammerer and Kroh, 1995; Strong andurgess, 2008). In the case of vinasse obtained from mezcal andine distillation, the color is generally attributed to highly recal-

itrant and inhibitory phenolic compounds (Borja et al., 1993a,b).lso, many researchers have used extracellular enzymes producedy ligninolytic fungi to degrade recalcitrant molecules such as phe-olic compounds and melanoidins present in vinasse.

Recalcitrant aromatic compounds, color and COD remaining ininasse after anaerobic treatment can be successfully removedy ligninolytic fungi (Benito et al., 1997; Kumar et al., 1998;aghukumar and Rivonkar, 2001; Singh et al., 2010); especially,

f an easily assimilable carbon and energy source is added (Benitot al., 1997; Fahy et al., 1997; Kumar et al., 1998).

Coulibaly et al. (2003) reviewed the utilization of fungi foriotreatment of wastewater. They covered the post-treatment bynaerobic digestion of wastewaters that have been pretreated with

ignolyitic fungi. Among several advantages of fungal pretreatmenthey highlighted the degradation of toxic and recalcitrant pollu-ants, the possible production of enzymes as added-value products,s well as single cell protein. Unfortunately most research of

technology 157 (2012) 524–546 535

fungal pretreatment has been performed at lab scale. They stronglyrecommend to foster research at pilot and commercial scale.

Pleurotus sajor-caju CCB020, a ligninolyitic fungus, was used ina sugar-cane vinasse (COD0 = 42 g/L and BOD0 = 11.3 g/L) biodegra-dation study (Ferreira et al., 2011). Reductions of 82.8% in COD,75.3% in BOD, 99.2% color, and 99.7% in turbidity were observed.A significant vinasse toxicity reduction was further determinedby bioassays with Pseudokirchneriella subcapitata, Daphnia magna,Daphnia similis and Hydra attenuata.

Dedeles et al. (2010) investigated the mechanism of color reduc-tion of diluted molasses distillery slop using crude manganeseperoxidase (MnP) from homogenized mycelia of the ligninolyiticfungus Ganoderma lucidum. Free, homogenized mycelia with addi-tion of 2.5% glucose lead to effective color reductions on days1–10. Greatest color reductions occurred on day 10 using MnP plus100 mg/L Mn(II) as MnSO4·5H2O. MnP activity was low withoutMn(II) in the nitrogen-deficient assay medium. 5 �M H2O2 but notthe chelators oxalic acid and dl-lactate improved MnP activity inphenol red oxidation system at pH 4.5–5.0 and 30 ◦C.

Winery wastewaters (COD0 = 0.665 to 12.6 g/L) were inoculatedwith the ligninolyitic fungus Trametes pubescens MB 89 to establishthe feasibility of fungal submerged culture and the usefulness ofa second biological treatment stage using methanogenic archaea(Strong, 2008). Fungal pre-treatment decreased the organic mat-ter content (COD) and increased the acidic pH values in all cases.Five of the wastewater samples showed an increase in laccase syn-thesis, but the concentrations were low. Fungal pretreatment wasbeneficial to methanogenic digestion of winery wastewaters thathad higher initial phenolic compound and color concentrations,but no for wastewaters with low initial phenolic compound andcolor concentrations. Anaerobic digestion of fungally-treated andraw samples generally showed little difference with regard to totalCOD removal and final pH.

Melamane et al. (2007) studied the combination of fungalpre-treatment with white rot fungus (Trametes pubescens) andanaerobic digestion in order to remove COD and phenolic com-pounds from wine distillery wastewater (COD0 = 15 g/L). In the firststage a 53% COD removal efficiency was reached. In the anaerobicdigestion the total COD removal efficiency was much higher thanthe first stage (89.5%). The anaerobic digestion treatment was ableto withstand shocks of organic loading rates due to the addition ofCaCO3 and K2HPO4 who acted as buffers.

In another work, vinasses from a distillery industry(COD0 = 40 g/L and BOD0 = 5.2 g/L) were treated by P. chrysosporiumin submerged culture (Potentini and Rodriguez-Malaver, 2006).The effect of two temperatures (25 and 39 ◦C) on removals of COD,total phenol concentration, and color were measured. The removalefficiencies of COD, phenolic concentration and color were notaffected by temperature (47.48%, 54.72%, 45.10% at 25 ◦C and54.21%, 59.41%, 56.81% at 39 ◦C, respectively).

Vinasse from sugar fermentation (COD0 = 7.2, 14.4 and 21.6 g/L)was treated with several fungi such as Coriolus versicolor, Funa-lia trogii, Phanerochaete chrysosporium and Pleurotus pulmonarius(Kahraman and Yesilada, 2003). Also the effect of cotton stalk ondecolorizing and COD removing capability of the four fungi wasdetermined. In the concentration range assayed (10%, 20% and 30%)vinasse was effectively decolorized by C. versicolor and F. trogii. Cot-ton stalk addition seemed to stimulate the discoloration activity ofall fungi. The utilization of cotton stalk was advantageous sinceit performed both as an attachment support and as a source ofnutrients.

Trametes sp., I-62 (CECT 20197) was used in order to degrade

the soluble organic matter and color present in vinasse fromfermentation of sugar-cane molasses (COD0 = 55.5 g/L) (Gonzálezet al., 2000). A dilution of 20% (v/v) with the culture mediumwas tested. After 7 days of fungal treatment, maximum effluent

536 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546

Table 6Selected fungal treatment of vinasses.

Process and experimental design Results Remarks Ref.

