Bioresource Technology 220 (2016) 609–614

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Ethanol fermentation characteristics of recycled water by Saccharomyces

cerevisiae in an integrated ethanol-methane fermentation process
Xinchao Yang a,b, Ke Wang a, Huijun Wang a, Jianhua Zhang a, Zhonggui Mao a,⇑
a
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
b
School of Biological Science and Technology, University of Jinan, Jinan 250022, China

h i g h l i g h t s

Fermentation time was shortened by 40% when mixed water was used as process water.

The maximal ethanol production rate increased from 1.07 g/L/h to 2.01 g/L/h.

The intracellular trehalose per DCW in mixed water was higher than that of in tap water.

This process could minimize wastewater discharge and water consumption.

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

Article history: An process of integrated ethanol-methane fermentation with improved economics has been studied
Received 4 June 2016 extensively in recent years, where the process water used for a subsequent fermentation of carbohydrate
Received in revised form 9 August 2016 biomass is recycled. This paper presents a systematic study of the ethanol fermentation characteristics of
Accepted 10 August 2016
recycled process water. Compared with tap water, fermentation time was shortened by 40% when mixed
Available online 12 August 2016
water was employed. However, while the maximal ethanol production rate increased from 1.07 g/L/h to
2.01 g/L/h, ethanol production was not enhanced. Cell number rose from 0.6  108 per mL in tap water to
Keywords:
1.6  108 per mL in mixed water but although biomass increased, cell morphology was not affected.
Ethanol
Saccharomyces cerevisiae
Furthermore, the use of mixed water increased the glycerol yield but decreased that of acetic acid, and
Recycled water the final pH with mixed water was higher than when using tap water.
Anaerobic digestion Ó 2016 Elsevier Ltd. All rights reserved.
Thin stillage

1. Introduction (Saha et al., 2005). Therefore substantial increases in ethanol pro-

The growing need to expand the use of renewable energy
sources in a sustainable manner has boosted the production of bio-
fuels worldwide (Moraes et al., 2015). However, biofuel production
processes generate large volumes of wastewater, especially stil-
lage, which is the aqueous by-product from the distillation of etha-
nol following fermentation of carbohydrates (Wilkie et al., 2000).
The production of ethanol from biomass, whether from sugar
crops, starch crops, dairy products, or cellulosic materials, results
in the concurrent production of stillage which has a considerable
pollution potential. Among the available feedstocks, cassava is of
particular interest due to its low cost, high productivity, and lack
of competition for arable land. In the conventional cassava ethanol
fermentation process, massive amounts of freshwater are con-
sumed and 8–15 L of distillery waste is produced per L ethanol
⇑ Corresponding author.
E-mail address: maozg@jiangnan.edu.cn (Z. Mao).
duction will also require effective solutions for stillage
management.
In the conventional ethanol fermentation process, distillery
waste is usually treated by solid-liquid separation followed by
anaerobic-aerobic digestion, and the effluent from this is further
treated using physical or chemical methods to achieve the national
discharge standard (Jördening and Winter, 2005; Kim et al., 1997).
Anaerobic digestion of corn-ethanol thin stillage and its biogas
generation potential have been evaluated in laboratory studies
since the 1980s, as an alternative approach to recover more energy
from the corn as well as treating the stillage (Lee et al., 2011). After
anaerobic digestion, the treated stillage could potentially be reused
within the plant as process water. Recycling of digested thin stil-
lage would not only eliminate the energy-intensive evaporator
but also reduce the fresh water requirements for ethanol produc-
tion (Alkan-Ozkaynak and Karthikeyan, 2011). Anaerobic digestion
of thin stillage offers not only the benefit of reduced energy input
for co-product drying, but also enables energy recovery through

