Journal of Biotechnology 157 (2012) 524–546

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Treatment of mezcal vinasses: A review

Vania Robles-González a , Juvencio Galíndez-Mayer a , Noemí Rinderknecht-Seijas b ,
Héctor M. Poggi-Varaldo c,∗
a
ENCB-IPN, México D.F., Mexico
b
ESIQIE-IPN, México D.F., Mexico
c
Environmental Biotechnology and Renewable Energy Rand D Group, Department of Biotechnology and Bioengineering, Centro de Investigación y de Estudios Avanzados del IPN,
Apdo. Postal 14-740, México D.F. 07000, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: Mexican distilleries produce near eight million liters of mezcal per year, and generate about 90 million
Received 14 February 2011 liters of mezcal vinasses (MV). This acidic liquid waste is very aggressive to the environment because
Received in revised form 5 August 2011 of its high content of toxic and recalcitrant organic matter. As a result, treatment is necessary before
Accepted 7 September 2011
discharge to water bodies. It is interesting, yet disturbing; verify that there is a significant gap on the
Available online 16 September 2011
treatment of MV. However, there is an abundant body of research on treatment of other recalcitrant toxic
effluents that bear some similarity to MV, for example, wine vinasse, vinasses from the sugar industry,
Keywords:
olive oil, and industrial pulp and paper wastewaters. The objective of this review is to critically organize
Aerobic
Anaerobic the treatment alternatives of MV, assess their relative advantages and disadvantages, and finally detect
Biohydrogen production the trends for future research and development.
Biological treatment Experience with treatment of this set of residuals, indicates the following trends: (i) anaerobic digestion,
Chemical treatment complemented by oxidative chemical treatments (e.g. ozonation) are usually placed as pretreatments,
Co-composting (ii) aerobic treatment alone and combined with ozone which have been directed to remove phenolic
Color removal compounds and color have been successfully applied, (iii) physico-chemical treatments such as Fenton,
Fungal treatment electro-oxidation, oxidants and so on., which are now mostly at lab scale stage, have demonstrated a
Mezcal vinasse
significant removal of recalcitrant organic compounds, (iv) fungal pretreatment with chemical treatment
Physical treatment: Recalcitrant effluents
followed by oxidative (O3 ) or anaerobic digestion, this combination seems to give attractive results,
Review
(v) vinasses can be co-composted with solid organic wastes, particularly with those from agricultural
activities and agro-industies; in addition to soil amenders with fertilizing value to improve soil quality
in typical arid lands where agave is cultivated, it seems to be a low cost technology very well suited for
rural regions in underdeveloped countries where more sophisticated technologies are difficult to adopt,
due to high costs and requirements of skilled personnel.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction The Mexican industrial standard NOM-070-SCFI-1994 (NOM-

1.1. Mezcal industry in Mexico
Mexican mezcal production in 2006 and 2007 was 8 and 6 mil-
lions liters, respectively, according to the Consejo Mexicano Regu-
lador de la Calidad del Mezcal A.C. (COMERCAM, Mexican Council
for Quality Regulation of Mezcal A.C.; http://www.comercam.org/).
There are seven states in Mexico that can produce Mezcal, a Denom-
ination of Origin distillate: Oaxaca, Durango, Guerrero, San Luis
Potosi, Zacatecas, Guanajuato, and Tamaulipas (Fig. 1). The State
of 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).
070-SCFI, 1994) (http://www.profeco.gob.mx/) defines mezcal as
the regional alcoholic beverage obtained by distillation and rectifi-
cation of broths that are directly prepared by fermentation of sugars
contained in juices extracted from the mature hearts (or heads or
“piñas”) of agaves. These mature “piñas” are previously hydrolyzed
(usually by cooking in ovens) and the extracts are subjected to
alcoholic fermentation with yeasts. In general, Mezcal process con-
sists of four stages, i.e., cooking of agave hearts, milling the cooked
hearts, juice or must fermentation, and distillation/rectification. In
the distillation/rectification stage, between 8 and 15 L of distillery
slops (also called mezcal vinasses, MV) are generated for each liter
of mezcal (Robles-González et al., 2010; Jiménez et al., 2005; Preeti
and Aniruddha, 2006). In 2007, the volume of MV represented
approximately 90 million liters.
MV contains a variety of organic substances such as acetic and
lactic acids, glycerol, phenols, polyphenols, melanoidins, as well

0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2011.09.006
 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546 525

to water bodies. It is interesting, yet disturbing, to verify that there

Nomenclature is a significant gap on the treatment of MV. However, there is an
abundant research on treatment of other recalcitrant toxic effluents
AC aromatic compounds that bear some similarity to MV, for example, wine vinasse, vinasses
AD anaerobic digestion from the sugar industry, olive oil, and industrial pulp and paper
AnSBR anaerobic sequencing batch reactor wastewaters. The objective of this review is to critically organize
AOP advanced oxidation process the treatment alternatives of MV, assess their relative advantages
APT anaerobically pre-treated and disadvantages, and finally detect the trends for future research
AFBR anaerobic fluidized bed reactor and development.
as specific surface area, area per unit volume of a pack- The scope of this review encompasses the following topics:
ing or support (i) Mezcal vinasses characteristics and environmental impact; (ii)
BM batch mode or batch operation Biological treatment of vinasses, covering experiences with anaer-
BOD0 initial BOD obic digestion which are the most used method in the treatment
Bv volumetric organic loading rate of vinasse and aerobic treatment that has studied mainly for
Bx specific organic loading rate, i.e., per unit of mass of removal of color (melanoids) and some toxic compounds (phe-
biomass nols and polyphenols) present in the vinasse; (iii) Co-composting
CF coagulation/flocculation with organic solid wastes, and fungal treatment, under the head-
COD0 initial COD ing of Biological Treatment; (iv) Physico-chemical treatment, with
CV cotton gin compost with vinasse focus on ozonation and Fenton oxidation, this type of treatment
CW cotton waste addresses the removal of organic matter recalcitrant to biological
EF electro Fenton degradation. Whenever available, data from MV treatment will be
EO electrochemical oxidation presented and discussed; otherwise, references from similar recal-
EGSB expanded granular sludge bed reactor citrant effluents will be included.
GI germination index
GM grape marc 1.2. Characteristics of mezcal vinasses
HRT hydraulic retention time
kO3 consumption ratio of ozone Mezcal vinasses mainly consist of distillery slops that is the
MnP manganese peroxidase main contributor to volume and the organic load, although there
MP melanoidin products are minor components that usually contribute to the effluent vol-
MV mezcal vinasse ume and load variability. Among those components, it can be found
OM organic matter the effluents from fermenter cleaning (low in volume although the
OT operation time organic load can be around 5,000 mg COD/L), condensates, cool-
RO reverse osmosis ing water, etc. (Robles-González, 2011; Duarte et al., 1997). Since
TOC0 soluble initial soluble total organic carbon cooling water may represent a high flowrate but esentially a very
TPT-V thermally pretreated vinasse low pollutant load, it is recommended its segregation from MV.
U units of enzyme activity Tables 1–3 display typical characteristics of MV and vinasses from
UASB upflow anaerobic sludge (granular) blanket reactor sugar cane molasses fermentation/distillation and those originated
UF ultrafiltration in the alcohol production from wine.
US ultrasound Vinasses generaly contain high concentrations of dissolved
V vinasse solids; up to 50% of this parameter can be reducing sugars (Sangave
VBM vinasse from fermented beet molasses et al., 2007a), non volatile compounds coming from the fermen-
VFSM vinasse from fermented sugar-cane molasses tation broth, phenolic and polyphenolic compounds (Sales et al.,
VOA volatile organic acids 1987; Capasso et al., 1992; Robles-González et al., 2010), rela-
VWD vinasses from wine distillation tively high concentrations of mineral salts that reflect on a high
WS wheat straw electrolytic conductivity (250–300 dS m−1 ), and ash. Vinasses are
Y yield acid with a pH that usually ranges from 3.5 to 5, dark colored
Yı̌ pseudoyield (brownish, ascribed to the presence of melanoids) (García et al.,
1997; Jiménez et al., 2003; Coca et al., 2005). The organic pollutant
Greek letters
load is very high with extremely elevated values of biochemical
arom removal efficiency of aromatic compounds
oxygen demand (35,000–50,000 mg O2 /L) and chemical oxygen
COD removal efficiency of organic matter on COD basis
demand (70,000–150,000 mg/L). Biodegradability indices in the
color removal efficiency of color
range 0.2–0.5 mg BOD/mg COD are very common (Robles-González
phen removal efficiency of phenolic compounds
et 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 soil
as inorganic species such as sulphates and phosphates salts. It may cause severe environmental impact.
has been pointed out that vinasses composition and character-
istics may vary, depending upon the feedstock and the process 1.3. Pollution and environmental impact of vinasses
used for distillate production (Duarte et al., 1997; Robles-González,
2011; Robles-González et al., 2010). In spite of that, MV shares Uncontrolled discharge of vinasses onto soils can negatively
the main following features: low pH in the range of 3–5, and impact soil quality. For instance, the high content of soluble salts
high contents of organic matter (35,000–50,000 mg O2 /L as BOD in MV can lead to soil salinity and sodicity (Tejada et al., 2009;
or 100,000–150,000 mg O2 /L as COD) which is usually toxic and Shojaosadati et al., 1999). This, in turn, can seriously deteriorate
recalcitrant. Vinasses are of environmental concern because its dis- soil structure, porosity, and fertility (Tejanda and González, 2005).
charge to water bodies or onto soils may have a negative impact on Low pH of vinasses can be associated to heavy metal remobiliza-
the ecosystem. As a result, treatment is necessary before discharge 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 –
Mezcal and Mezcal; 4. Michoacan – Tequila and Sikua; 5. Tamaulipas – Mezcal and Tequila; 6. Oaxaca – Mezcal; 7. Durango – Mezcal and Sotol; 8. Guererro – Mezcal; 9. San
Luis Potosi – Mezcal; 10. Zacatecas – Mezcal; 11. Sonora – Bacanora; 12. Chihuahua – Sotol; 13. Coahuila–Sotol (http://www.elmezcal.org/general/map-of-spirits-made-in-
mexico-wahaka-tequila-sotol-and-more).

clog pores in soils, thus leading to development of anaerobic condi-
tions that not only are noticeable for their bad aesthetic symptoms,
but can also contribute to lower the soil pH and remobilization of
heavy metals mentioned previously (García et al., 1997; Jiménez
et al., 2005) and phytotoxicity to crops due to accumulation of a
variety of substances generated in the fermentation of vinasses
such as acetic acid, lactic acid, glycerol, and ammonia nitrogen
(Yavuz, 2007). Also, phenolic and polyphenolic compounds present
in vinasses (Table 3) can inhibit seed germination and damage sev-
eral crops, as well as to negatively impact soil microbial activity
(Díaz et al., 2002a,b; Kannabiran and Pragasam, 1993; Mattiazzo
and de Glorie, 1987).
Since vinasses leave the factory at temperatures around
50–80 ◦ C, if not cooled before discharge to water bodies, they
can increase water temperature and to diminish dissolved oxy-
gen below its critical level for fish survival (Jiménez et al., 2005;
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 cause
eutrophication in water bodies, reservoirs, and channels (Vlyssides
et al., 1997.)
Furthermore, presence of putrescible organic compounds such
as indol, 3-methyl indol as well as other sulphur-containing sub-
stances are associated to serious aesthetic problems as well as
possible toxicity (Pant and Adholeya, 2007a.)
Currently, several countries have enacted more stringent stan-
dards for discharge of effluents from alcohol distilleries. For
example, in 2005, the Indian environmental authorities made the
decision to convert the distillery industry in a zero-discharge indus-
try in a few years (Pant and Adholeya, 2007a). This approach should

Table 1
Characteristics of mezcal vinasses from different manufacturing facilities in the State of Oaxaca, México.