Vinasses from: Brandy distillation (BD) anddistillation spirits (DS); laboratory scale (LS);Microorganism used: Trametes pubescens MB89; T = 28 ◦C; pH 4.5; phenols concentrationBD = 35–280 mg/L; DS = 290–320 mg/L

The fungal culture showed higher percentagesof �phen = 87%, �color = 88% and �COD = 83% thanthose of the enzyme tests �Phe = 61and�color = 12

The fungal and the enzymatic (laccase)treatment were tested separatelyIncreased color in the tests performed with theenzyme (�color = 160%)

1

VFSMMicroorganism used: Trametes sp. I-62 (CECT20197); LS

�color = 73% and �COD = 61.7% after 7 days ofculture

Use of 20% diluted vinasses 2

VBM previously subjected to conventionalanaerobic–aerobic treatment; Trametesversicolor; LS; variation of the carbon source,nutrients and initial pH

Removals in the effluent:�color = 82%, �COD = 77% y �N–NH4

+ = 36%Best results were obtained supplementingsucrose as co-substrate and KH2PO4

Color adsorbed onto mycelium = 5–10% On theother hand, there was no influence on CODremoval

3

VFSM Flavodon flavus; Immobilization of thefungus in polyurethane foam; Ecotoxicity testsusing Oreochromis mossamb cus; Repeated BM,LS

Vinasse without dilution was obtained:�color = 10%Vinasse diluited at 50% �coluor = 60 y 73%, in 5 y7 days respectively68% decrease in the concentration of polycyclicaromatic compounds (benzo (a) pyrene) after 5days of treatment

The immobilization of the fungus is effectivebleaching using for up to three batch cycles.No damage was found by the vinasse treated inthe liver of fish compared to untreated vinasseDetected benzo(a)pyrene in the vinasse andthis appears to be one of the causes of toxicity

4

Anaerobically digested VMSMS Aspergillusheteromorphus; production of laccase; LS

The use of anaerobically digested SMDW andligninocellulosic biomass increased laccaseproduction (6.6 UI mL−1)

5

VFSM Phanerochaete chrysosporiumCulture time 32 daysT = 25 y 39 ◦C;LS

�COD = 47.48%; �Phe = 54.72%; �color = 45.10% at25 ◦C

�COD = 4.21%; �Phe = 59.41% �color = 56.81% at39 ◦C

Two operating conditions 25 and 39 ◦C bestresults at 39 ◦C

6

VFSM; Pretreatment at 30 ◦C and 10 days ofculture with Geotrichum candidumPostreatment using Coriolus versicolor,Phanerochaete chrysosporium and sterilemycelia; LS

A 10 days pretreatment with Geotrichumcandidum resulted in �COD = 53.17% and�phe = 47.82%Coriolus versicolor immobilized in apacked-bed reactor reduced �COD = 50.3%giving a global �COD = 77%

It is technically feasible to bioremediate spentwash using a multi-stage treatment process(Pretreatment step with Geotrichum candidum)

7

Anaerobically digested VFSM frombiomethanation plants; fungi Coriolusversicolor andPhanerochaete chrysosporium; LSInfluent diluted 25%, 12.5% and 25% (v/v) withwater

Optimum growth and discoloration occurredat 35–40 ◦C, pH 5.0, glucose 3–5% (w/v) andVFSM diluted with water 6.25% (v/v)C. versicolor: �color = 71.5%, �COD = 90.0%P. chrysosporium: �color = 53.5%, �COD = 73.0%

Using additional labile carbon sourceDecolorization and COD removal decreasedsignificantly at the higher concentrations ofanaerobically digested VFSM

9

Notes: VBM, vinasse from fermented beet molasses; VFSM, vinasse from fermented sugar molasses; �color, removal efficiency of color; �phen, removal efficiency of phenoliccompounds; �N–NH4

+, removal efficiency of nitrogen; �COD, removal efficiency of COD; LS Laboratory scale; BM, batch mode or batch operation.References: 1. Strong and Burgess, 2008; 2. González et al., 2000; 3. Benito et al. (1997); 4. Raghukumar et al. (2004); 5. Singh et al. (2010); 6. Potentini and Rodriguez-Malaver(

d6ibioc

(credoffibdtg5r7

2006); 7. Fitzgibbon et al. (1995); 8. Fahy et al. (1997); 9. Kumar et al. (1998).

iscoloration values and COD reduction were obtained, 73.3% and1.7%, respectively, at 7 d of incubation. A significant increase

n laccase production was observed, but no MnP activity coulde detected. After the period of treatment a significant decrease

n a number of pyrolysis product (mainly furan derivatives) wasbserved that could be related to the vinasses color-removal asso-iated with melanoidin degradation.

García et al. (1997) working with vinasses from molassesCOD0 = 75 gO2/L) obtained 66% and 70% phenolic removal effi-iencies employing Aspergillus terreus and Geotrichum candidum,espectively, after five days fermentation. Jiménez et al. (2003)mployed four different types of fungus (Penicillium sp., Penicillumecumbens, Penicillium lignorum y Aspergillus niger) for the discol-ration, phenolic removal and COD of vinasses from distillation ofermented molasses. A decrease in the effluent color starting therst incubation day for all the fungi tested has been observed;est results were obtained with Penicillum decumbens with 40%iscoloration. Decolorization was ascribed to the degradation oro tannins and some phenolic compounds adsorption onto fun-

al mycelium. The maximum COD removal efficiency achieved was2.1% with Penicillium sp and 50.5% with Penicillum decumbens. Theemoval efficiency of phenolic compounds was similar in all cases,0%.

Untreated vinasses (COD0 = 80.5 g/L) and vinasses previouslytreated with Penicillium decumbens were (COD0 = 23.0 g/L) post-treated by anaerobic digestion (Jiménez et al., 2006). Thepre-treatment of vinasses with P. decumbens reduced 67.7% of theinitial content of phenolic compounds present in this substrate,decreasing considerably its biotoxicity and enhancing the maxi-mum specific growth rate and kinetic constant for the anaerobicdigestion (AD) process for pre-treated vinasses by 9.6 and 6.9 timesrespectively, than those obtained in the AD of untreated vinasses.In another work, Jiménez et al. (2005) studied the growth, sub-strate (as COD) and phenolic (as gallic acid) compounds removalpresent in vinasses using Penicillitum decumbens. Batch tests wereperformed where the initial concentration of COD and gallic acidwere 42.6 g/L and 0.21 g/L, respectively. P. decumbens without theneed for any nutrient supplements in the medium was capableof significantly degrading the phenolic compounds in vinasses.After 3 days 74% of removal phenolic compounds was obtainedwhereas the best result in color removal (41%) was obtained afterthe day 4 of treatment. Biomass yield coefficient was found to be

0.35 g VSS/gCOD. The authors also developed a kinetic model thataccurately described the variation of substrate and biomass con-centrations with time in the aerobic degradation process of vinasseswith P. decumbens.