http://dx.doi.org/10.1016/j.biortech.2016.08.040
0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
610 X. Yang et al. / Bioresource Technology 220 (2016) 609–614
the production of methane, with a prospective improvement in
net-energy balance for ethanol production.
In recent years, many researchers have studied the potential for
the use of thin stillage as dilution water to minimize the use of
fresh water for substrate suspension during fermentation. Sho-
jaosadati and coworkers studied modifications of the conventional
ethanol fermentation process using biomass and stillage recycle,
where recycle of 15–70% of stillage from a previous fermentation
was tested successfully, permitting a 13–47% decrease in water
consumption and 13–47% reduction of stillage volume
(Shojaosadati et al., 1996). For the corn-ethanol process, up to half
of the thin stillage is directly recycled as process water, and the
remainder is evaporated to produce a condensed, soluble, syrup
containing 30% solids, requiring a substantial proportion of plant
energy inputs (van Leeuwen et al., 2012). It would be much more
advantageous to use the nutrients in thin stillage for the cultiva-
tion of beneficial microbes or microbial products (Rasmussen
et al., 2014). A portion of thin stillage may be used as make-up
water for upstream processes (cooking and fermentation), which
will further help to reduce the volume of stillage produced, as well
as reduce consumption of water, steam and some chemicals. How-
ever, stillage strength will actually rise with its increased use in
backset water, as the chemical oxygen demand produced will not
change, while accumulation of fermentation products and non-
fermentable sugars can inhibit the fermentation process (Sun
et al., 2013). Typically, between 15 and 30% of the thin stillage is
recycled as backset for upstream processes, although a maximum
Fig. 1. Flowchart of the integrated ethanol–methane fermentation process.
recycle of 50% may be possible (Bothast and Schlicher, 2004;
Wilkie et al., 2000).
The integrated method of ethanol-methane fermentation has
been studied in recent years. As shown in Fig. 1, some of the
organic matter within thin stillage is converted to methane by
anaerobic digestion. After further treatment the digestate is reused
as part of the process water for ethanol fermentation, forming a
closed-circuit circulation process. The overall technology reduced
energy consumption and water consumption, reducing waste
water emissions. Zhang and co-works (2010b) studied a full recy-
cling process incorporating two-stage anaerobic treatment of dis-
tillery wastewater from bioethanol production from cassava.
Furthermore, Zhang et al. (2010a) studied the effect on ethanol
production of utilizing waste anaerobic digestion distillate effluent
in an integrated ethanol-methane fermentation process. Wang and
co-workers (2013) established and assessed a novel cleaner pro-
duction process for corn-grain fuel ethanol. Subsequently, Wang
et al. (2014a) studied the reuse of a mixture of anaerobic digestion
effluent and thin stillage for cassava ethanol production. In this
novel integrated process, a portion of thin stillage is mixed with
the effluent from anaerobic digestion of residual thin stillage, pro-
ducing a ‘mixed’ water which is then reused as process water for
the following ethanol fermentation batch (Wang et al., 2014a). This
novel integrated process reduced both energy consumption and
wastewater production.
However, the ethanol fermentation characteristics of mixed
water were not studied systematically. The major goal of this study
 X. Yang et al. / Bioresource Technology 220 (2016) 609–614 611

was to determine the ethanol fermentation characteristics of 2.5. Ethanol fermentation