Parameter IMF-1a IMF-2b TMFc

pH 3.7 3.6 3.8

Alkalinity (mg de CaCO3 /L) ND ND ND
Conductivity (mS/cm) 2.6 ± 0.02 3.9 ± 0.03 4.2 ± 0.05
Color (475 nm) 4.6 ± 0.3 6.0 ± 0.2 10.6 ± 0.5
COD (mg O2 /L) 56,230 ± 162 60,560 ± 1004 122,860 ± 2270
BOD5 (mg O2 /L) 26,500 ± 710 22,000 ± 2830 33,600 ± 2260
Phenol (mg gallic acid/L) 478 ± 24 521 ± 16 542 ± 48
Fructose (mg/L) 14.8 ± 2.3 25.4 ± 4.2 50.0 ± 6.4
Nitrogen Kjeldahl (mgNH3 -N/L) 660 ± 37 843 ± 97 5,650 ± 503
Total solid (mg/L) 26,830 ± 1120 43,450 ± 1490 94,7130 ± 4055
Total suspended solids (mg/L) 3130 ± 168 3905 ± 156 8400 ± 504
Volatile suspended solids (mg/L) 1130 ± 88 2500 ± 100 6850 ± 411
Fixed suspended solids (mg/L) 2000 ± 80 1405 ± 56 1550 ± 93
Phosphate (mg/L) 290 ± 5 850 ± 14 1705 ± 30
Sulphate (mg/L) 308 ± 14 947 ± 12 842 ± 14

Robles-González (2011).
a
Notes: IMF-1: Industrial mezcal factory 1.
b
IMF-2: Industrial mezcal factory 2.
c
TMF: Traditional or handcraft mezcal factory.
 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546 527

Table 2 rely in a sort of “kidney” scheme, including in-plant treatment of

Typical characteristics of vinasses obtained from different substrates.
effluents, reuse, and recycling similar to zero-discharge pulp mills
Parameters FSMa DGAb that are in operation in Europe (Asghar et al., 2008; Gavrilescu and
pH 3.8–4.7 1,2,3,7
3.7–4.15,6 Puiţe, 2007; Ritchlin and Johnston, 1998.)
Alkalinity (mg 5800–60001,2 4501 In Mexico there is still no standard that specifically regulates
CaCO3 /L) the discharge of mezcal vinasses or alcohol industry wastewater
Turbidity (NTU) 30,000 in general. So far, the applicable environmental regulation is the
Total solid (mg/L) 63,000–79,0001,2 21,410–100,0004,5
Mexican Official Standard NOM-001-ECOL-1997 (NOM-ECOL-001,
Total volatile solids 50,0004
(mg/L) 1997) (Secretaría de Medio Ambiente y Recursos Naturales, SEMAR-
Total suspended solids 3000–11,0002,3,7 39006 NAT), which sets the maximum limits of pollutants in wastewater
(mg/L) discharges waste in water bodies, soil, wetlands, etc., and possi-
Total volatile supended 2500–90002,3 –
bly additional requirements (particular discharge conditions) that
solids (mg/L)
BOD5 (mgO2 /L) 31,500–75,0001,3 11,150–42,2285,6 can be imposed by the environmental agency in Mexico SEMAR-
COD (mgO2 /L 59,000–80,5001,2,7 24,500–120,0004,5 NAT. Despite the lack of specificity, maximum permissible limits
Kjeldahl N (mg N/L) 1600–18001,2 30,0004 for BOD5 , total suspended solids, and other pollutants of the Mex-
Ammonia N (mg N/L 150–70001,7 ican regulation are much lower than the corresponding parameter
Total N (mg/L) 9753 16004
values in raw MV. Therefore, a significant extent of MV treatment
NO3 -N (mg/L) 9978 –
Sulphate (mg/L) 31001 46004 is required.
Phosphate (mg/L) 20–3333,8 614
Total organic volatile 85002 13305
acids (acetic acid 2. Biological treatment
basis) (mg/L)
Sodium (mg/L) – 0.0324 As previously discussed, treatment of vinasses prior to their
Calcium (mg/L) – 0.64
discharge or recycling is mandatory from regulatory and envi-
Potassium (mg/L) 30,0008 1.924
Iron (mg/L) 203,0008 0.0354 ronmental standpoints in Mexico (environmental regulations
Zinc (mg/L) 15,0008 0.00184 NOM-003-SEMARNAT-1997 (NOM-003-SEMARNAT, 1997) “Estab-
Total phenol 450 mg gallic acid/L 477 mg caffeic acid/L lishing the permissible maximum levels of contaminants for
469 Total phenol/L2,3 735 mg gallic acid/L6,9 treated wastewater is reused in the public services” and NOM-
Total O-diphenol 343
(mg/L)
001-ECOL-1996 “Establishing the maximum permissible levels of
TOC (mg/L) 26,000–32,0003,7 36,2756 contaminants in wastewater discharges into national waters”).
Fixed suspended solids – 10006 Among the most commonly treatment methods used we can find
(mg/L) biological and physico-chemical techniques (Maiorella et al., 1983).
Total dissolved solids 49,000–510007 –
Their main goal is to reduce vinasse’s pollution impact by removing
(mg/L)
Reducing sugars (mg/L) 4000–5000 7
– the degradable organic matter, removing or degrading or at least
transforming major toxic organic substances to compounds that
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. Sangave
may be more susceptible to biodegradation, and desirably convert-
et al., 2007a; 8. Madejón et al., 2001a,b; 9. Beltrán et al., 1999a. ing pollutants to resources such as bioenergy or higher added-value
a
FSM: vinasse from distillation of fermented sugar molasses. metabolites. In this way, a large menu of techniques have been
b
DGA: vinasse from distillation of grape alcohol; the number after A: means ‘Ref- explored and applied, such as lagoons, anaerobic bioreactors of
erence for vinasse type in column A’; the number after B: means ‘Reference for
the fluidized bed type, UASB (abbreviation of upflow anaerobic
vinasse type in column B’.
sludge blanket reactor), packed ones, etc. in the biological treat-
ment category, as well as evaporation, combustion, and controlled
discharge onto soils to reclaim vinasses fertilizer value (Durán-de-
Bazúa et al., 1991; Gemtos et al., 1999; Harada et al., 1996; Madejón
Table 3 et al., 2001a,b; Rao, 1972; Sheehan and Greenfield, 1980).
Concentration of phenolic compounds in vinasses.

Phenolic compounds Basis of Type of vinasses Ref.

2.1. Anaerobic digestion
concentration (mg/L) expression

4300 Total phenols From distillation of 1

Anaerobic digestion has been one of the most employed system
grape alcohol
244 Caffeic acid From distillation of 2
for vinasses treatment because of low operational costs, aeration
grape alcohol savings, low sludge production and the obtaining of by-products
450 Gallic acid From distillation of 3 as methane gas (Durán-de-Bazúa et al., 1991; Harada et al., 1996;
fermented sugar Jiménez et al., 2006; Lalov et al., 2001). In this regard, a variety
molasses
of bioreactor configurations has been tested. For example Moletta
477 Caffeic acid From ethyl alcohol 4
production line (2005) reported work with bioreactors with suspended biomass
735 Gallic acid From distillation of 5 growth or flocs (suspended biomass as used in anaerobic con-
grape alcohol tact digesters, sequential batch anaerobic reactors and anaerobic
843 Total phenols From distillation of 6
lagoons) and immobilized biomass (i.e., granular sludge anaerobic
grape alcohol
469 Total phenols From the fermentation 7
reactors); he also mentioned the use of a combination of the two
of sugar molasses approaches systems: granular sludge bed reactors with anaerobic
210 Gallic acid From distillation of 8 filters, known as hybrid digesters. A summary of selected works on
grape alcohol anaerobic treatment of vinasses is shown in Table 4.
480–540 Gallic acid Mezcal vinasses 9
Anaerobic digestion is successful in dealing with the degradable
References: 1. Beltran-de-Heredia et al. (2005a); 2. Benitez et al. (2003); 3. Jiménez fraction of organic matter in vinasses; however, there is always
et al. (2003); 4. Martín et al. (2002); 5. Beltrán et al. (1999a); 6. Beltrán et al. (1999b);
an important recalcitrant compounds fraction (i.e., brown poly-
7. García et al. (1997); 8. Jiménez et al. (2005); Robles-González (2011).
mers melanoidins and other compounds) that still remain after
528 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546

Table 4
Selected results on anaerobic treatment of vinasses.

Process and experimental design Results Remarks Ref.

Vinasses from Winery distillation (VWD)
Anaerobic fluidized bed (AFBR); Supports: (i)
corrugated plastic and (ii) open pore
sintered-glass media; Laboratory scale (LS)
VWD
Anaerobic packed bed reactor; inert media:
small floating support of polyethylene
(Bioflow 30:  = 0.93 g/cm3 ; as = 320 m2 /m3 );
LS
VWD
Anaerobic moving bed biofilm reactor (ABBR);
filled with cylinders of polyethylene
( = 0.84 g/cm3 ; LS
VFSM vinasse from fermented sugar-cane
molasses
AFBR; carrier support was zeolite particles of
0.25–0.50 and 0.50–0.80 mm diameter; LS
VWD
AFBR filled with porous support media; LS
VFSM
AFBR; carrier support zeolite and activated
carbon; LS
VFSM anaerobic column reactor (ACR);
Support material studied: coconut coir; LS
Mezcal vinasses
AFBR, LS, 35 ◦ C
Bv = 1.96, 2.73, 5.70, 10.70 y 30.40 kg COD/m3 d,
(i) COD = 76% at organic load rate
(Bv ) = 6.29 kg COD/m3 d
(ii) COD = 96% at Bv = 5.88 kg
COD/m3 d
COD = 80% at Bv = 30 kg COD/m3 d
COD = 81–89% at Bv = 29.6 kg
COD/m3 d
COD = 90% at Bv = 20 kg COD/m3 d
COD = 96.5% at Bv = 5.88 kg
COD/m3 d and HRT = 2.55 days
COD = 81.5% at Bv = 32 kg COD/m3 d
COD = 70% at Bv = 10 kg COD/m3 d
COD = 64% at Bv = 23.25 kg
COD/m3 d; HRT = 8 d
COD = 85% at Bv = 1.96 COD/m3 d
[CH4 ] = 83%, v/v
COD = 61% at Bv = 30.4 COD/m3 d
[CH4 ] = 49% v/v
(i)Adequate for wastewater treatments 1
containing organic compounds easily
assimilated or that do not require high rates of
COD removal
(ii)Promotes cell transport and ensures contact
between the microorganisms and the
substrate, suitable for effluent toxic and
recalcitrant compounds
Excellent support for use in a high rate 2
anaerobic fixed bed
Bioflow 30 showed high retention capacity of
biomass (4.6 g dry solids per support) at the
end of the experiment, biomass accounted for
57 g solids/L reactor
At the end of the experiment 83.4% of the total 3
biomass was attached to the support
66% of the bioreactor was filled with
cylindrical polyethylene support
Biomass concentration attached to zeolite in 4
reactor was 40–45 g volatile solids/L
Experiments were carried out 30 ± 2 ◦ C
Experiments were carried out at 55 ◦ C 5
Previously colonization of the support used in
AFBR in semicontinuous anaerobic fixed-bed
reactors treating VWD
Bioreactor acidogenic crash at Bv = 40.5 kg
COD/m3 d, corresponding HRT to 0.37 days,
dramatic decrease of COD removal and no
production of biogas
The activated carbon + natural zeolite showed 6
good qualities as AFBR support
Coconut coir as the support material appears 7
to be a cost effective and promising technology
for treatment distillery effluent, however
disadvantages are the operation at long
HRTand moderate efficiencies moderate
COD = 64%
Methanogenesis deteriorated at Bv > 10.7 kg 8
COD/m3 d as revealed by decreases in the
removal efficiency (60–70%) as well as biogas
production, along with a jump in the alfa factor
up to values 0.51–0.64.

Notes: AFBR, anaerobic fluidized bed reactor; VWD, vinasses from wine destilation; VFSM, vinasse from fermented sugar molasses; Bv , volumetric organic loading rate; HRT:
hydraulic retention time; LS: laboratory scale; COD , removal efficiency of COD.
References: 1. Pérez-García et al. (2005); 2. Thanikal et al. (2007); 3. Sheli and Moletta (2007); 4. Fernández et al. (2007); 5. Pérez et al. (1999); 6. Fernández et al. (2001); 7.
Acharya et al. (2008); 8. Robles-González (2011).