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Strong (2010) conducted lab scale treatment tests on vinassesrom alcoholic beverage derived from the distillation of fermented

arula fruit using several white rot fungus (Trametes pubescensB 89, Ceriporiopsis subvermispora, Pycnoporus cinnabarinus and

hanerochaete chrysosporium). The marula vinasses exhibited ahemical oxygen demand (COD) of 27 g/L, a pH of 3.8, a high concen-ration of phenolic compounds (866 mg/L) and a high suspendedolids content (10.5 g/L), all of which could adversely affect biolog-cal treatment. Full-strength wastewater was treated in shake-flaskure cultures of the four white rot fungi. Trametes pubescens per-ormed the best with regards to degrading phenolic compounds,OD and color. Laccase production was only detected in the T.ubescens and C. subvermispora cultures. In a second phase, sixastewater concentrations (100%, 80%, 60%, 40%, 20% and 10%) atH 4.5 were evaluated with batch cultures of T. pubescens. CODnd phenolic removal efficiencies did not vary with concentrationCOD: 71–77%; phenolic compounds: 87–92%). A maximum lac-ase activity (1063 units/L) was obtained in the 80% wastewateroncentration.

Robledo-Narvaez et al. (2006) reported that after anaerobic pre-reatment, a post-treatment with fungal pellets of a defined mixedulture of Lentinus edodes and Trametes versicolor immobilized onak sawdust and activated carbon increased the overall removalf the organic matter present in effluents from the pulp and paperndustry. The removal efficiencies of color, lignin and COD were4%, 28% and 40%, respectively. A post-treatment of similar efflu-nts using a pure culture of Trametes versicolor immobilized inood saw-dust (hybrid pellets), produced higher removal efficien-

ies of color (54%) and lignin-like compounds (69%); but a lesseremoval of COD (32%) (Ortega-Clemente et al., 2007). Usually, inhe anaerobic phase, the removal of biodegradable organic mat-er was about 90% or more, while in the fungal stage, about 60%f the recalcitrant matter not eliminated in the first stage wasemoved.

Since some compounds present in vinasse can stimulate lac-ase production in ligninolytic fungi (Strong and Burgess, 2007), atrategy for vinasse treatment is the use of fungi for laccase produc-ion. For example, Singh et al. (2010) used anaerobically pretreatedinasse and lignocellulosic biomass (rice straw, wheat straw andugarcane bagasse) to produce laccase from Aspergillus heteromor-hous. Pretreated vinasse was a good inducer of the enzyme, and theddition of lignocellulosic material increased laccase production.

Using vinnase from wine distillation, Strong and Burgess (2007)eported the laccase production by Trametes pubescens MB 89 grow-ng in shake-flasks and a bubble lift reactor. The procedure usedmproved the vinasse quality removing 79%, 80% and 71% of COD,otal phenols and color, respectively. In another work, Strong andurgess (2008) evaluated wine-related vinasses as a substrate toroduce laccase enzyme using Trametes pubescens. The enzymaticole in phenolic compounds degradation and color change werevaluated using crudely purified laccase. The best result obtainedn the fungal treatment gave removals of of 83%, of 87% and of 88%or COD, phenolic compounds, and color, respectively. Laccase syn-hesis was greater than 1500 U/L in all treated wastewaters, with a

aximum of 8997 U/L.Aguiar et al. (2010) cultivated three strains of Pleurotus and Tri-

hoderma reesei on pre-treated bagasse and vinasses as sources ofarbon. Under 2% H2O2, 1.5% NaOH and autoclave treatment thereater fibre breakage was obtained in these condition increasinghe cellulose level up to 1.2 times and decreasing 8.5 times theemicellulose content and high ligninolytic activity for all cultures.. reesei produced laccase, peroxidase and MnP; the MnP activity

as 1.9–4.8 times higher than that of Pleurotus.

A culture media that consisted of either vinasses or a syntheticulture media, with supplementation and without of banana waste,as used as substrate in cultures of the white rot fungi P. ostreatus

technology 157 (2012) 524–546 537

APK-1 for laccase production (Shanmugam et al., 2009). Vinassesculture medium was a better laccase-inducer medium than the syn-thetic culture medium; the addition of banana waste in the mediumenhanced the enzyme production. Laccase production in batch testsby two white rot fungi (Coriolus versicolor, Funalia trogii) under dif-ferent nutritional conditions were reported (Kahraman and Gurdal,2002). Various synthetic culture media and natural culture medium(vinasse and molasses wastewater) were tested as well as cottonstalk supplements. Tests with cotton stalk showed that vinasseculture medium was a better laccase-inducer medium than thesynthetic culture medium. In another experiment, Kahraman andYesilada (2001) reported that white-rot fungi Coriolus versicolorand Funalia trogii produced laccase in media with diluted olive-oil mill wastewater and vinasse. Addition of spent cotton stalksenhanced the laccase activity with a maximum after 12 d of culti-vation.

Studies made by Acharya et al. (2010) indicate that anaerobicallypretreated vinasse can be used as a viable nutrient source for cellu-lase production by the non ligninolytic fungus Aspergillus ellipticusin solid-state fermentation.

Finally, it is important to remark that most current studies withfungi have been carried out at a laboratory level. It is also worthnoting that pure enzyme production is a process that raises thetreatment costs. Thus, it is necessary to find new and economicmethods to produce large amounts of ligninolytic or other enzymes,as well as performing large scale studies as another way to decreaseproduction costs.