mixed water to test its suitability for reuse and assess any eco-
nomic benefits arising.
2. Materials and methods
2.1. Microorganism
A commercial strain of S. cerevisiae TG1348 for ethanol produc-
tion was obtained from Henan Tian Guan Co., Ltd., (Henan,
Nanyang, China).
2.2. Seed medium
One loopful of S. cerevisiae was inoculated into a 500 mL
Erlenmeyer flask holding 200 mL of a solution containing (g/L):
glucose 20, yeast extract 8.5, (NH4)2SO4 1.3, MgSO47H2O 0.1 and
CaCl22H2O 0.06. The flask was incubated on a shaker (200 rpm)
at 30 °C for 18 h to produce seed medium.
2.3. Operation of the process
Tap water was used for the first ethanol fermentation batch. At
the end of the fermentation the resulting ‘beer’ was distilled. Part
of the residual liquor was centrifuged (4000g for 30 min) to
obtain the ‘thin stillage’ supernatant which was stored at 20 °C
before use. The residual liquor was fed into a two-stage anaerobic
digester. Digestate (60%) was combined with thin stillage (40%)
and employed as ‘mixed’ process water for the following ethanol
fermentation batch. Table 1 shows the characteristics of the differ-
ent process waters (thin stillage, digestate, mixed water, tap
water).
2.4. Fermentation medium
Fermentation medium was composed of (g/L): glucose 100,
MgSO47H2O 0.1, CaCl22H2O 0.06, and urea 0.5, dissolved in the
appropriate process water. Urea was sterilized separately to avoid
Maillard reaction. The pH was adjusted to 5.0 using 30% (w/w)
H2SO4 or 10% (w/v) NaOH. Thin stillage and digestate were pro-
vided by Wujiang Yongxiang Alcohol Manufacturing Co. Ltd.
(Wujiang, Jiangsu, China). Tap water was used as a process water
control.
Triplicate fermentations were carried out in 250 mL flasks con-
taining 135 mL of medium. Urea was added before inoculating. A
10% (v/v) inoculum of seed medium was added to each flask. All
fermentations were carried out at 30 °C without shaking.
2.6. Analysis
Glucose, ethanol, glycerol and acetic acid concentrations in fer-
mentation media were determined by high-performance liquid
chromatography (HPLC). Samples were centrifuged (10,000g for
10 min) and the supernatant passed through a 0.20 lm filter prior
to analysis. Standards and prepared samples (20 lL) were injected
into a Bio-Rad HPX-87H Aminex ion exclusion column. The column
was operated at 65 °C and sulfuric acid (0.005 mol/L) was used as
mobile phase at a flow rate of 0.6 mL/min. A refractive index detec-
tor (Shodex RI-101, Shodex, Tokyo, Japan) was employed. Data
were processed using Chromeleon software (Dionex, CA, USA).
For mixed water, yeast cells were counted using a hemocytometer.
Cell number was converted to dry cell weight (DCW) using a factor
of 2.5  1011 g dry mass per cell (Devantier et al., 2005). For tap
water (control), cell growth was determined by absorbance at
600 nm. Note that, lmax ¼ dx  1 . X (X: mass concentration of cells,
dt X
t: time).
2.7. Trehalose content
Trehalose was extracted from cells that were chilled and
washed with cold 0.5 M trichloroacetic acid, and the trehalose con-
centration estimated using the anthrone method (Wang et al.,
2014b). Samples for dry weight analysis were washed with dis-
tilled water and dried at 80 °C for 12 h.
3. Results and discussion
3.1. Characteristics of different process water
Before fermentation, there was an initial content of glycerol,
acetic acid and lactic acid in the mixed process water, as shown
in Table 1. Compared with tap water, mixed water is rich in amino
acids. Its total metal ion content was a maximum of 119.3 mmol/L,
whereas tap water contained negligible amounts.

Table 1
Comparison of different process water in terms of pH, glycerol, acetic acid, lactic acid, metal ion and amino acid.