anaerobic treatment (Durán-de-Bazúa et al., 1991). In this regard,
other treatment techniques such as advanced oxidation processes
(AOP) (Lucas et al., 2010; Martín et al., 2002; Sreethawong and
Chavadej, 2008; Zeng et al., 2009) can be used in combination with
anaerobic digestion to enhance removal of biologically-recalcitrant
components.
2.1.1. Anaerobic fluidized-bed reactor
This process has been used in the treatment of high strength
wastewaters and can effectively operate at very high organic load-
ing rates and high feed rates. Biomass does not wash-out from the
bioreactor because of its immobilization on support particles; with
an adequate start-up it is relatively easy to the develop a bed of very
active bioparticles that is “fluidized” inside the bioreactor, that is,
suspended by drag forces of upcoming effluent.
Anaerobic fluidized bed bioreactors display all the typical ben-
efits of anaerobic digestion (Chen et al., 1988; Iza, 1991; Pérez
et al., 1997–1999). In addition to those, more specific advantages
of this bioreactor configuration can be quoted (Collivignarelli et al.,
1991; Durán-de-Bazúa et al., 1991; Holst et al., 1997; Meunier and
Williamson, 1981; Tay and Zhang, 2000): it can carry a high con-
centration of biomass attached to a dense carrier, which cannot
be easily washed out from the bioreactor and increases overall
pollutant depuration rate; a very large surface area for biomass
attachment and wastewater/biocatalyst contact is provided; typi-
cally high mass transfer rates are achieved for substrate from bulk
liquid to bioparticles and for products from bioparticles to the
bulk liquid phase enhanced by liquid recirculation and by either
in 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
 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546
strength wastewaters; it has the ability to control and optimize the
biofilm thickness; the biomass carrier can be chosen for a specific
application to enhance removal; recirculation of treated effluent
means that the reactor shows an excellent hydraulic pattern that
avoids plugging, shortcircuiting and dead zones; for the same rea-
son an excellent dilution of influent with the effluent is achieved,
which provides alkalinity (and consequently, some neutralization)
and reduces the concentrations of pollutants and toxic substances
(important for high organic wastewaters and/or toxic wastewaters
such as vinasses). Last, but not least, anaerobic fluidized bed biore-
actors exhibit a fast start-up (even when non anaerobic inocula are
used), good stand-by features, and an outstanding process stabil-
ity (Durán-de-Bazúa et al., 1991; Garibay-Orijel et al., 2005; Iza,
1991; Pérez et al., 1997; Poggi-Varaldo and Rinderknecht-Seijas,
1996; Switzenbaum, 1983). These are the reasons why the anaer-
obic reactors have been scaled up at pilot and industrial levels.
Robles-González (2011) treated mezcal vinasses in a lab scale,
mesophilic anaerobic fluidized bed reactor with organic loading
rates of 2.0, 2.7, 5.7, 10.7, and 30.4 kg COD/m3 .d. The carrier medium
was 6/20 mesh granular activated carbon. Removal efficiency of
organic matter was in the range 61.5–84.8% as COD. Methanogen-
esis started to deteriorate at the loading rate 30.4 kg COD/(m3 .d)
as revealed by decreases in the removal efficiency (60–70%) as
well as biogas production, along with a jump in the alfa factor up
to values 0.51–0.64. Methane content in biogas was in the range
48.9–82.9%; higher values corresponded to lower organic loading
rates of operation. In summary, these experiments confirmed the
technical feasibility of anaerobic treatment of mezcal vinasses in
fluidized bed bioreactors.
Pérez et al. (1999) performed studies in distillation wines
vinasses degradation with a termophilic fluidized bed reac-
tor (55 ◦ C) at lab scale, they used SIRAN carbon as support
medium (previously colonized in a semicontinuous fixed bed).
They reported 96.5% COD removal operating at organic volumetric
loads BV = 5.88 kg COD/m3 .d. When the bioreactor was challenged
at BV = 32 kg COD/m3 .d, a substrate to biomass ratio Bx = 0.55 kg
COD/kg VS.d, and 2.55 h hydraulic retention time (HRT), a volumet-
ric methane production of 1.08 m3 /(m3 .d) was obtained during an
operation period of 94 days. The increase of organic load provoked
the decrease in the contaminants removal (COD) and in biogas pro-
ductivity. Durán-de-Bazúa et al. (1991) reported the operation of
a 300 L capacity, mesophilic pilot anaerobic fluidized bed reactor
loaded with 700 ␮m diameter spent ion exchange resin as support
medium. They studied three HRT (4, 3 and 2 d, based on the biore-
actor volume) and they reported up to 70% COD removal efficiency
(at BV = 34 kg COD/m3 .d and HRT of 2 days), and 7 m3 (STP)/m3 .d
biogas volumetric productivity with 70–80% methane. In a another
study, Sheli and Moletta (2007) treated wine distillation vinasses
in a bioreactor with a 66% poliethylene support material (density
0.84 g/mL); it was mixed during 1.25 min periods with a submerged
pump in the bottom, and they obtained 81.3–89.2% COD removal
efficiency at BV = 29.6 kg COD/m3 .d. At the end of the experiment
an increase in the biomass attached to the carrier was observed
(83.4%).
2.1.2. Anaerobic filters
Anaerobic filters are packed columns with a type of static
medium support colonized by an anaerobic microbial consortium.
These bioreactors can work in upflow and downflow mode; the
latter achieves in general better, sustained and more reliable oper-
ation. The downflow anaerobic filter has the capability to minimize
clogging of the packed bed when operated with effluents with high
concentrations of suspended solids (Nicolella et al., 2000). Braun
and Huss (1982) treated vinasses from molasses fermentation in a
pilot scale anaerobic filter reactor during a year operation, at load-
ing rates Bv up to 50 g VS/m3 .d, with HRT approximately 1 day.
529
They reported a biogas yield between 430 and 460 L/kgVS with a
13–20.5 m3 /(m3 d) biogas volumetric productivity. In experiments
on treatment of winery wastewater in a lab scale upflow anaerobic
filter 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 yield
0.30–0.35 m3 -CH4 /kg COD, the best results were obtained working
with multiple feed points. In another study, Thanikal et al. (2007)
treated vinasses in a lab scale anaerobic reactor packed with small
pieces of low density poliethylene (0.93 g/cm3 , Bioflow 30) as sup-
port medium. They found COD removal efficiencies over 80% even
at 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 a
fixed biomass of 57 g solids/L reactor. Bories et al. (1988) treated
vinasses 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 of
concentration from 5000 to 15,000 mg COD/L) in a mesophilic, con-
tinuous lab scale packed bed anaerobic reactor using polyester
cloth as support medium, Hamoda and Kennedy (1986) obtained
a 87% COD removal and 4 m3 /(m3 (reactor volume).d) methane
production rate at BV = 24 kg COD/m3 .d and HRT of 5 h. Farhadian
et al. (2007) used an upflow anaerobic fixed bed reactor filled
with different support materials to treat beet sugar effluents;
they observed that COD removal efficiency depended on the type
support employed. Best results were obtained using a standard
industrial corrugated packing (75–93%) whereas lower results cor-
responded to a PVC packing (65–77%).
2.1.3. Upflow anaerobic sludge blanket and related reactors
Since 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 in
continuous and upflow regime, it can accept high feed rates, it
possesses an internal biogas collection system, and it can work
under mesophilic and termophilic conditions (Wiegant and De
Man, 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 available
in the open literature; poor mixing and existence of dead zones
when operated at low flowrates; active biomass wash-out when
the reactor is subjected to hydraulic loading upsets or when granu-
lar sludge entraps gases; granular sludge destruction due to toxicity
episodes; extreme susceptibility to suspended solids in the influ-
ent, which accumulate in the reactors and pose a major problem
for the operation of the reactor and reduce the reactor capacity
(Nicolella et al., 2000). To some extent, some disadvantages such as
that of poor mixing have been overcome with the modification of
the so called expanded granular sludge bed reactor (EGSB) that is an
UASB with effluent recycling. In this way, several advantages of the
anaerobic fluidized bed bioreactor were incorporated to the EGSB
(Garibay-Orijel et al., 2005; Nicolella et al., 2000; Poggi-Varaldo and
Rinderknecht-Seijas, 1996).
Harada et al. (1996) treated alcohol distillation spentwash in
a UASB reactor under termophilic conditions (55 ◦ C) at BV = 28 kg
COD/m3 .d, COD influent concentration of 10 kg COD/m3 . They
observed up to 45 and 80% of COD and BOD removal efficiency,
respectively. In studies performed by Espinosa et al. (1995) in a
lab 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 with
a 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
530 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546
COD removal efficiency(58%) and biogas production (from 10.7
to 14.8 NL/d). Shivayogimath and Ramanujam (1999) tested a lab
scale hybrid UASB reactor (UASB at the bottom, and packed with
polypropilene rings in the upper zone as a separation gas–liquid
and biomass retention system) for the treatment of a distillery
spentwash. At BV = 36 kg COD/m3 .d and HRT of 6 h a 80% COD
removal was achieved; the produced biogas had 80% methane and
the methane pseudoyield was up to 0.40 m3 (NPT) CH4 /kg CODfed ,
where the pseudoyield is defined as the amount of methane gen-
erated per unit mass of substrate fed or applied (as g or kg COD or
BOD fed) and the amount of methane is expressed in m3 at 273 K
and 101.325 kPa absolute pressure.
The variation and low COD removal efficiencies obtained in
some of the reported investigations could be a consequence of the
fermented material origin, the distillation process (Harada et al.,
1996) as well as due to the presence of some organic compounds
(phenolic compounds) that have been reported as being toxic and
recalcitrants for the methanogenic systems (Borja et al., 1993a;
Benitez et al., 2003; Field and Lettinga, 1989).
2.1.4. Biohydrogen production
In recent years a wide group of investigators have proposed
and chosen hydrogen as an alternative renewable energy source.
Consequently, scientists have tried to look for new alternatives
to generate hydrogen from organic compounds present in liquid
or solid agroindustrial wastes using microorganisms (Fountoulakis
and Manios, 2009; Kapdan and Kargi, 2006; Valdez-Vazquez and
Poggi-Varaldo, 2009; Wang and Zhao, 2009). It has been shown that
organic compounds present in vinasses could be used as substrate
for hydrogen-producing microorganisms, particularly in dark fer-
mentation approach. In this regard, Lay et al. (2010) used vinasses
from distillation of fermented molasses fermentation as growth
medium for the hydrogen production in a lab scale, complete mix
reactor, operating at HRT of 3–24 h. Their bioreactor was seeded
with activated municipal wastewaters treatment plant sludges
where the presence of Clostridium acetobutylicum and Clostridium
pasteurianum bacterial genuses were identified. A production of up
to 390 mmol H2 /(L.d = was observed using Bv = 320 g COD/(L.d) at
a HRT of 3 h. In another study Yu et al. (2002) treated vinasses
from rice fermentation in a lab scale upflow reactor at a HRT of
2–24 h, COD 14–36 gCOD/L, pH 4.5–6.0 and temperature 20–55 ◦ C.
The obtained biogas composition showed 53–61% hydrogen con-
centration and 37–45% carbon dioxide in the biogas. The optimum
hydrogen production rate was 9.33 LH2 /(gVSS.d) with 2 h HRT and
the corresponding yield ranged 1.37–2.14 mol H2 /mol-hexose at
34 g/L COD, pH 5.5 and 55 ◦ C.
2.1.5. Ozonation combined with anaerobic digestion
Vinasse from bioethanol manufacturing (COD0 = 68.56 g/L) was
subjected to a very short ozonation pre-treatment (15 min) in batch
laboratory-scale reactors at 35 ◦ C (Siles et al., 2011). Under this
condition 50% of reduction of phenols was obtained. The stable con-
centration of organic carbon concentration showed that phenols
were transformed into other simple forms. Anaerobic biodegrad-
ability of raw and pre-treated vinasses were similar (values close
to 80% as COD). However the pre-ozonation process enhanced the
methane yield coefficient (14% increase) and methane production
rate (by 41.16%). The integrated chem-biological process proved to
be a viable option for the treatment of vinasses.
Martín et al. (2002) tested the anaerobic treatment of
vinasse from ethyl alcohol production line (COD0 = 97.5 g/L;
BOD0 soluble = 42.228 g/L; TOC0 soluble = 36.275 g/L) pretreated with
ozone; ozone/UV light and Ozone/UV light/TiO2 . The last pretreat-
ment decreased COD by 32%, although TOC removal was very low
(7%). In the anaerobic digestion stage, pretreated vinasses gave an
increased yield coefficient (1.12 mL CH4 /mg TOC) and also a higher
specific rate of methane production (20%) compared to those from
raw vinasse.
Sangave et al. (2007b) examined the effect of the ozonation
and ultrasound (US) on aerobic biodegradability of two vinasses
from the fermentation of sugar-cane molasses. Thermally (TPT-V)
and anaerobically pre-treated (APT) were tested. COD was diluted
down to 10–12 g/L; COD reductions of 45.6% and 13% were reported
for ozonation and US, respectively. In both cases, the subsequent
aerobic oxidation rate was enhanced. The aerobic biodegradability
increased 25 times with the ozonation treatment of TPT-V, how-
ever the use of US and ozone did not improve the biodegradability
of the APT. Overall, ozonation peformed better than US regarding
the increase of both COD removal and aerobic biodegradability.
In another work, Alvarez et al. (2005) treated cherry stillage in
an anaerobic sequencing batch reactor (AnSBR); influent COD was
varied between 5 AND 50 g/L. Different cycle times were selected
to test specific organic loading rates (Bx ), from 0.3 to 1.2 g COD/(g
VSS.d). They observed COD and TOC removal efficiencies higher
than 80% for influent COD up to 28.5 g/L and low Bx . Yet, volatile
organic acids (VOA) were accumulated and methane production
deteriorated at influent COD higher than 10 g/L; the AnSBR showed
signs of instability and could not operate efficiently at Bx > 0.3 g
COD/(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 shown
to be a suitable treatment technology as the following advantages
compared to the single AnSBR treatment.
2.2. Aerobic treatment
There are several studies on aerobic treatment of vinasses
mainly for removal of color (melanoids) and, particularly, toxic
substances such as phenols and polyphenols. In general, aerobic
treatment is not recommended for wastewaters with high contents
of organic matter such as vinasses, because of the high energy costs,
limitation of oxygen transfer in the aeration, and the high amount
of biomass generated (waste sludge, ca. 50% of BOD is transformed
in biomass or sludge) that may represent huge additional costs of
sludge treatment and disposal (Jiménez et al., 2005; Metcalf and
Eddy, 2004.)
Aerobic treatment of vinasses has used microbial consor-
tia (mixed cultures with bacterial predominance, sampled from
wastewater 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 (Fadil
et al., 2003; García et al., 1997).
Either single aerobic process or aerobic treatment combined
with 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 light
and H2 O2 have been explored and reported (Beltran-de-Heredia
et al., 2005a; Benitez et al., 2003). Most of these studies have been
carried out at lab scale. Also, there have been efforts for reclaiming
bacterial 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 methods
Robles-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 range
4.5–11% COD (contact times up to 1.5 h), whereas the removal of
aromatic compounds and phenols (expressed as gallic acid) were
within the ranges 16–32% and 48–83%, respectively. In the aerobic
 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546
post-treatment, the maximum removal of COD was 84% that cor-
responded to ozonated vinasses with previous 1.5 h of ozonation.
The overall removal efficiency (COD) of the combined treatment
ozonation + aerobic degradation reached a maximum value of 87%,
where the major contribution corresponded to the biological stage.
Beltran-de-Heredia et al. (2005a) carried out batch, lab scale exper-
iments with aerobic pre-treatment and chemical oxidation with
Fenton reagent and hydrogen peroxide for the removal of organic
matter as COD and phenols present in vinasses from wine distil-
lation (inlet COD0 = 18.5 g/L). An aerobic seed sampled from a full
scale wastewater treatment plant was used, the inoculum was pre-
viously acclimatized to vinasses (in the acclimatization step COD
was 5–18 g/L). Removal efficiencies were 75% and 54% in the aero-
bic pre-treatment for COD and phenols, respectively, whereas the
values observed for the chemical oxidation with Fenton-H2 O2 were
94% and 79%.
Benitez et al. (2003) studied the oxidation of the organic
substrate present in wastewaters generated in wine vinasses
(COD0 = 24.5 g/L and BOD0 = 11.15 g/L) by both an ozonation pro-
cess and an aerobic activated sludge system. The ozonation was
carried out in a subsequent first discontinuous and a second con-
tinuous periods. Organic matter removals ranged from 5% to 25.2%
as COD; total aromatic compounds removal varied between 16.8%
and 51.4%. The effects of the inlet ozone partial pressure, the
hydraulic retention time in the continuous reactor (inlet COD0
around 21.7 g/L and 0.454 Abs at 254 nm for aromatic compounds
(AC)) and the presence of UV radiation and H2 O2 in addition to
ozone (COD0 = 3.8 g/L and 0.05 Abs for AC) were evaluated. It was
found that AC removals increased with the increase of ozone partial
pressure (16.8–31.2%) and the COD removal was low (5.0–7.5%).
The increase of ozone contact time from 3 to 9 h also favored
pollutant removals with up to 25.2% COD and 51.4% AC. The use
of ozone + UV + H2 O2 was much more efficient than ozone alone
(COD0 = 3.8 g/L); the first approach lead to removals of 58.4% and
76.9% of COD and AC, respectively. Kinetic fittings for batch and
continuous periods lead to the evaluation of the apparent rate con-
stants for pollutant degradation as 216 L/(mol O3 h) and 232 L/(g
COD h), respectively. In the aerobic degradation (COD0 = 19.56 g/L)
by the activated sludge system COD removals from 31% to 85% were
achieved at hydraulic retention times between 24 and 72 h. The aer-
obic treatment satisfactorily fitted the Contois model for organic
matter degradation.
Sangave et al. (2007a,b) conducted lab-scale experiments in
order to assess the effect of ozone as pre-aerobic treatment and
post-aerobic treatment for the treatment of vinasses from sugar
cane. The treatment was carried out by ozonation, aerobic biolog-
ical degradation processes alone and by using the combinations
of these two processes. Seed for the aerobic treatment step was
sampled from a full scale wastewater treatment plant and further
acclimatized to vinasses before tests. Ozone removed up 27% of
organic matter as COD during the pretreatment step itself. In the
combined process, pretreatment of the influent led to enhanced
rates of subsequent biological oxidation step. Nearly a 2.5 times
increase in the initial oxidation rate was reported. Post-treatment
with ozone led to further removal of COD along with the com-
plete discoloration of the effluent. The 3-step integrated process
(ozone–aerobic oxidation–ozone) achieved 79% COD reduction
along with discoloration of the effluent sample as compared to
34.9% COD reduction for non-ozonated vinasses over a similar treat-
ment period.
In another research, wine vinasses were treated separately first
by means of a chemical ozonation (COD0 = 34–36 g/L) and a biologi-
cal aerobic degradation in an activated sludge system, and secondly
by a combined process which consisted of an aerobic pretreatment
followed by an post-ozonation treatment (Benítez et al., 2000).
Processes were continuous in all cases. Ozonation effected low
531
COD removals in the range 4.4-16%, with increased when both
the hydraulic retention time and the ozone partial pressure were
increased. An ozonation model based on a mixed flow reactor for
the 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 O3
consumed, respectively. The activated sludge stage results fitted a
modified Contois model with kinetic parameters K−1 = 5.43 L/g VSS
and qmax = 6.29 g COD/(g VSS.h). Finally, the authors reported that
the combined process significantly improved the efficiency of the
ozonation stage compared to the ozonation alone; this was also
reflected 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 treatment
of 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% and
82% 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 fitted
to a Contois’ model kinetics.The post-ozonation stage was effective
for the removal of polyphenols and A254 .
2.2.2. Treatment using bacterial consortia and bacterial pure
cultures
Vinasses discoloration has been frequently performed using
fungi 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 cultures
having the capacity to reduce color and remove the COD from the
vinasses. Mohanaa et al. (2007) isolated and characterized a bac-
terial consortium that consisted of Pseudomonas aeruginosa PAO1,
Stenotrophomonas matophila and Proteus mirabilis employing 16s
rDNA 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 using
an effluent supplemented with 0.5% glucose, 0.1% KH2 PO4 , 0.05%
KCl and 0.05% MgSO4 ·7H2 O.
Sirianuntapiboon et al. (2004) tested an acetogenic bacterial
strain BP103 and achieved 32.3% and 73.5% color removal from
raw vinasses from sugar molasses distillation and anaerobically-
treated vinasses, respectively. Both effluents were supplemented
with 3.0% glucose, 0.5% malt extract, 0.1% KH2 PO4 , 0.05% KCl and
0.05% MgSO4 ·7H2 O. Without the nutrient supplementation the dis-
coloration decreased. The same effect was observed when the strain
was evaluated in a sequencing batch reactor at 7 days HRT using 10
times diluted anaerobically-treated vinasses supplemented with
30 g/L glucose (Tondee and Sirianuntapiboon, 2008). COD removal
efficiency was significant (65.2%) as well as removal of other
pollutant parameters (BOD5 , 82.8%; TKN, 32.1%; and melanoidin
pigment, 50.2%).
Chavan et al. (2006) isolated a Pseudomonas sp. strain from soil
near a sugar-cane alcohol distillation factory (COD0 = 146.38 g/L
and BOD0 = 70.84 g/L, 10% vinasse dilution was necessary). Color
before 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, temperature
in the range 30–35 ◦ C and with the optimum medium compo-
sition 4 g/L glucose, 0.2 g/L KH2 PO4 and 0.009 g/L MgSO4 ·7H2 O.
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; Tondee
and Sirianuntapiboon, 2008), as well as by the production of
enzymes, such as sorbose oxidase, manganese peroxidase depen-
dant, and glucose oxidase. The latter, for instance, is activated in the
532 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546
presence of glucose, and yields H2 O2 as a by-product (Scheleissner
et al., 1997). H2 O2 , in turn, can oxidize organic matter thus yielding
simpler compounds like volatile acids (Kumar and Viswanathan,
1991), facilitating their further degradation to carbon dioxide and
to biomass production. This, in turn, results in removal of COD
(Ghosh et al., 2002; Watanabe et al., 1982) and reduction of color
(Ohomomo et al., 1988).
Adsorption could be another mechanism of color removal,
although it has been reported that previous acclimatization of cells
to a medium containing melanoidins products (MP) may decrease
the adsorption activity (Sirianuntapiboon and Prasertsong, 2008).
Contribution of volatilization to color and COD removal is unlikely,
since concentration of volatile compounds in vinasse is negligi-
ble. Dahiya et al. (2001a) achieved 76% discoloration employing
a Pseudomonas fluorescens strain immobilized in a porous cellu-
lose carrier under non sterile conditions; color reduction was 90%
under sterilized medium for the wastewaters supplemented with
glucose 0.5% (w/v) provenient from molasses fermentation and dis-
tillation process. They discussed that this difference might be also
due to the role of the high temperatures reached during the ster-
ilization process that could have caused the pigment degradation
into low molecular weight compounds. Kumar and Viswanathan
(1991) isolated and acclimatized an unidentified bacterial strain
by continuous increasing the distillery wastewater concentration.
This strain was capable of removing 80% COD of the influent in 4–5
days.
In the precedent cases the vinasses had to be supplemented with
glucose (0.4–30 g/L) and other essential nutrients, the discoloration
was possible after 3–10 days. This could mean an economical
drawback for these treatments since the mezcal vinasse quan-
tity generated each year is approximately 90,000,000 L, whereas
the annual amount of vinasses in India is nearly 40 billions liters
(Raghukumar et al., 2004).
2.2.3. Alternative sources of protein
Due to the costs of wastewater treatment the obtaining of by-
products as part of the process is becoming, without doubt, an
important factor that could enhance the economic feasibility of
the treatment. For instance, microbial biomass is a by-product that
could be used as unicellular protein source for animal feed. On the
one hand, this is of particular interest in countries where high qual-
ity fodder and proteins for animal feed are scarce, such as Mexico,
India, and others. On the other hand, even in developed countries
where conventional, quality animal feeds are available, microbial
biomass protein could play a significant role in abating animal hus-
bandry costs.
Several researchers have carried out studies to solve the envi-
ronmental problems provoked by the vinasses high organic loads
looking for producing unicellular biomass as alternative source
of protein. Research included bacterial, fungal, and algal biomass.
Durán-de-Bazúa et al. (1991) treated vinasses from sugar molasses
fermentation in lab and pilot tests in aerobic rotating disk bioreac-
tor system. At lab scale they used diluted vinasses and obtained
microbial biomass with a raw protein content between 18 and
27%, and 1.8 kg wet biomass/kg COD removed system yield; COD
removal efficiency obtained was 65% and 70% and BOD removal
efficiency was ca. 95%. At pilot plant scale they employed non-
diluted vinasses with a concentration 60 to70 kg COD/m3 ; a similar
biomass yield to that the lab scale tests was obtained, with a
small increase in the protein content (20% and 30%). However, it
was observed that organic matter removal efficiency significantly
decreased compared to lab tests (40–50% and 60–70% for COD and
BOD5 removal efficiency, respectively). The raw protein percent-
ages were similar to those found by Díaz et al. (2003c). In effect,
the latter reported 30.1% when using Candida utilis in a lagoon
system. Shojaosadati et al. (1999) isolated a fungus species of the
genus Hansenula from an alcohol plant effluent. In batch tests with
this strain and vinasses they observed a concentration of 5.7 g/L
biomass, with 39.6% raw protein and 31% COD removal efficiency
with no addition of external nutrients source. When supplementing
nutrients 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 use
of Rhizopus microsporus (var. oligosporus) as protein ingredient
in feed for aquaculture. They cultivated the microorganisms in
vinasses from sugar-cane alcohol distillation; vinasses were sup-
plemented with nitrogen and phosphorus. It was found a high
fungal growth at pH 5.0 and 30 ◦ C, the obtained biomass contained
ca. 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 beet
vinasse; they reported biomass concentrations of 3.5 and 4.8 g/L
and productivities of 0.15–0.24 g/(L.d). The biomass concentration
and productivity increased to 8 g/L and 0.7 g/(L.d), respectively,
when they used a photobioreactor with Schlösser medium with
2 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 is
presented in Table 5. Land disposal of raw vinasse may impair
the physical, chemical and biological properties of soil, which is
reflected in the increase of soil loss, reduced vegetation cover
and increased values of exchangeable sodium (Tejada et al.,
2008). These problems can be overcome by co-composting the
vinasse with other solid wastes. Because no information about
co-composting of mezcal-vinasse was found in literature, several
examples of this procedure that used other kinds of vinasse are
discussed.
Using a mixture of vinasse and cotton waste, Díaz et al. (2003a)
investigated the influence of vinasse addition and incubation time
on 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 added
vinasse, at moderate operating times (20–35 days), showed high
biodegradability and minimum losses of nitrogen.
Three mixtures of a concentrated vinasse and solid wastes
(grape husks, squeezed olive paste and cotton wastes) were
co-composted in static windrows (Madejón et al., 2001a). The com-
posts obtained were used in field experiments to study the effect
of their application as deep fertilizer on three crops: corn, sugar-
beet and sunflower. At the end of the experimental period, soil
oxidizable-C, total humic extract-C and humic acids-C contents sig-
nificantly increased in soils treated with composts when compared
with an unamended control and inorganic fertilizer treatments.
Organic fertilization also increased the Kjeldahl-N content of the
soil. A slight increase of soil salinity was observed both in the
composts and the inorganic fertilizer treatments. Nevertheless, this
increase did not cause sodium hazard to the soil.
In another study, Díaz et al. (2003b) composted mixtures of
vinasse and grape husks in a lab scale reactor at 55 ◦ C. They found
that the best properties of the co-compost were obtained with
additions of vinasse in proportions between 10% and 20%. Higher
vinasse proportions increased the NKT losses, the acidity, and the
salinity 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 respect
to a control soil. The use of this product would contribute to soil