3. Physico-chemical treatment

It has been observed that after vinasse bio treatment, an impor-tant fraction of non-degraded recalcitrant organic matter stillremains (Durán-de-Bazúa et al., 1991; Sangave et al., 2007a). There-fore, alternative or complementary treatment methods, such asphysical and chemical processes have been used (Manisankar et al.,2004; Vlyssides et al., 1997; Yavuz, 2007). A common thread inmost of these methods is their relatively high cost, elevated reagentdoses, and in some cases other pollutants are generated (Fanninet al., 1987; Lucas et al., 2009; Pena et al., 2003). Table 7 presentsa summary from literature on physical-chemical treatments usedfor vinasse treatment.

3.1. Ozone treatment

Because microorganisms used in conventional biological treat-ments could not have the enzymes required for the completebiodegradation of recalcitrant compounds, chemical oxidationprior biological treatment has been introduced to transform ordegrade these compounds into smaller molecules (Gogateand andPandit, 2004).

Chemical oxidation with ozone has desirable characteristicsin the pre and post-treatment of industrial wastewater contain-ing recalcitrant compounds. Some of its properties are: (i) it isa strong oxidant able to degrade recalcitrant organic compounds(phenols), resulting in byproducts more susceptible to biodegrada-tion (Heredia et al., 2000), (ii) it is a gas relatively soluble in water,readily available and highly reactive with compounds possessingdouble bonds, which frequently are associated with color (Rehmanet al., 2006; Sangave et al., 2007a; Sreethawong and Chavadej,2007), (iii) it can promote the formation of highly reactive hydroxylradicals, which can even have more oxidative power that the same

ozone (Sangave et al., 2007a).

Traditionally, ozone has been used for disinfection of waterand wastewater, as well as for oxidation of organic and inor-ganic compounds, including the removal of taste, smell and color

538 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546

Table 7Physico-chemical treatments used in depuration of vinasses.

Process and experimental design Results Remarks Ref.

Vinasse from distillation of grape alcohol(VDGA);Laboratory scale (LS); Treatment usingO3; parameters: pH 5.4, 7.0, 10; T = 10, 20 y30 ◦C @ Q = 30 L/h, [O3]i = 20 mg/L, f = 10

Optimum conditions pH 10, T = 30 ◦C, doseof O3 = 0.58 g O3/L mixed wastewater

Dilution of vinasse with municipal wastewater(1:10, v/v)

1

VDGA; LS; Batch mode (BM); oxidation usingFenton-H2O2, Reaction time effect on: �COD,�pphe, �Aro; condition T = 25 ◦C, pH 3.5,[FeSO4·7H2O]0 = 0.01–0.1 mol/L, addition of0.1–0.65 mol/L of H2O2

�arom = 90% and �phen = 90% at 5 min ofcontact

Dilution of vinasse with municipal wastewater(1:10, v/v), aerobically pretreated

2

�COD = 50–80%, maximum removal wasobserved at 30 min

VDGA; LS; BM; 1st stage Fenton-H2O2, theexperimental variables studied were:[FeSO4·7H2O] and [H2O2]

Optimum conditions �COD = 74% at[H2O2]0 = 0.5 mol/L,[H2O2]0/[Fe2+]0 = 15 mol/mol

Used Ca(OH)2 as the precipitating agent in thecoagulation/flocculation stage

3

2nd stage coagulation/flocculation.(CF)Vinasse from beet molasses (VBM); Pilot plantscale (PP); electrolysis treatment (anodePt/TiO2)

Optimum conditions �COD ≈ 89% at[NaCl] = 4%, T = 42 ◦C, pH 9.5, Q = 30 mL/min,under these conditions was achieved�COD ≈ 89%.

Wastewater was mixed with NaCL and thenfed to an electrolytic cell. Cl2 and otheroxidants were produced; they oxidized organicpollutants to CO2 and water

4

Vinasses from fermented sugar molasses(VFSM); LS; electrodialysis treatment, Constantvoltage of 17 V, concentrated solution(brine) = 5 g NaCl/L of electrolyte solution = 21 gKNO3/L

[K] was reduced from 10 to 2.5 g/L Aim: to reduce the concentration of potassiumto prevent crystallization (as K2SO4)

5

Pre-treated distillery wastewater (PTDW)(evaporator and centrifuges); condensed liquidphase is used as PTDW; LS; BM,

�COD = 93.5% for PTDW EC tested at current density of 20 mA/cm2 with0.2 M Na2SO4.

6

Two electrochemical methods wereinvestigated: electrocoagulation (EC) andelectro-Fenton (EF)

EC studies EF carried out at current density of 60 mA/cm2,(supporting electrolyte), [H2O2] = 60 g/L, pH 4

Poor results �COD = 14.3%EF studies EF process was found to be very effective�COD = 92.6% y �TOC = 88.7%

VFSM; LS; Catalytic thermal treatment, CuOcatalyst; 1 L reactor; BM; T = 100–140 ◦C; CuOmass loading in the range of 2–5 Kg/m3

Optimum conditions �COD = 60% at pH0 = 2;T = 140 ◦C y 3 Kg/m3 catalyst loading

Thermal catalysis used as pretreatmentprocess Initial pH0 had strong impact on �COD

The residue can be used as a fuel and the ashcan be blended with organic manure and usedin agriculture

7

Stillage wastewater; LS; EO process; Twomaterials tested in the anode: graphiteparticles and titanium sponge; cathode madefrom Ti/RuO2

Anode made from titanium sponge showedbest results acidic conditions (pH 1)resulted in the increased oxidation oforganic pollutants

Maximum current efficiency decreased withoperation time due to passivation of theelectrode surface

8

Additives H2O2 and NaCl promoted high�COD = 89.62% and �color = 92.24%

Anaerobically digested VFSM; LS;coagulation/flocculation (CF) andelectrochemical oxidation (EO) treatment

CF: Optimum conditions CF was sensitive to pH and coagulantconcentration

9

Applying two complementary process: CF usedFeCl3 as coagulant followed by EO usingTi/RuPb(40%)Ox anode and Ti/PtPd(10%)Ox

cathode

�COD = 84%; color and turbidityremoval ≈ 99% at pH 8.4; coagulantconcentrationof 20 g/L