Parameter and unit Thin stillage Digestate Mixed watera Tap water
pH 4.30 7.90 6.46 7.42
Glycerol (g/l) 5.36 NDb 2.14 ND
Acetic acid (g/l) 0.51 0.19 0.32 ND
Lactic acid (g/L) 5.62 ND 2.25 ND
Na+ (mmol/L) 55.89 94.35 78.97 1.42
K+ (mmol/L) 33.27 21.14 26.00 0.09
Mg2+ (mmol/L) 7.24 8.24 7.84 0.44
Ca2+ (mmol/L) 3.87 1.13 2.23 0.54
Al3+ (mmol/L) 3.85 2.11 2.81 N.D.
Fe3+ (mmol/L) 1.40 0.11 0.63 0.13
Cu2+ (mmol/L) 0.30 0.01 0.13 0.03
Zn2+ (mmol/L) 0.04 0.01 0.02 N.D.
Mn2+ (mmol/L) 0.15 0.01 0.07 0.005
Li+ (mmol/L) 0.98 0.32 0.58 0.23
Phe (mg/L) ND 74.2 (Wang et al., 2013) 44.52 ND
Asp (mg/L) 0.02 28.20 (Wang et al., 2013) 16.93 ND
Gly (mg/L) 0.02 17.00 (Wang et al., 2013) 10.21 ND
Pro (mg/L) 0.05 12.7 (Wang et al., 2013) 7.64 ND
Glu (mg/L) 0.09 4.51 (Wang et al., 2013) 2.74 ND
a
Mixed water of thin stillage (40%) and digestion (60%).
b
ND: not detectable.
612 X. Yang et al. / Bioresource Technology 220 (2016) 609–614
3.2. Performance of ethanol fermentation
A shorter fermentation time was achieved when mixed water
was used as the process water (Fig. 2); glucose was depleted after
30 h and 50 h respectively with mixed water and tap water
(Fig. 2a). Thus fermentation time was reduced by 40% when using
mixed water compared with tap water. Although mixed water
increased the fermentation rate, ethanol production was not
increased (Fig. 2a, b). In addition, using mixed water gave rise to
raised glycerol levels (Fig. 2c), but reduced acetic acid production
(Fig. 2d).
The results shown in Fig. 2 suggest that other components of
mixed water could accelerate the depletion of glucose. This obser-
vation can readily be explained. Growth of yeast is faster and the
biomass produced is greater in media containing complex ingredi-
ents when compared to minimal medium (Narendranath et al.,
2001). During ethanol fermentation in the integrated process,
methanogenic bacteria present in the digestate component of
mixed water are decomposed and release intracellular substances.
Nitrogenous compounds including protein give rise to ammonium
ion which has previously been found at a relatively high concentra-
tion in the two-stage anaerobic digestate derived from thin stillage
(759 mg/L on average; Wang et al., 2014a).
Ammonium ion in mixed water could promote yeast growth
and hence increase the fermentation rate (Wang et al., 2012). In
our study, mixed water contained high levels of phenylalanine,
aspartic acid and glycine (Table 1). Trace amounts of amino acids
were found by other workers in all stillages tested (Wilkie et al.,
2000). These amino acids may promote rapid yeast growth during
the log phase. Improved growth in complex media has been
thought to be entirely due to the availability of greater amounts
Fig. 2. Representative profiles of metabolism of S. cerevisiae with mixed water (d) or tap water (j). Data presented are the average of three independent cultivations and
error bars represent standard deviations.
of nutrients, although it is now recognized that some components
of complex media play non-nutritional roles in promoting the
growth and survival of yeast (Thomas et al., 2002). Buffering capac-
ity was created during the anaerobic digestion process in mixed
water: the main source is proteins, which when hydrolyzed release
ammonia and salts of weak organic acids (Foxon et al., 2010),
resulting in a small increase in pH at the end of fermentation com-
pared with tap water.
Yeast cells have evolved defense strategies against several types
of stress damage. Factors such as trehalose and heat shock pro-
teins, which stabilize or repair denatured proteins, were increased
in stressed yeast cells (Zhao and Bai, 2009). Trehalose is a non-
reducing disaccharide consisting of two glucose monomers (a-D-
glucopyranosyl-1,1-a-D-glucopyranoside; Gibney et al., 2015).
Because trehalose is relatively inert and very stable, it is thought
to act as a protectant for subcellular structures against osmotic
and other stress (Eleutherio et al., 2014; Jain and Roy, 2009). Mixed
process water contains complex constituents, which result in
osmotic and other stress for cells. This is the most likely explana-
tion for the increase in intracellular trehalose when mixed water
was employed.
Acetic acid is another factor promoting ethanol fermentation in
mixed water, stimulating ethanol production in some circum-