Table 5
Use of vinasses in co-composting.
Substrate, process and operational
conditions
Vinasse from wine; Lab scale (LS);
aerobic conditions at 55 ◦ C for 43 days;
determination of ratio grape
marc/vinasse for an optimum
composting
Mixture of depotassified beet vinasse
(45%, dry weight) and cotton gin waste
(55%) using two different aeration
systems: static aerated pile (process A)
and windrows (process B); Pilot scale
Mixtures of depotassified beet vinasse
with three different residues were
co-composted in static windrows,
under cover, with aeration and
controlled conditions
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
Field experiments to study compost
effects on three crops
Mixtures of vinasse (V) with solid
residue cotton waste (CW) at: 0%, 11%,
40%, 69% and 80% (V/CW, w/w wet
weight); times 1, 7, 23, 38 and 45 days
LS at 55 ◦ C; Observed the effect of
operation time (OT) and vinasse added
over pH; OM; Kjeldahl-N; C/N ratio,
biodegradability and germination
index (GI)
Pile composting, 12 weeks
Mixtures of rice straw, soybean residue
and enriched with rock phosphate
supplemented with vinasse. Buffalo’s
manure was used as inoculum,
V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546 533
Features of the process; compost Remarks Ref.
characteristics; effects of application
pH did not show great differences among the Higher vinasse ratio the higher the 1
mixtures (7.9–8.31). salinity of the product
Organic matter (OM) content decreased in all Recommended 10–20% vinasse in the
mixtures mix might as the best for the
optimization of the process and high
quality compost
At lower vinasse ratio higher OM losses and
higher values of biodegradability
Increasing the ratio of vinasse in the mix was
an increase in the losses of Kjeldahl-N; P
content decreased. Possible limit to vinasse
addition because of the optimum range of C/P
ratio is 75–150
A faster increase of temperature in the process The decrease of NH4 + –N content could 2
B (54 ◦ C at 7 days) than in process A (45 ◦ C at be used as a criteria of composting
21 days) was observed maturity
In both systems high NO3 − –N content at the Decreased phytotoxicity
beginning of composting was observed; it
decreased during and slightly increased during
maturation phase
Increase of the NH4 + –N in both processes High salinity; compost should be used
during the thermophilic phase. At the end of at moderate doses due to the relative
the process a decrease in NH4 + –N content was high values of electrolytic conductivity
observed (21.7 and 12.9 dS/cm for A and B
processes respectively)
Mass porosity did not change in process A, Windrow process recommended
whereas it increased in process B
High amounts of macronutrients, particularly
N and K
Low concentrations of heavy metals
Composts presented low concentrations of Compost had a moderte positive effect 3
micronutrients on plant nutrition and yield, and on
soil chemical fertility
Risk of use for its high content of salts present No serious risks of salinization or
in vinasse is decreased with the combination sodification for coarse textured or well
of urea with compost Vinasse and P2 O5 drained soils under irrigation
No phytotoxicity detected
Composts increased crops yields with respect
to the control without vinasses
pH and Kjeldahl-N were influenced by both OT Conditions recommended: 4
and V ratio; more sensitive to OT. Kjeldahl-N
increased with OT and V ratio
OM and GI were similarly affected by OT and V. For OM, V: 0–11%; OT: 38–45 d
GI significantly increased with OT but
drastically decreased with V ratio
C/N decreased with V ratio For GI, V: 11–40% OT: 38–45 days
The addition of V increased the concentrations For C/N, V: 0–40%; OT: 7–23 d
of K, Ca and Mg decrease in the concentration
of P
Minimal Kjedahl-N losses with 11–40%
of V and OT 7–23 d
Decrease in P could limit the addition
of vinasse since optimal C/P (75–150)
would not be met
Sucession of microbial populations was Temperature > 55 ◦ C was enough to 5
observed and inferred by physicochemical sanitisize the produced composts
changes
Mineralization of organic matter was
promoted by microbial activities led to organic
matte
534 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546