EO with anode of Ti/RuPb(40%)Ox effectivelyremoved the organic material, color andturbidity

EO: �COD = 99%; color and turbidity removal100%

VFSM; PPS; Ultrafiltration (UF) and reverseosmosis (RO) treatment

RO process UF and RO processes successfully removedcolor and other contaminants

10

UF evaluated the pressure variation onthin-film composite polyamide

�COD = 96.8%; �TDS = 97.9%

UF effectively separated the SS at pressure10 atm�COD = 63.2%; �SS = 96.5%

Notes: VDGA, vinasse from distillation of grape alcohol; VBM, vinasse from fermented beet molasses; VFSM, vinasse from fermented sugar molasses; T, temperature, Q,wastewater flow, [O3]i, ozone concentration in the reactor inlet, f, domestic wastewater volume/volume of vinasse, �COD, removal efficiency of COD, �phen, removal efficiencyof polyphenols, �arom, removal efficiency of aromatic, �COT, removal efficiency of total organic carbon, �TDS, removal efficiency of total dissolved solids, �color, removal efficiencyof color, LS, laboratory scale, PP, pilot plant scale, BM, batch mode, SS, suspended solids.References: 1. Beltrán et al. (1999a); 2. Beltrán et al. (1999b); 3. Beltran-de-Heredia et al. (2005b); 4. Vlyssides et al. (1997); 5. Decloux et al. (2002); 6. Yavuz (2007); 7.C rthy

(whlf

haudhari et al. (2007); 8. Piya-areetham et al. (2006); 9. Zayas et al. (2007); 10. Mu

Gottschalk et al., 2000; Pena et al., 2003; Ried et al., 2007). Many

orks have shown that the additional use of ultraviolet radiation,ydrogen peroxide, iron oxide, or titanium dioxide and tin cata-

ysts increase the degradation efficiency due to the generation ofree hydroxyl radicals that produce organic radicals (R•) capable

and Chaudhari (2009).

of oxidizing organic compounds, in a kind of chain reaction that

leads to the final destruction of biodegradable or not biodegrad-able organic compounds (Martín et al., 2002; Sangave et al., 2007a;Sreethawong and Chavadej, 2007; Takahashi et al., 2007; Zeng et al.,2009).

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V. Robles-González et al. / Journa

.1.1. Ozone productionOzone is an unconventional chemical reagent, in the sense that it

ust be generated in situ during wastewater treatment. The ozoneroduction is based on the overall chemical reaction:

O2Electricity−→ 2O3 (1)

This process must operate with extremely dry air, reasonablyree of impurities, or with pure oxygen. In this case, the ozoneroduction yield increases significantly (EPA, 1986).

In ozone production, air or pure oxygen is introduced into thenterior of electrical shock tubes. Potential differences of about5–25 KV are applied, causing the breakdown of oxygen molecules

nto reactive atoms of oxygen, which combine with new O2olecules to form ozone (Sangave et al., 2007a). In order to reduce

he costs associated with the use of ozone it is necessary to optimizehe process efficiency. Thus, it is recommended stillage condition-ng before ozonation (Coca et al., 2005; Rehman et al., 2006; Riedt al., 2007).

.1.2. Factors affecting the ozone oxidation efficiencyThe oxidizing action of ozone is strongly dependent on pH. At

ow pH values, ozone reacts with specific functional groups throughirect selective reactions. At high pH values, hydroxyl ions catalyzehe decomposition of ozone to produce free radicals, which mayave a higher oxidation potential than ozone itself (Beltrán et al.,001; Coca et al., 2005; Rehman et al., 2006). However, the stillagelkalinity is mainly due to bicarbonate and carbonate ions (Durán-e-Bazúa et al., 1991; Jiménez et al., 2006). Bicarbonate is a wellnown scavenger of free radicals formed by the ozone breakdown.ncreased color and COD elimination have been observed after alka-inity removal (Coca et al., 2005). These results are consistent withhe fact that the bicarbonate is an inhibitor of the oxidizing actionf hydroxyl radicals.

Ozone reactivity is also a function of temperature; thus, theemoval efficiency of color and COD increases with the processemperature, up to a maximum of about 40 ◦C (Coca et al., 2005).owever, lower temperatures (about 25 ◦C) are preferred because

he operational costs of ozonation also increase with tempera-ure.

There are several studies on vinasse ozonation, mostly in theontext of combined ozonation + biological treatment. Selectedesults and references were already discussed in Section 2.2.1nd displayed in Table 7, whereas combined ozonation-anaerobicigestion works are summarized in Section 2.1.5. Flow diagrams ofombined vinasse treatment based on anaerobic digestion followedy ozonation post-treatment are outlined in Fig. 2. The left branch

n Fig. 2 is convenient when vinasse presents a low biodegradabil-ty and/or it contains significant amounts of toxic and recalcitrantompounds. A pre-ozonation step could effectively help in remov-ng the latter in such a way that the pre-treated vinasse is moremenable to the biological process, i.e., anaerobic digestion. On thether hand, the right branch of the flow diagram could be recom-ended when the vinasse exhibits a significant biodegradability.

he anaerobic digestion first step would remove most organic mat-er; the post-treatment with ligninolyitc fungi would be useful forhe removal of recalcitrant organic compounds that could not beegraded removed by bacterial consortia.

.1.3. Treatment of vinasses using ozone and ozone plus catalystsTreatment of vinasses by ozonation has been performed in

rder to oxidize recalcitrant compounds (like color pigments

elanoidins) or remove organic matter and make it more amenable

o biodegradation. Ozone has been widely used as a chemical pre-reatment step and a variety of catalysts have been employed to

ake more effective the oxidizing action of ozone.

technology 157 (2012) 524–546 539

Santos et al. (2003) used ozonation at acid and alkaline pHin order to reduce phenolic compounds present in vinasses fromethanol distillation processs (COD0 = 103 g/L and BOD0 = 41.2 g/L).The process in acid conditions improved the removed of pheno-lic compounds and more readily biodegradable organic matter.Alfafara et al. (2000) investigated the chemical degradation ofmelanoidins present in the distillery vinasses using ozone treat-ment. Ozone had positive effects on the discoloration (efficiencyof 80%) and biodegradability improvement (40%) in 40 h contacttime. Yet, removal of COD was low (16%). A slight depolymeriza-tion was observed from the decrease in molecular weight of organicmatter.