Table 5 (Continued)

Substrate, process and operational Features of the process; compost Remarks Ref.
conditions characteristics; effects of application

External sources of microorganisms enhanced

Beet vinasse was mixing with
vermicompost (constituted by a green
forages)
Application tests, evaluation of
changes on physical, chemical and
biological properties of soils
Beet vinasse alone (V) and mixtures of
crushed cotton gin compost with
vinasse (CV)
Tests for evaluation of effects on plant
cover, soil’s physical, chemical and
biological properties
Mixtures of sugarbeet molasses
distillery slops V and olive mill husks
In-vessel composting process.
Evaluation of operating conditions and
extent of the stabilization time
Mixtures of sugar beet vinasses (V)
with cotton gin (CW) and grape marc
(GM)
Supplemented with phosphate (P) and
Leonardite (L) in order to prevent great
losses of Nitrogen
Pile composting
Pile A: GM (76%) + V (20%) + L (1%) + P
(3%)
Pile B: CW (76%) + V (20%) + L (1%) + P
(3%)
Mixtures of vinasses from alcohol
distillery and GM, wheat straw (WS).
Wet pre-treatments with 7–15% NaOH
on total dry matter at 70 ◦ C
Mixture A: 100 g wet GM, 16 g WS, and
300 ml V
Mixture B: only changed 16 g for 30 g
of WS
the biodegradation of recalcitrant substances
The compost maturity was satisfactory after 84
days
The adittion of vinasse plus vermicompost had Mixing of beet vinasse with 6
a positive effect on the soil’s parameters vermicomposts resulted a feasible
evaluated (physical, chemical and biological strategy for protecting soil’s properties
properties) and recovering semiarid areas
The loss of amended soil decreased by 31.2%;
plant cover increased 68.7% compared with
unamended soil
The porcentaje of plant cover increased 86% in The negative effect of only-V on the 7
CV-amended soils and decreased 58.3% with soilı̌s properties was ascribed to high
only-V, compared to the control soil concentrations cations such as Na+ and
fulvic acid present in the fresh vinasses
Only-V addition on the soil had negative effect Application of CV to soil helps in its
on the soil’s physical, chemical and biological restoration
properties
CV had a positive effect on the soil’s physical
(structural stability and bulk density), and
biological properties (microbial biomass, soil
respiration, and enzymatic activities) with
respect to the control soil
OT of 30–40 d and the addition of 0–10% V Mixtures of 0–40% of V and up to 40 8
associated to best results in terms of OM days of OT were evaluated
biodegradation, compost maturity and
limitation of Kjeldahl-N losses
CW and GM were adequate for co-composting 9
of V
Lower losses of organic matter was observed
with V co-composted with grape marc
The composts tested had high fertilizer values,
high levels of stability and absence of
phytotoxicity
No lignin degradation; cellulose degradation
only observed in the pile A
NaOH concentration 7–15% increased Increase in the digestibility of the two 10
digestibility from 37% to higher than 50% solid components and utilization of the
valuable compounds contained in V
Contact times of 3 h adequate Mixture A was superior to mixture B.
Quality of the mixture was significantly NaOH conc. < 5–6% did not improve
affected when increased the WS from 20.5% to digestibility
32.6%
Pretreated mixtures had acceptable values of
digestibility, crude fibre, and crude protein;
better property profiles than untreated GM
aloine and WS alone
High Na, K, Fe, Cu and Zn content of both
mixtures 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. Díaz et al. (2003b); 9. Madejón et al. (2001b); 10. Vaccarino et al. (1993).

protection, and to the eventual restoration of arid lands (Tejada
et al., 2009).
An inspection of works in Table 5 suggests that the best com-
posts are those with low ratios of vinasses in the mixtures (10–20%)
(Díaz et al., 2002a,b, 2003b; Madejón et al., 2001b). In gen-
eral, for those mixtures, high decreases of biodegradable organic
matter, adequate nutrient profiles, and positive effects on soil
properties and crop yields are reported (Madejón et al., 2001a).
Furthermore, the best composts formulated with vinasses have
presented a high fertilizing value as well as an adequate stability
and absence of phytotoxicty (Díaz et al., 2002a,b; Madejón et al.,
2001b).
For higher ratios of vinasses in the mixtures, undesirable high
salinity is observed, associated to high concentrations of cations
such as Na,K,P, Mg y Ca; this issue certainly could limit compost
usefulness and application (Díaz et al., 2002a,b, 2003a).
 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546
Overall, application of composts from co-composting of vinasses
and other wastes to soils has helped in minimizing soil losses as
well as increasing the vegetal cover (Tejada et al., 2007, 2009).
The co-composting of vinasse with agricultural and agro-industrial
solid wastes is worth investigating because it can provide a low-
cost treatment technology suitable for rural regions where more
sophisticated technologies are difficult to implement, due to high
costs and requirements of skilled personnel.
2.4. Fungal treatment
The decomposition of organic matter is usually carried out
by fungi, generally Basidiomycetes found in forests and meadows.
These molds colonize the surface soil, layers of humus, dead plants,
and grass wastes. The protective layer of lignin is converted in ligno-
cellulose by nonspecific extracellular oxide-reductases produced
by fungi (Hatakka, 2001; Heinzkill and Messner, 1997; Steffen et al.,
2000). Once that lignin was removed, the plant polysaccharides are
available as a carbon and energy source. Four ligninolytic enzymes
have been described; lignin peroxidase (LiP), manganese peroxi-
dase (MnP), versatile peroxidase (VP), and laccase. The first three
enzymes contain a hemo group as cofactor. All of them use H2 O2
as the first electron acceptor.
Laccase uses O2 as the final electron acceptor (Papinutti et al.,
2003; Thurston, 1994), and plays an important role in remediation
because it acts over many organic pollutants such as: polycyclic aro-
matic hydrocarbons, phenols, pesticides, endocrine disrupters, and
dyes (Dahiya et al., 2001b; López et al., 2004; Majeau et al., 2010;
Yuzhu and Viraraghavan, 2001). Laccase enzyme has also been used
for bleaching pulp and vinasse, and for the discoloration of effluents
from the textile industry (Couto and Toca-Herrera, 2007; Rodríguez
et al., 1999; Strong and Burgess, 2007; Wesenberg et al., 2003).
Table 6 shows a summary of several vinasse treatments using fungi,
mostly ligninolytic. It can be seen that most studies have relied on
supplementation with glucose or another easily degradable carbon
source. This is a drawback for fungal treatment application at full
scale.
As it was mentioned above, one of the main features of vinasse
is its color as well as the content of phenolic compounds; the lat-
ter are known to be toxic and their concentration can be relatively
high (Table 3, ca. 500 mg gallic acid/L). So, efforts for degrading
these substances by using aerobic treatment with fungi have been
made. Unlike the characteristic brown color of stillage generated
by the fermentation of molasses, which is attributed to recalcitrant
melanoidines generated by the condensation of reducing sugars
and amines or amino acids (Cammerer and Kroh, 1995; Strong and
Burgess, 2008). In the case of vinasse obtained from mezcal and
wine distillation, the color is generally attributed to highly recal-
citrant and inhibitory phenolic compounds (Borja et al., 1993a,b).
Also, many researchers have used extracellular enzymes produced
by ligninolytic fungi to degrade recalcitrant molecules such as phe-
nolic compounds and melanoidins present in vinasse.
Recalcitrant aromatic compounds, color and COD remaining in
vinasse after anaerobic treatment can be successfully removed
by ligninolytic fungi (Benito et al., 1997; Kumar et al., 1998;
Raghukumar and Rivonkar, 2001; Singh et al., 2010); especially,
if an easily assimilable carbon and energy source is added (Benito
et al., 1997; Fahy et al., 1997; Kumar et al., 1998).
Coulibaly et al. (2003) reviewed the utilization of fungi for
biotreatment of wastewater. They covered the post-treatment by
anaerobic digestion of wastewaters that have been pretreated with
lignolyitic fungi. Among several advantages of fungal pretreatment
they highlighted the degradation of toxic and recalcitrant pollu-
tants, the possible production of enzymes as added-value products,
as well as single cell protein. Unfortunately most research of
535
fungal pretreatment has been performed at lab scale. They strongly
recommend to foster research at pilot and commercial scale.
Pleurotus sajor-caju CCB020, a ligninolyitic fungus, was used in
a 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 determined
by 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 manganese
peroxidase (MnP) from homogenized mycelia of the ligninolyitic
fungus Ganoderma lucidum. Free, homogenized mycelia with addi-
tion of 2.5% glucose lead to effective color reductions on days
1–10. Greatest color reductions occurred on day 10 using MnP plus
100 mg/L Mn(II) as MnSO4 ·5H2 O. MnP activity was low without
Mn(II) in the nitrogen-deficient assay medium. 5 ␮M H2 O2 but not
the chelators oxalic acid and dl-lactate improved MnP activity in
phenol red oxidation system at pH 4.5–5.0 and 30 ◦ C.
Winery wastewaters (COD0 = 0.665 to 12.6 g/L) were inoculated
with the ligninolyitic fungus Trametes pubescens MB 89 to establish
the feasibility of fungal submerged culture and the usefulness of
a 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 was
beneficial to methanogenic digestion of winery wastewaters that
had higher initial phenolic compound and color concentrations,
but no for wastewaters with low initial phenolic compound and
color concentrations. Anaerobic digestion of fungally-treated and
raw samples generally showed little difference with regard to total
COD removal and final pH.
Melamane et al. (2007) studied the combination of fungal
pre-treatment with white rot fungus (Trametes pubescens) and
anaerobic digestion in order to remove COD and phenolic com-
pounds from wine distillery wastewater (COD0 = 15 g/L). In the first
stage a 53% COD removal efficiency was reached. In the anaerobic
digestion the total COD removal efficiency was much higher than
the first stage (89.5%). The anaerobic digestion treatment was able
to withstand shocks of organic loading rates due to the addition of
CaCO3 and K2 HPO4 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. chrysosporium
in 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 removal
efficiencies of COD, phenolic concentration and color were not
affected by temperature (47.48%, 54.72%, 45.10% at 25 ◦ C and
54.21%, 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 on
decolorizing and COD removing capability of the four fungi was
determined. 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 of
all fungi. The utilization of cotton stalk was advantageous since
it performed both as an attachment support and as a source of
nutrients.
Trametes sp., I-62 (CECT 20197) was used in order to degrade
the soluble organic matter and color present in vinasse from
fermentation of sugar-cane molasses (COD0 = 55.5 g/L) (González
et al., 2000). A dilution of 20% (v/v) with the culture medium
was tested. After 7 days of fungal treatment, maximum effluent
536 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546

Table 6
Selected fungal treatment of vinasses.

Process and experimental design Results Remarks Ref.

Vinasses from: Brandy distillation (BD) and
distillation spirits (DS); laboratory scale (LS);
Microorganism used: Trametes pubescens MB
89; T = 28 ◦ C; pH 4.5; phenols concentration
BD = 35–280 mg/L; DS = 290–320 mg/L
VFSM
Microorganism used: Trametes sp. I-62 (CECT
20197); LS
VBM previously subjected to conventional
anaerobic–aerobic treatment; Trametes
versicolor; LS; variation of the carbon source,
nutrients and initial pH
VFSM Flavodon flavus; Immobilization of the
fungus in polyurethane foam; Ecotoxicity tests
using Oreochromis mossamb cus; Repeated BM,
LS
Anaerobically digested VMSMS Aspergillus
heteromorphus; production of laccase; LS
VFSM Phanerochaete chrysosporium
Culture time 32 days
T = 25 y 39 ◦ C;LS
◦
VFSM; Pretreatment at 30 C and 10 days of
culture with Geotrichum candidum
Postreatment using Coriolus versicolor,
Phanerochaete chrysosporium and sterile
mycelia; LS
Anaerobically digested VFSM from
biomethanation plants; fungi Coriolus
versicolor and
Phanerochaete chrysosporium; LS
Influent diluted 25%, 12.5% and 25% (v/v) with
water
The fungal culture showed higher percentages
of phen = 87%, color = 88% and COD = 83% than
those of the enzyme tests Phe = 61and
color = 12
color = 73% and COD = 61.7% after 7 days of
culture
Removals in the effluent:
color = 82%, COD = 77% y N–NH4 + = 36%
Vinasse without dilution was obtained:
color = 10%
Vinasse diluited at 50% coluor = 60 y 73%, in 5 y
7 days respectively
68% decrease in the concentration of polycyclic
aromatic compounds (benzo (a) pyrene) after 5
days of treatment
The use of anaerobically digested SMDW and
ligninocellulosic biomass increased laccase
production (6.6 UI mL−1 )
COD = 47.48%; Phe = 54.72%; color = 45.10% at
25 ◦ C
COD = 4.21%; Phe = 59.41% color = 56.81% at
39 ◦ C
A 10 days pretreatment with Geotrichum
candidum resulted in COD = 53.17% and
phe = 47.82%
Coriolus versicolor immobilized in a
packed-bed reactor reduced COD = 50.3%
giving a global COD = 77%
Optimum growth and discoloration occurred
at 35–40 ◦ C, pH 5.0, glucose 3–5% (w/v) and
VFSM diluted with water 6.25% (v/v)
C. versicolor: color = 71.5%, COD = 90.0%
P. chrysosporium: color = 53.5%, COD = 73.0%
The fungal and the enzymatic (laccase) 1
treatment were tested separately
Increased color in the tests performed with the
enzyme (color = 160%)
Use of 20% diluted vinasses 2
Best results were obtained supplementing 3
sucrose as co-substrate and KH2 PO4
Color adsorbed onto mycelium = 5–10% On the
other hand, there was no influence on COD
removal
The immobilization of the fungus is effective 4
bleaching using for up to three batch cycles.
No damage was found by the vinasse treated in
the liver of fish compared to untreated vinasse
Detected benzo(a)pyrene in the vinasse and
this appears to be one of the causes of toxicity
5
Two operating conditions 25 and 39 ◦ C best 6
results at 39 ◦ C
It is technically feasible to bioremediate spent 7
wash using a multi-stage treatment process
(Pretreatment step with Geotrichum candidum)
Using additional labile carbon source 9
Decolorization and COD removal decreased
significantly at the higher concentrations of
anaerobically digested VFSM

Notes: VBM, vinasse from fermented beet molasses; VFSM, vinasse from fermented sugar molasses; color , removal efficiency of color; phen , removal efficiency of phenolic
compounds; 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
(2006); 7. Fitzgibbon et al. (1995); 8. Fahy et al. (1997); 9. Kumar et al. (1998).