Sreethawong and Chavadej (2008) used ozonation in theabsence and presence of alumina balls as the support media tobe coated with the iron oxide catalyst in order to reduce the col-ored compounds present in distillery wastewater from sugar canemolasses (COD0 = 106.5 g/L and BOD0 = 31.6 g/L, vinasse diluted bydistilled water by 1:20). They observed that removal efficiencies ofCOD and color increased with increasing either the input power ofthe ozone generator or the ozone mass flow rate. In the presence ofthe Fe2O3 catalyst, ozone resulted more effective in reducing bothCOD and color. The authors ascribed this to possible the forma-tion and action of free hydroxyl radicals. Zeng et al. (2009) used O3plus SnO2 catalyst in order to reduce color present in fermentedmolasses (Color absorbance unit of 2.14 at 475 nm after 10 timesdilution, COD0 = 95.0 g/L and BOD0 = 22.0 g/L). Decolorization of thiswastewater was 60.24% when ozonated for 60 min in the presenceof SnO2 catalyst. In contrast, discoloration was just 43.04% with justozonation.

Lucas et al. (2010) reported the treatment of winery wastewa-ter (COD0 = 4.65 g/L) in a pilot-scale, buble column photo oxidizingreactor. At the natural pH 4.0 of the influent, the photolytic actionof UV-C radiation, O3/UV, O3/UV/H2O2 on the COD removal waslow-to-moderate (12%, 21% and 35% respectively after 180 min).In the treatment O3/UV/H2O2 at pH 10 they found a significantimprovement of COD removal efficiency (57%).

3.2. Fenton’s oxidation

3.2.1. Principles of the methodOxidation with Fenton’s reagent (H2O2 plus Fe2+ in acidic aque-

ous solution) is a method widely used for destruction of organiccompounds. It is based on the generation of free hydroxyl radicals(•OH), which have a high oxidation potential (•OH/H2O = +2.73 V).

The mechanism of oxidation of organic compounds by Fenton’sreagent is very complex, but is thought to occur through the fol-lowing stages (Beltran-de-Heredia et al., 2005b):

H2O2 + Fe2+ K1−→Fe3+ + OH− + •OH (2)

P + H2O2K2−→Pox (3)

P + •OHK3−→Pox (4)

H2O2 + OH• K4−→HO•2 + H2O (5)

OH• + Fe2+ K5−→Fe3+ + OH− (6)

Fe3+ + H2O2K6−→Fe2+ + H+ + HO•

2 (7)

Fe3+ + HO•2

K7−→O2 + Fe2+ + H+ (8)

On the other hand, under alkaline conditions Fe3+ forms Fe(OH)3 which is a highly insoluble compound in equilibrium withFeO (OH). Ferric hydroxide forms a precipitate, which can facili-tate the separation of effluent’s suspended matter. In consequence,

540 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546

a

References: 1.Martín et al., 2002; 2.Siles et al., 2011; 3. Kumar et al., 1998; 4. Durán-de-Bazúa et al., 1991...”Notes: AB: Anaerobic biodegradability; BLSR: Batch lab-scale reactor.

b

3 Absorbance at 254 nm was used for assessing color removal Reference: Sangave et al., 2007a

Raw vinasses

Pre-treatment using ozone oxidation ηCOD =24.8% , Ozonation time 2 h (1)ηphenols 50% Ozonation time 15 min(2)

Anaerobic treatment (BLSR) (1) AB of raw and pre-treated vinasse was similar

ηCOD≈ 80%

Enhanced the methane yield coefficient by 13.6% and methane production rate in 41.6% (2)

Anaerobic treatment (3) ηCOD= 70 % (4)

Post-treatment using White-rot fungi (vinasses diluted from anaerobic treatment

12.5% v/v) (3) ηColor =73-90%

ηCOD=53.5-71.5%

Raw vinasses

Ozone pretreatment

ηCOD= 27%

Aerobic treatment ηCOD=53%

After 12 h of aerobic

oxidation

Ozone post-treatment

ηCOD= 79% ηcolour= 92%3

F erobit

ttIc

3

((T(em(rHaow

Fwm[

ig. 2. Flow diagrams of successful combined treatments of vinasses: (a) using anareatment combined with ozonation.

his treatment can also reduce the phenolic compounds by adsorp-ion on the ferric gel formed (Beltran-de-Heredia et al., 2005b).t seems that the ultimate mechanism of removal of phenols hasontributions from direct oxidation and coagulation.

.2.2. Treatment of vinasses using Fenton processVinasses from fermentation of sugar cane molasses

COD0 = 12–39 g/L) was treated using Fenton and photo-Fenton80 min of UV radiation was applied) (Hadavifar et al., 2010).he photo-Fenton exhibited the highest COD removal efficienciesrange 18–97%) compared to Fenton alone (range 5–47%). Yangt al. (2008) used Fenton reaction in order to remove organicatter and color from pre-treated (aerobic process) wine vinasses

COD and BOD after aerobic pre-treatment, 0.636 and 0.090 g/L,espectively). Concentrations of 450 and 300 mg/L of FeSO4 and2O2, respectively, significantly reduced the values of COD, BODnd color at 30 min of reaction time. The hydroxyl radicals couldxidize most refractory organics present in the vinasses; some loweight molecular organics remained after treatment.