discoloration values and COD reduction were obtained, 73.3% and
61.7%, respectively, at 7 d of incubation. A significant increase
in laccase production was observed, but no MnP activity could
be detected. After the period of treatment a significant decrease
in a number of pyrolysis product (mainly furan derivatives) was
observed that could be related to the vinasses color-removal asso-
ciated with melanoidin degradation.
García et al. (1997) working with vinasses from molasses
(COD0 = 75 gO2 /L) obtained 66% and 70% phenolic removal effi-
ciencies employing Aspergillus terreus and Geotrichum candidum,
respectively, after five days fermentation. Jiménez et al. (2003)
employed four different types of fungus (Penicillium sp., Penicillum
decumbens, Penicillium lignorum y Aspergillus niger) for the discol-
oration, phenolic removal and COD of vinasses from distillation of
fermented molasses. A decrease in the effluent color starting the
first incubation day for all the fungi tested has been observed;
best results were obtained with Penicillum decumbens with 40%
discoloration. Decolorization was ascribed to the degradation or
to tannins and some phenolic compounds adsorption onto fun-
gal mycelium. The maximum COD removal efficiency achieved was
52.1% with Penicillium sp and 50.5% with Penicillum decumbens. The
removal efficiency of phenolic compounds was similar in all cases,
70%.
Untreated vinasses (COD0 = 80.5 g/L) and vinasses previously
treated with Penicillium decumbens were (COD0 = 23.0 g/L) post-
treated by anaerobic digestion (Jiménez et al., 2006). The
pre-treatment of vinasses with P. decumbens reduced 67.7% of the
initial 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 anaerobic
digestion (AD) process for pre-treated vinasses by 9.6 and 6.9 times
respectively, 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 removal
present in vinasses using Penicillitum decumbens. Batch tests were
performed where the initial concentration of COD and gallic acid
were 42.6 g/L and 0.21 g/L, respectively. P. decumbens without the
need for any nutrient supplements in the medium was capable
of significantly degrading the phenolic compounds in vinasses.
After 3 days 74% of removal phenolic compounds was obtained
whereas the best result in color removal (41%) was obtained after
the day 4 of treatment. Biomass yield coefficient was found to be
0.35 g VSS/gCOD. The authors also developed a kinetic model that
accurately described the variation of substrate and biomass con-
centrations with time in the aerobic degradation process of vinasses
with P. decumbens.
 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546
Strong (2010) conducted lab scale treatment tests on vinasses
from alcoholic beverage derived from the distillation of fermented
marula fruit using several white rot fungus (Trametes pubescens
MB 89, Ceriporiopsis subvermispora, Pycnoporus cinnabarinus and
Phanerochaete chrysosporium). The marula vinasses exhibited a
chemical oxygen demand (COD) of 27 g/L, a pH of 3.8, a high concen-
tration of phenolic compounds (866 mg/L) and a high suspended
solids content (10.5 g/L), all of which could adversely affect biolog-
ical treatment. Full-strength wastewater was treated in shake-flask
pure cultures of the four white rot fungi. Trametes pubescens per-
formed the best with regards to degrading phenolic compounds,
COD and color. Laccase production was only detected in the T.
pubescens and C. subvermispora cultures. In a second phase, six
wastewater concentrations (100%, 80%, 60%, 40%, 20% and 10%) at
pH 4.5 were evaluated with batch cultures of T. pubescens. COD
and phenolic removal efficiencies did not vary with concentration
(COD: 71–77%; phenolic compounds: 87–92%). A maximum lac-
case activity (1063 units/L) was obtained in the 80% wastewater
concentration.
Robledo-Narvaez et al. (2006) reported that after anaerobic pre-
treatment, a post-treatment with fungal pellets of a defined mixed
culture of Lentinus edodes and Trametes versicolor immobilized on
oak sawdust and activated carbon increased the overall removal
of the organic matter present in effluents from the pulp and paper
industry. The removal efficiencies of color, lignin and COD were
44%, 28% and 40%, respectively. A post-treatment of similar efflu-
ents using a pure culture of Trametes versicolor immobilized in
wood saw-dust (hybrid pellets), produced higher removal efficien-
cies of color (54%) and lignin-like compounds (69%); but a lesser
removal of COD (32%) (Ortega-Clemente et al., 2007). Usually, in
the anaerobic phase, the removal of biodegradable organic mat-
ter was about 90% or more, while in the fungal stage, about 60%
of the recalcitrant matter not eliminated in the first stage was
removed.
Since some compounds present in vinasse can stimulate lac-
case production in ligninolytic fungi (Strong and Burgess, 2007), a
strategy for vinasse treatment is the use of fungi for laccase produc-
tion. For example, Singh et al. (2010) used anaerobically pretreated
vinasse and lignocellulosic biomass (rice straw, wheat straw and
sugarcane bagasse) to produce laccase from Aspergillus heteromor-
phous. Pretreated vinasse was a good inducer of the enzyme, and the
addition of lignocellulosic material increased laccase production.
Using vinnase from wine distillation, Strong and Burgess (2007)
reported the laccase production by Trametes pubescens MB 89 grow-
ing in shake-flasks and a bubble lift reactor. The procedure used
improved the vinasse quality removing 79%, 80% and 71% of COD,
total phenols and color, respectively. In another work, Strong and
Burgess (2008) evaluated wine-related vinasses as a substrate to
produce laccase enzyme using Trametes pubescens. The enzymatic
role in phenolic compounds degradation and color change were
evaluated using crudely purified laccase. The best result obtained
in the fungal treatment gave removals of of 83%, of 87% and of 88%
for COD, phenolic compounds, and color, respectively. Laccase syn-
thesis was greater than 1500 U/L in all treated wastewaters, with a
maximum of 8997 U/L.
Aguiar et al. (2010) cultivated three strains of Pleurotus and Tri-
choderma reesei on pre-treated bagasse and vinasses as sources of
carbon. Under 2% H2 O2 , 1.5% NaOH and autoclave treatment the
greater fibre breakage was obtained in these condition increasing
the cellulose level up to 1.2 times and decreasing 8.5 times the
hemicellulose content and high ligninolytic activity for all cultures.
T. reesei produced laccase, peroxidase and MnP; the MnP activity
was 1.9–4.8 times higher than that of Pleurotus.
A culture media that consisted of either vinasses or a synthetic
culture media, with supplementation and without of banana waste,
was used as substrate in cultures of the white rot fungi P. ostreatus
537
APK-1 for laccase production (Shanmugam et al., 2009). Vinasses
culture medium was a better laccase-inducer medium than the syn-
thetic culture medium; the addition of banana waste in the medium
enhanced the enzyme production. Laccase production in batch tests
by 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 cotton
stalk supplements. Tests with cotton stalk showed that vinasse
culture medium was a better laccase-inducer medium than the
synthetic culture medium. In another experiment, Kahraman and
Yesilada (2001) reported that white-rot fungi Coriolus versicolor
and Funalia trogii produced laccase in media with diluted olive-
oil mill wastewater and vinasse. Addition of spent cotton stalks
enhanced the laccase activity with a maximum after 12 d of culti-
vation.
Studies made by Acharya et al. (2010) indicate that anaerobically
pretreated vinasse can be used as a viable nutrient source for cellu-
lase production by the non ligninolytic fungus Aspergillus ellipticus
in solid-state fermentation.
Finally, it is important to remark that most current studies with
fungi have been carried out at a laboratory level. It is also worth
noting that pure enzyme production is a process that raises the
treatment costs. Thus, it is necessary to find new and economic
methods to produce large amounts of ligninolytic or other enzymes,
as well as performing large scale studies as another way to decrease
production costs.
3. Physico-chemical treatment
It has been observed that after vinasse bio treatment, an impor-
tant fraction of non-degraded recalcitrant organic matter still
remains (Durán-de-Bazúa et al., 1991; Sangave et al., 2007a). There-
fore, alternative or complementary treatment methods, such as
physical and chemical processes have been used (Manisankar et al.,
2004; Vlyssides et al., 1997; Yavuz, 2007). A common thread in
most of these methods is their relatively high cost, elevated reagent
doses, and in some cases other pollutants are generated (Fannin
et al., 1987; Lucas et al., 2009; Peña et al., 2003). Table 7 presents
a summary from literature on physical-chemical treatments used
for vinasse treatment.
3.1. Ozone treatment
Because microorganisms used in conventional biological treat-
ments could not have the enzymes required for the complete
biodegradation of recalcitrant compounds, chemical oxidation
prior biological treatment has been introduced to transform or
degrade these compounds into smaller molecules (Gogateand and
Pandit, 2004).
Chemical oxidation with ozone has desirable characteristics
in the pre and post-treatment of industrial wastewater contain-
ing recalcitrant compounds. Some of its properties are: (i) it is
a 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 possessing
double bonds, which frequently are associated with color (Rehman
et al., 2006; Sangave et al., 2007a; Sreethawong and Chavadej,
2007), (iii) it can promote the formation of highly reactive hydroxyl
radicals, which can even have more oxidative power that the same
ozone (Sangave et al., 2007a).
Traditionally, ozone has been used for disinfection of water
and 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 7
Physico-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 using
O3 ; parameters: pH 5.4, 7.0, 10; T = 10, 20 y
30 ◦ C @ Q = 30 L/h, [O3 ]i = 20 mg/L, f = 10
VDGA; LS; Batch mode (BM); oxidation using
Fenton-H2 O2 , Reaction time effect on: COD ,
pphe , Aro ; condition T = 25 ◦ C, pH 3.5,
[FeSO4 ·7H2 O]0 = 0.01–0.1 mol/L, addition of
0.1–0.65 mol/L of H2 O2
VDGA; LS; BM; 1st stage Fenton-H2 O2 , the
experimental variables studied were:
[FeSO4 ·7H2 O] and [H2 O2 ]
2nd stage coagulation/flocculation.(CF)
Vinasse from beet molasses (VBM); Pilot plant
scale (PP); electrolysis treatment (anode
Pt/TiO2 )
Vinasses from fermented sugar molasses
(VFSM); LS; electrodialysis treatment, Constant
voltage of 17 V, concentrated solution
(brine) = 5 g NaCl/L of electrolyte solution = 21 g
KNO3 /L
Pre-treated distillery wastewater (PTDW)
(evaporator and centrifuges); condensed liquid
phase is used as PTDW; LS; BM,
Two electrochemical methods were
investigated: electrocoagulation (EC) and
electro-Fenton (EF)
VFSM; LS; Catalytic thermal treatment, CuO
catalyst; 1 L reactor; BM; T = 100–140 ◦ C; CuO
mass loading in the range of 2–5 Kg/m3
Stillage wastewater; LS; EO process; Two
materials tested in the anode: graphite
particles and titanium sponge; cathode made
from Ti/RuO2
Anaerobically digested VFSM; LS;
coagulation/flocculation (CF) and
electrochemical oxidation (EO) treatment
Applying two complementary process: CF used
FeCl3 as coagulant followed by EO using
Ti/RuPb(40%)Ox anode and Ti/PtPd(10%)Ox
cathode
VFSM; PPS; Ultrafiltration (UF) and reverse
osmosis (RO) treatment
UF evaluated the pressure variation on
thin-film composite polyamide
Optimum conditions pH 10, T = 30 ◦ C, dose
of O3 = 0.58 g O3 /L mixed wastewater
arom = 90% and phen = 90% at 5 min of
contact
COD = 50–80%, maximum removal was
observed at 30 min
Optimum conditions COD = 74% at
[H2 O2 ]0 = 0.5 mol/L,
[H2 O2 ]0 /[Fe2+ ]0 = 15 mol/mol
Optimum conditions COD ≈ 89% at
[NaCl] = 4%, T = 42 ◦ C, pH 9.5, Q = 30 mL/min,
under these conditions was achieved
COD ≈ 89%.
[K] was reduced from 10 to 2.5 g/L
COD = 93.5% for PTDW
EC studies
Poor results COD = 14.3%
EF studies
COD = 92.6% y TOC = 88.7%
Optimum conditions COD = 60% at pH0 = 2;
T = 140 ◦ C y 3 Kg/m3 catalyst loading
Anode made from titanium sponge showed
best results acidic conditions (pH 1)
resulted in the increased oxidation of
organic pollutants
Additives H2 O2 and NaCl promoted high
COD = 89.62% and color = 92.24%
CF: Optimum conditions
COD = 84%; color and turbidity
removal ≈ 99% at pH 8.4; coagulant
concentrationof 20 g/L
EO: COD = 99%; color and turbidity removal
100%
RO process
COD = 96.8%; TDS = 97.9%
UF effectively separated the SS at pressure
10 atm
COD = 63.2%; SS = 96.5%
Dilution of vinasse with municipal wastewater 1
(1:10, v/v)
Dilution of vinasse with municipal wastewater 2
(1:10, v/v), aerobically pretreated
Used Ca(OH)2 as the precipitating agent in the 3
coagulation/flocculation stage
Wastewater was mixed with NaCL and then 4
fed to an electrolytic cell. Cl2 and other
oxidants were produced; they oxidized organic
pollutants to CO2 and water
Aim: to reduce the concentration of potassium 5
to prevent crystallization (as K2 SO4 )
EC tested at current density of 20 mA/cm2 with 6
0.2 M Na2 SO4 .
EF carried out at current density of 60 mA/cm2 ,
(supporting electrolyte), [H2 O2 ] = 60 g/L, pH 4
EF process was found to be very effective
Thermal catalysis used as pretreatment 7
process Initial pH0 had strong impact on ␩COD
The residue can be used as a fuel and the ash
can be blended with organic manure and used
in agriculture
Maximum current efficiency decreased with 8
operation time due to passivation of the
electrode surface
CF was sensitive to pH and coagulant 9
concentration
EO with anode of Ti/RuPb(40%)Ox effectively
removed the organic material, color and
turbidity
UF and RO processes successfully removed 10
color and other contaminants

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 efficiency
of polyphenols, arom , removal efficiency of aromatic, COT , removal efficiency of total organic carbon, TDS , removal efficiency of total dissolved solids, color , removal efficiency
of 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.
Chaudhari et al. (2007); 8. Piya-areetham et al. (2006); 9. Zayas et al. (2007); 10. Murthy and Chaudhari (2009).

(Gottschalk et al., 2000; Peña et al., 2003; Ried et al., 2007). Many
works have shown that the additional use of ultraviolet radiation,
hydrogen peroxide, iron oxide, or titanium dioxide and tin cata-
lysts increase the degradation efficiency due to the generation of
free hydroxyl radicals that produce organic radicals (R•) capable
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).
 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546
3.1.1. Ozone production
Ozone is an unconventional chemical reagent, in the sense that it
must be generated in situ during wastewater treatment. The ozone
production is based on the overall chemical reaction:
Electricity
3O2 −→ 2O3
This process must operate with extremely dry air, reasonably
free of impurities, or with pure oxygen. In this case, the ozone
production yield increases significantly (EPA, 1986).
In ozone production, air or pure oxygen is introduced into the
interior of electrical shock tubes. Potential differences of about
15–25 KV are applied, causing the breakdown of oxygen molecules
into reactive atoms of oxygen, which combine with new O2
molecules to form ozone (Sangave et al., 2007a). In order to reduce
the costs associated with the use of ozone it is necessary to optimize
the process efficiency. Thus, it is recommended stillage condition-
ing before ozonation (Coca et al., 2005; Rehman et al., 2006; Ried
et al., 2007).
3.1.2. Factors affecting the ozone oxidation efficiency
The oxidizing action of ozone is strongly dependent on pH. At
low pH values, ozone reacts with specific functional groups through
direct selective reactions. At high pH values, hydroxyl ions catalyze
the decomposition of ozone to produce free radicals, which may
have a higher oxidation potential than ozone itself (Beltrán et al.,
2001; Coca et al., 2005; Rehman et al., 2006). However, the stillage
alkalinity is mainly due to bicarbonate and carbonate ions (Durán-
de-Bazúa et al., 1991; Jiménez et al., 2006). Bicarbonate is a well
known scavenger of free radicals formed by the ozone breakdown.
Increased color and COD elimination have been observed after alka-
linity removal (Coca et al., 2005). These results are consistent with
the fact that the bicarbonate is an inhibitor of the oxidizing action
of hydroxyl radicals.
Ozone reactivity is also a function of temperature; thus, the
removal efficiency of color and COD increases with the process
temperature, up to a maximum of about 40 ◦ C (Coca et al., 2005).
However, lower temperatures (about 25 ◦ C) are preferred because
the operational costs of ozonation also increase with tempera-
ture.
There are several studies on vinasse ozonation, mostly in the
context of combined ozonation + biological treatment. Selected
results and references were already discussed in Section 2.2.1
and displayed in Table 7, whereas combined ozonation-anaerobic
digestion works are summarized in Section 2.1.5. Flow diagrams of
combined vinasse treatment based on anaerobic digestion followed
by ozonation post-treatment are outlined in Fig. 2. The left branch
in Fig. 2 is convenient when vinasse presents a low biodegradabil-
ity and/or it contains significant amounts of toxic and recalcitrant
compounds. A pre-ozonation step could effectively help in remov-
ing the latter in such a way that the pre-treated vinasse is more
amenable to the biological process, i.e., anaerobic digestion. On the
other hand, the right branch of the flow diagram could be recom-
mended when the vinasse exhibits a significant biodegradability.
The anaerobic digestion first step would remove most organic mat-
ter; the post-treatment with ligninolyitc fungi would be useful for
the removal of recalcitrant organic compounds that could not be
degraded removed by bacterial consortia.
3.1.3. Treatment of vinasses using ozone and ozone plus catalysts
Treatment of vinasses by ozonation has been performed in
order to oxidize recalcitrant compounds (like color pigments
melanoidins) or remove organic matter and make it more amenable
to biodegradation. Ozone has been widely used as a chemical pre-
treatment step and a variety of catalysts have been employed to
make more effective the oxidizing action of ozone.
539
Santos et al. (2003) used ozonation at acid and alkaline pH
in order to reduce phenolic compounds present in vinasses from
ethanol 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.
(1) Alfafara et al. (2000) investigated the chemical degradation of
melanoidins present in the distillery vinasses using ozone treat-
ment. Ozone had positive effects on the discoloration (efficiency
of 80%) and biodegradability improvement (40%) in 40 h contact
time. Yet, removal of COD was low (16%). A slight depolymeriza-
tion was observed from the decrease in molecular weight of organic
matter.
Sreethawong and Chavadej (2008) used ozonation in the
absence and presence of alumina balls as the support media to
be coated with the iron oxide catalyst in order to reduce the col-
ored compounds present in distillery wastewater from sugar cane
molasses (COD0 = 106.5 g/L and BOD0 = 31.6 g/L, vinasse diluted by
distilled water by 1:20). They observed that removal efficiencies of
COD and color increased with increasing either the input power of
the ozone generator or the ozone mass flow rate. In the presence of
the Fe2 O3 catalyst, ozone resulted more effective in reducing both
COD and color. The authors ascribed this to possible the forma-
tion and action of free hydroxyl radicals. Zeng et al. (2009) used O3
plus SnO2 catalyst in order to reduce color present in fermented
molasses (Color absorbance unit of 2.14 at 475 nm after 10 times
dilution, COD0 = 95.0 g/L and BOD0 = 22.0 g/L). Decolorization of this
wastewater was 60.24% when ozonated for 60 min in the presence
of SnO2 catalyst. In contrast, discoloration was just 43.04% with just
ozonation.
Lucas et al. (2010) reported the treatment of winery wastewa-
ter (COD0 = 4.65 g/L) in a pilot-scale, buble column photo oxidizing
reactor. At the natural pH 4.0 of the influent, the photolytic action
of UV-C radiation, O3 /UV, O3 /UV/H2 O2 on the COD removal was
low-to-moderate (12%, 21% and 35% respectively after 180 min).
In the treatment O3 /UV/H2 O2 at pH 10 they found a significant
improvement of COD removal efficiency (57%).
3.2. Fenton’s oxidation
3.2.1. Principles of the method
Oxidation with Fenton’s reagent (H2 O2 plus Fe2+ in acidic aque-
ous solution) is a method widely used for destruction of organic
compounds. It is based on the generation of free hydroxyl radicals
(• OH), which have a high oxidation potential (• OH/H2 O = +2.73 V).
The mechanism of oxidation of organic compounds by Fenton’s
reagent is very complex, but is thought to occur through the fol-
lowing stages (Beltran-de-Heredia et al., 2005b):
K1
H2 O2 + Fe2+ −→Fe3+ + OH− + • OH (2)
K2
P + H2 O2 −→Pox (3)
K3
P + • OH−→Pox (4)
K4
H2 O2 + OH• −→HO• 2 + H2 O (5)
K5
OH• + Fe2+ −→Fe3+ + OH− (6)
K6
Fe3+ + H2 O2 −→Fe2+ + H+ + HO• 2 (7)
K7
Fe3+ + HO• 2 −→O2 + Fe2+ + H+ (8)
On the other hand, under alkaline conditions Fe3+ forms Fe
(OH)3 which is a highly insoluble compound in equilibrium with
FeO (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 Raw
vinasses