Beltran-de-Heredia et al. (2005b) investigated the integrated

enton coagulation/flocculation treatment for the depuration ofine distillery wastewater (COD0 = 15–16.5 g/L). They reported aaximum COD removal efficiency 74% at 0.5 mol/L H2O2 and a ratio

H2O2]:[Fe2+] = 15 mol/mol, 3 h of contact time.

c digestion combined with either ozonation or ligninolytic fungi; (b) using aerobic

3.2.3. Treatment of vinasses using electrochemical processesElectrochemical treatment of pre-treated vinasses from alco-

hol distillery wastewater (COD0 = 4.985 g/L, TOC0 = 1.507 g/L) usingiron electrode with and without the presence of H2O2 was investi-gated (Yavuz, 2007). Nearly 93% of the COD was removed whereasTOC removal efficiency was 88.7% in the electro-Fenton treatmentwith the addition of 0.3 M NaSO4 and 60,000 mg/L H2O2 at pH 4. Aspecific energy consumption of 0.53 kWh/g CODremoved was regis-tered at a current density of 60 mA/cm2. Control experiments withelectrocoagulation alone were found ineffective.

Rincon et al. (2009) treated vinasses from ethanol distilleries(COD0 = 21.4 g/L) using an electro-flotation/oxidation procces. Bestresults achieved were a COD reductions ca. 58% using galva-nized steel electrodes, high pH, current density 20 mA/cm2 and60,000 mg/L H2O2.

4. Perspectives and conclusion

Mezcal vinasses are of concern due to their potential negativeenvironmental impact if discharged in an uncontrolled manner. Thevinasse composition is related to the treatment received by thefermented wort before its distillation. Usually, it is directly led to

distillation; but occasionally, after filtrating, centrifuging or decant-ing the fermented broths, the yeast biomass is recovered to be usedas animal feed (Fig. 3). Then, the supernatant liquid is distilled.Obviously, depending upon the feedstock and the process used for

V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546 541

Agave Conditioning processes

Milling

Bagasse (fiber)

Composts from co-composting with

vinasse (1,2,3,4)

Fermentation

Distillation

Vinasse

CH4 Production(20 to 28)

Bio-H2(33 to 37)

Microbial fuel cells

Filtration

Liquid

Biomass Alternative source of protein

Juice extraction Dilution until 28-30

°Brix

Mezcal

Alternative single cell protein source; Candida(5); Spirulina maxima(6);

single cell protein(7); Hansenula(8); Rhizopus

microspores(9); Aspergillus awamori var. kawachi (10)

Polyphenolic antioxidants (18)sugarcane wax (19)

Laccase from Aspergillus

heteromorphus (11)Laccase from Coriolus versicolor and Funalia

trogii (12) Laccase from Trametes pubescens MB 89 (13);

Plant growth hormonesfrom Funalia trogii ATCC 200800 and T. versicolor

ATCC 200801(14); Lignin peroxidase, Mn-

peroxidase Laccase (15); Ligninases (16);

Cellulases (17)

Bio-energies

Anaerobic digestion

Bio-Products

Composts from co-composting with

agro-industrial waste (1 to 4)

Electricity (29 to 32)

Enzymes(38 to 40)

Fig. 3. Possible biorefinery approach for mezcal vinasses.References: 1. Díaz et al. (2002a); 2. Díaz et al. (2002b); 3. Madejón et al. (2001a,b); 4. Díaz et al. (2003c); 5. Tauk (1982); 6. Barrocal et al. (2010); 7. Durán-de-Bazúa et al.(1991); 8. Shojaosadati et al. (1999); 9. Nitayavardhana and Khanal (2010); 10. Morimura et al. (1994); 11. Singh et al. (2010); 12. Kahraman and Yesilada (2001); 13. Strongand Burgess (2007); 14. Yürekli et al. (1999); 15. Pant and Adholeya (2007b); 16. Ferreira et al. (2010); 17. Acharya et al. (2010); 18. Díaz et al. (2011); 19. Nuissier et al. (2008);20. Poggi-Varaldo et al. (2005); 21. Moletta (2005); 22. Harada et al. (1996); 23. Shivayogimath and Ramanujam (1999); 24. Jiménez et al. (2003); 25. Pérez et al. (1997); 26.H hanak3 6); 35( (2003)

datnbc

tsottd

amoda and Kennedy (1986); 27. Borja et al. (1993b); 28. Lo and Liao (1986); 29. Mo2. Zhang et al. (2009); 33. Munoz-Páez et al. (2011); 34. Valdez-Vazquez et al. (2002011); 38. Aguiar et al. (2010); 39. Singh et al. (2010); 40. Kahraman and Yesilada

istillate production, the composition of the vinasses obtained willlso vary, and consequently, their treatment’s results. Because ofhe variation in concentration and diversity of vinasses’ compo-ents, the results obtained after the treatment of these wastes coulde highly variable. The use of combined waste treatment methodsould deal with these variations.

For this reason, in the case of vinasses, we strongly recommendhe use of combined biotic and abiotic treatments. A plausiblecheme, although not a panacea, could consist of preliminary

zonation followed by anaerobic digestion; a polishing stage forhe so treated vinasses with ligninolytic fungi or a final ozona-ion treatment could be recommended depending on the particularischarge regulations as it was shown in Fig. 2.

rishna et al. (2010); 30. Poggi-Varaldo et al. (2009); 31. Vazquez-Larios et al. (2010);. Espinoza-Escalante et al. (2009); 36. Lay et al. (2010); 37. Escamilla-Alvarado et al..

Available information on mezcal vinasses treatment is scarce.Fortunately, there is an abundant body of research on treatment ofother recalcitrant toxic effluents that bear some similarity to mez-cal vinasses, such as wine vinasse, vinasses from the sugar industry,olive oil, and pulp and paper wastewaters. Experience with treat-ment of this set of residuals, indicates the following trends: (i)anaerobic digestion, complemented by oxidative chemical treat-ments (e.g. ozonation) are usually placed as pretreatments, (ii)aerobic treatment alone and combined with ozone which have

been directed to remove phenolic compounds and color have beensuccessfully applied, (iii) physico-chemical treatments such as Fen-ton, electro-oxidation, oxidants and so on., which are now mostlyat lab scale stage, have demonstrated a significant removal of

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42 V. Robles-González et al. / Journa

ecalcitrant organic compounds, (iv) fungal pretreatment withhemical treatment followed by oxidative (O3) or anaerobic diges-ion, this combination seems to give attractive results, (v) vinasses

ay be co-composted with solid organic wastes, particularly withhose from agricultural activities and agro-industry.

Vinasses treatments that could generate added-value by-roducts and abate the costs of pollution control, such as bioenergynd enzymes, are in distinct stages of development. Anaerobicigestion of vinasses is attractive and already a commercial tech-ology, with reported costs of 5000–25,000D\m3 of the digesterr 0.52D\m3 of treated vinasse (Moletta, 2005). Lucas et al. (2010)eported a cost of 1.31D\m3 using an AOP O3/UV/H2O2 for a wineryastewater (TOC0 of 1.254 g/L) in a pilot scale bubble column for a

ontact time of 150 min. The wastewater was extremely diluted, so,he costs for treating more concentrated vinasses would probablye one order of magnitude to 30 fold superior to the reported cost.

On the other hand, unicellular protein, biohydrogen and enzymeroduction from vinasses could be promising ways to improve theconomic feasibility of vinasses treatment, although they are inhe earlier stages of research and yields and costs issues shoulde addressed before future commercial application. As preliminary

nformation that could be important for feasibility studies, currentrices of pure hydrogen for fuel cells, enzymes cellulase and lac-ase, and biomass/protein are US$ 440/kg, US$ 12/kg (industrialrade) and US$ 206–2000/g (purified enzyme), and US$ 1.5–3.0iomass/kg, respectively (Infra de México; Enmex; Sigma–Aldrich;ena Bioscience; Tianjin Shareglory Chemical Industrial Co., Ltd.;hanghai Genon Biotech Co., Ltd.).

The co-composting of vinasse with agricultural and agro-ndustrial solid wastes, although attractive and feasible, is notommonly practiced. Yet, it is worth investigating and implement-ng because it can provide a low-cost treatment technology suitableor rural regions in underdeveloped countries where more sophis-icated technologies are difficult to adopt, due to high costs andequirements of skilled personnel. Furthermore, the final productf co-composting, i.e., soil amenders, would be very valuable formproving the quality of soils, increasing crop yields, and in gen-ral to achieve sustainability of agricultural practices in the aboveentioned rural regions that very often are semi-arid.Integration of several processes revised in this work as well as

thers in a biorefinery approach could lead to maximization of theesources-from-vinasse. A possible biorefinery setup for reclaimingezcal vinasses and obtaining a variety of added-value products is

hown in Fig. 3. This approach is based on the Principle of Cascadingnd production of bioenergy as gas biofuels. Other arrangementsre possible by integrating generation of liquid biofuels (ethanol,utanol, biodiesel). After the stages of agave conditioning and pre-rocessing, agave bagasse is generated as a by-product from theeparation from the juice. Bagasse could be used as a substratef solid fermentation processes with strains such as Trichodermaeseei and ligninolytic fungi in order to produce enzymes of comer-ial interest (cellulases, laccase, ligninoperoxidase, etc.). Also, soilmenders could be obtained by co-composting of agave bagasseith mezcal vinasse. So, the streams of enzymes and soil amender

rom this branch could join the enzymes and soil amenders pro-uced from the vinasses branch.

Raw vinasses can be filtered to give two main streams: vinassesnd solids rich en yeast and microbial biomass. The latter cane processed to generate an alternative protein source for ani-al feed and other uses. Filtered vinasses could be subjected tovariety of biotechnological processes in order to give diverse

dded-value bioproducts (Fig. 3): special bioproducts (Díaz et al.,

011; Nuissier et al., 2008), enzymes (Acharya et al., 2010; Ferreirat al., 2010; Kahraman and Yesilada, 2001; Pant and Adholeya,007b; Singh et al., 2010; Strong and Burgess, 2007), protein (yeast,lgal, bacterial, fungal) (Barrocal et al., 2010; Durán-de-Bazúa et al.,

technology 157 (2012) 524–546

1991; Morimura et al., 1994; Nitayavardhana and Khanal, 2010;Tauk, 1982) and soil amenders by co-composting vinasses withagro-industrial and other wastes (Díaz et al., 2002a,b; Madejónet al., 2001a,b). Effluents from these fermentation processes, ifnot exhausted, could be fed to the Bioenergy branch of the Biore-finery (not shown in Fig. 3). In parallel, filtered vinasses couldbe used for methane and biohydrogen production either as sep-arate stages or as series biohydrogen-methane process (Borja et al.,1993b; Durán-de-Bazúa et al., 1991; Escamilla-Alvarado et al.,2011; Espinoza-Escalante et al., 2009; Harada et al., 1996; Hamodaand Kennedy, 1986; Jiménez et al., 2003; Lay et al., 2010; Lo andLiao, 1986; Moletta, 2005; Munoz-Páez et al., 2011; Pérez et al.,1997; Shivayogimath and Ramanujam, 1999; Valdez-Vazquez et al.,2006), as well as bioelectricity generation via microbial fuel cells(Mohanakrishna et al., 2010; Poggi-Varaldo et al., 2009; Vazquez-Larios et al., 2010; Zhang et al., 2009).

Treated vinasses from bioenergy stages can be directed to co-composting of agroindustrial and other wastes or post-treated withAOP or biological processes before discharge or reuse in irrigation(not shown in Fig. 3) in such a way to close the environmentalcircle. Crucial areas for further development and ensuring prof-itability of the biorefinery approach are, among others: hydrogenyield improvement and hydrogen purification and storage, scale-upof enzyme production from wastes, scale-up of processes yieldingspecial bioproducts, and boosting performance of microbial fuelcells by one order of magnitude as well as their scale up for har-vesting meaningful amounts of biopower.

Acknowledgements

CONACYT (Mexican Council of Science and Technology) granteda graduate fellowship to one of the authors (VR-G). JG-M and NR-Sacknowledge COFAA-IPN for support.

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