Pre-treatment using ozone oxidation Anaerobic treatment (3)

ηCOD =24.8% , Ozonation time 2 h (1) ηCOD= 70 % (4)
ηphenols 50% Ozonation time 15 min(2)

Post-treatment using White-rot fungi

(vinasses diluted from anaerobic treatment
12.5% v/v) (3)
ηColor =73-90%
Anaerobic treatment (BLSR) (1) ηCOD=53.5-71.5%
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)

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 Raw
vinasses

Aerobic Ozone
Ozone treatment post-treatment
pretreatment ηCOD=53%
After 12 h of ηCOD= 79%
ηCOD= 27% aerobic ηcolour= 92%3
oxidation

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

Fig. 2. Flow diagrams of successful combined treatments of vinasses: (a) using anaerobic digestion combined with either ozonation or ligninolytic fungi; (b) using aerobic
treatment combined with ozonation.

this treatment can also reduce the phenolic compounds by adsorp-
tion on the ferric gel formed (Beltran-de-Heredia et al., 2005b).
It seems that the ultimate mechanism of removal of phenols has
contributions from direct oxidation and coagulation.
3.2.2. Treatment of vinasses using Fenton process
Vinasses from fermentation of sugar cane molasses
(COD0 = 12–39 g/L) was treated using Fenton and photo-Fenton
(80 min of UV radiation was applied) (Hadavifar et al., 2010).
The photo-Fenton exhibited the highest COD removal efficiencies
(range 18–97%) compared to Fenton alone (range 5–47%). Yang
et al. (2008) used Fenton reaction in order to remove organic
matter and color from pre-treated (aerobic process) wine vinasses
(COD and BOD after aerobic pre-treatment, 0.636 and 0.090 g/L,
respectively). Concentrations of 450 and 300 mg/L of FeSO4 and
H2 O2 , respectively, significantly reduced the values of COD, BOD
and color at 30 min of reaction time. The hydroxyl radicals could
oxidize most refractory organics present in the vinasses; some low
weight molecular organics remained after treatment.
Beltran-de-Heredia et al. (2005b) investigated the integrated
Fenton coagulation/flocculation treatment for the depuration of
wine distillery wastewater (COD0 = 15–16.5 g/L). They reported a
maximum COD removal efficiency 74% at 0.5 mol/L H2 O2 and a ratio
[H2 O2 ]:[Fe2+ ] = 15 mol/mol, 3 h of contact time.
3.2.3. Treatment of vinasses using electrochemical processes
Electrochemical treatment of pre-treated vinasses from alco-
hol distillery wastewater (COD0 = 4.985 g/L, TOC0 = 1.507 g/L) using
iron electrode with and without the presence of H2 O2 was investi-
gated (Yavuz, 2007). Nearly 93% of the COD was removed whereas
TOC removal efficiency was 88.7% in the electro-Fenton treatment
with the addition of 0.3 M NaSO4 and 60,000 mg/L H2 O2 at pH 4. A
specific energy consumption of 0.53 kWh/g CODremoved was regis-
tered at a current density of 60 mA/cm2 . Control experiments with
electrocoagulation alone were found ineffective.
Rincon et al. (2009) treated vinasses from ethanol distilleries
(COD0 = 21.4 g/L) using an electro-flotation/oxidation procces. Best
results achieved were a COD reductions ca. 58% using galva-
nized steel electrodes, high pH, current density 20 mA/cm2 and
60,000 mg/L H2 O2 .
4. Perspectives and conclusion
Mezcal vinasses are of concern due to their potential negative
environmental impact if discharged in an uncontrolled manner. The
vinasse composition is related to the treatment received by the
fermented 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 used
as 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 Enzymes
processes (38 to 40)

Composts from
Milling co-composting with
vinasse
(1,2,3,4)

Juice extraction Bagasse (fiber)

Dilution until 28-30
°Brix

Filtration Biomass Alternative

Fermentation
source of protein

Distillation Liquid

Bio-energies
Mezcal Vinasse

Anaerobic Bio-H2
digestion (33 to 37)

Composts from
co-composting with CH4 Production Microbial fuel Electricity
Bio-Products
agro-industrial waste (20 to 28) cells (29 to 32)
(1 to 4)

Polyphenolic antioxidants (18) Laccase from Alternative single cell

sugarcane wax (19) Aspergillus protein source; Candida
heteromorphus (11) (5); Spirulina maxima(6);
Laccase from Coriolus single cell protein(7);
versicolor and Funalia Hansenula(8); Rhizopus
trogii (12) microspores(9); Aspergillus
Laccase from Trametes awamori var. kawachi (10)
pubescens MB 89 (13);
Plant growth hormones
from Funalia trogii ATCC
200800 and T. versicolor
ATCC 200801(14);
Lignin peroxidase, Mn-
peroxidase Laccase
(15); Ligninases (16);
Cellulases (17)

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. Strong
and 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.
Hamoda and Kennedy (1986); 27. Borja et al. (1993b); 28. Lo and Liao (1986); 29. Mohanakrishna et al. (2010); 30. Poggi-Varaldo et al. (2009); 31. Vazquez-Larios et al. (2010);
32. Zhang et al. (2009); 33. Muñoz-Páez et al. (2011); 34. Valdez-Vazquez et al. (2006); 35. Espinoza-Escalante et al. (2009); 36. Lay et al. (2010); 37. Escamilla-Alvarado et al.
(2011); 38. Aguiar et al. (2010); 39. Singh et al. (2010); 40. Kahraman and Yesilada (2003).

distillate production, the composition of the vinasses obtained will
also vary, and consequently, their treatment’s results. Because of
the variation in concentration and diversity of vinasses’ compo-
nents, the results obtained after the treatment of these wastes could
be highly variable. The use of combined waste treatment methods
could deal with these variations.
For this reason, in the case of vinasses, we strongly recommend
the use of combined biotic and abiotic treatments. A plausible
scheme, although not a panacea, could consist of preliminary
ozonation followed by anaerobic digestion; a polishing stage for
the so treated vinasses with ligninolytic fungi or a final ozona-
tion treatment could be recommended depending on the particular
discharge regulations as it was shown in Fig. 2.
Available information on mezcal vinasses treatment is scarce.
Fortunately, there is an abundant body of research on treatment of
other 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 been
successfully applied, (iii) physico-chemical treatments such as Fen-
ton, electro-oxidation, oxidants and so on., which are now mostly
at lab scale stage, have demonstrated a significant removal of
542 V. Robles-González et al. / Journal of Biotechnology 157 (2012) 524–546
recalcitrant organic compounds, (iv) fungal pretreatment with
chemical treatment followed by oxidative (O3 ) or anaerobic diges-
tion, this combination seems to give attractive results, (v) vinasses
may be co-composted with solid organic wastes, particularly with
those from agricultural activities and agro-industry.
Vinasses treatments that could generate added-value by-
products and abate the costs of pollution control, such as bioenergy
and enzymes, are in distinct stages of development. Anaerobic
digestion of vinasses is attractive and already a commercial tech-
nology, with reported costs of 5000–25,000 D\m3 of the digester
or 0.52 D\m3 of treated vinasse (Moletta, 2005). Lucas et al. (2010)
reported a cost of 1.31 D\m3 using an AOP O3 /UV/H2 O2 for a winery
wastewater (TOC0 of 1.254 g/L) in a pilot scale bubble column for a
contact time of 150 min. The wastewater was extremely diluted, so,
the costs for treating more concentrated vinasses would probably
be one order of magnitude to 30 fold superior to the reported cost.
On the other hand, unicellular protein, biohydrogen and enzyme
production from vinasses could be promising ways to improve the
economic feasibility of vinasses treatment, although they are in
the earlier stages of research and yields and costs issues should
be addressed before future commercial application. As preliminary
information that could be important for feasibility studies, current
prices of pure hydrogen for fuel cells, enzymes cellulase and lac-
case, and biomass/protein are US$ 440/kg, US$ 12/kg (industrial
grade) and US$ 206–2000/g (purified enzyme), and US$ 1.5–3.0
biomass/kg, respectively (Infra de México; Enmex; Sigma–Aldrich;
Jena Bioscience; Tianjin Shareglory Chemical Industrial Co., Ltd.;
Shanghai Genon Biotech Co., Ltd.).
The co-composting of vinasse with agricultural and agro-
industrial solid wastes, although attractive and feasible, is not
commonly practiced. Yet, it is worth investigating and implement-
ing because it can provide a low-cost treatment technology suitable
for rural regions in underdeveloped countries where more sophis-
ticated technologies are difficult to adopt, due to high costs and
requirements of skilled personnel. Furthermore, the final product
of co-composting, i.e., soil amenders, would be very valuable for
improving the quality of soils, increasing crop yields, and in gen-
eral to achieve sustainability of agricultural practices in the above
mentioned rural regions that very often are semi-arid.
Integration of several processes revised in this work as well as
others in a biorefinery approach could lead to maximization of the
resources-from-vinasse. A possible biorefinery setup for reclaiming
mezcal vinasses and obtaining a variety of added-value products is
shown in Fig. 3. This approach is based on the Principle of Cascading
and production of bioenergy as gas biofuels. Other arrangements
are possible by integrating generation of liquid biofuels (ethanol,
butanol, biodiesel). After the stages of agave conditioning and pre-
processing, agave bagasse is generated as a by-product from the
separation from the juice. Bagasse could be used as a substrate
of solid fermentation processes with strains such as Trichoderma
reseei and ligninolytic fungi in order to produce enzymes of comer-
cial interest (cellulases, laccase, ligninoperoxidase, etc.). Also, soil
amenders could be obtained by co-composting of agave bagasse
with mezcal vinasse. So, the streams of enzymes and soil amender
from this branch could join the enzymes and soil amenders pro-
duced from the vinasses branch.
Raw vinasses can be filtered to give two main streams: vinasses
and solids rich en yeast and microbial biomass. The latter can
be processed to generate an alternative protein source for ani-
mal feed and other uses. Filtered vinasses could be subjected to
a variety of biotechnological processes in order to give diverse
added-value bioproducts (Fig. 3): special bioproducts (Díaz et al.,
2011; Nuissier et al., 2008), enzymes (Acharya et al., 2010; Ferreira
et al., 2010; Kahraman and Yesilada, 2001; Pant and Adholeya,
2007b; Singh et al., 2010; Strong and Burgess, 2007), protein (yeast,
algal, bacterial, fungal) (Barrocal et al., 2010; Durán-de-Bazúa et al.,
1991; Morimura et al., 1994; Nitayavardhana and Khanal, 2010;
Tauk, 1982) and soil amenders by co-composting vinasses with
agro-industrial and other wastes (Díaz et al., 2002a,b; Madejón
et al., 2001a,b). Effluents from these fermentation processes, if
not exhausted, could be fed to the Bioenergy branch of the Biore-
finery (not shown in Fig. 3). In parallel, filtered vinasses could
be 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; Hamoda
and Kennedy, 1986; Jiménez et al., 2003; Lay et al., 2010; Lo and
Liao, 1986; Moletta, 2005; Muñoz-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 with
AOP or biological processes before discharge or reuse in irrigation
(not shown in Fig. 3) in such a way to close the environmental
circle. Crucial areas for further development and ensuring prof-
itability of the biorefinery approach are, among others: hydrogen
yield improvement and hydrogen purification and storage, scale-up
of enzyme production from wastes, scale-up of processes yielding
special bioproducts, and boosting performance of microbial fuel
cells 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) granted
a graduate fellowship to one of the authors (VR-G). JG-M and NR-S
acknowledge COFAA-IPN for support.
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