stances (Mohammad J. Taherzadeh, 1997; Thomas et al., 2002;
Zhao et al., 2009). It was found that acetic acid production was
lower when using mixed water (which itself contains acetic acid)
as process water (Table 2). However, the mechanism whereby
acetic acid is converted into ethanol requires clarification. Under
anaerobic conditions, glycerol generated by yeast reoxidizes
NADH, produced as a secondary fermentation product, to NAD+
(Guo et al., 2009) and therefore glycerol production increases with
 X. Yang et al. / Bioresource Technology 220 (2016) 609–614
Table 2
Growth rates and product yields for anaerobic growth of S. cerevisiae on glucose with
different water. Data presented are the average of three independent cultivations.
Parameter and unit Mixed watera
1
Maximal specific growth rate (h ) 0.77
Ethanol yield (mol/mol of glucose) 1.82
Glycerol yield (mmol/mol of glucose) 97.78
Acetic acid yield (mmol/mol of glucose) 8.72
Maximal ethanol production rate (g/L/h) 2.01
Maximal trehalose yield (g/g of DCW) 0.15
a
Mixture of thin stillage (40%) and digestion (60%).
biomass. Thus the accelerated glucose depletion in mixed water
shown in Fig. 2c could be explained by the enhanced rate of cell
production seen in Fig. 3a.
Cell number in mixed water increased up to 1.6  108 per mL at
the end of log phase, whereas the value was 0.6  108 per mL in tap
water (Fig. 3a). The difference in biomass reflected different glyc-
erol yield production. For both process waters, the final pH was
reached after 18 h of fermentation and remained unchanged there-
after. However, the final pH with mixed water was higher than
with tap water (Fig. 3b). Lactic acid production was higher when
using mixed water as process water (Fig. 3c). Intracellular tre-
halose increased gradually over 40 h for mixed water, while in
mixed water intracellular trehalose per DCW was 4–6-fold higher
than in tap water (Fig. 3d).
The higher rate of conversion of glucose (Fig. 2) was also
reflected in the higher maximum specific growth rate (lmax)
(Table 2) in mixed water, with a lmax of 0.77 h1 compared to
0.45 h1 in tap water. When glucose was depleted, ethanol and
glycerol yields per glucose molecule were consistent with the etha-
nol and glycerol production observed. The use of mixed water
Fig. 3. Representative profiles of metabolism of S. cerevisiae with mixed water (d) or tap water (j). Data presented are the average of three independent cultivations and
error bars represent standard deviations.
613
increased the glycerol yield but decreased acetic acid yield, while
the maximal ethanol production rate of 2.01 g/L/h was higher than
the 1.07 g/L/h found in tap water, reflecting faster glucose
Tap water metabolism.
0.45 In traditional fermentation processes, the whole stillage is cen-
1.89 trifuged to separate the liquid fraction, or thin stillage, from the
65.29 solid fraction, or the wet distillers’ grains. It has been reported that
13.85
1.07
acetic acid increased the levels of ethanol production and acceler-
0.02 ated the fermentation rate (Abbott and Ingledew, 2004). Our
results also suggest that acetic acid could promote the production
of ethanol.
3.3. Microscopic images of mixed water fermentation
Fig. 4 (Supplementary Data) shows electron microscopy images
of S. cerevisiae in the middle of logarithmic phase growing on the
medium prepared with either tap water or mixed water. Fig. 4a
shows the normal cell morphological structure when grown in
tap water. Fig. 4b showed the cells morphological structure when
grown in mixed water. Compared with tap water, cell number
increased, as counted using a hemocytometer, when mixed water
was used as process water. In addition, cellular morphology and
germination capacity were not affected when mixed water was
employed.
Our results show that cell grown is faster in mixed water than
in tap water. Greater amount of nutrients are present in mixed
water. Appreciable amounts of amino acids were present in the
thin stillage. These amino acids provide rich nutrition for the
growth of yeast, resulting in an increased cell number when mixed
water was used as process water compared to tap water. In this
process, ethanol is the required end product of fermentation by
Saccharomyces cerevisiae. During fermentation some weak acids
614 X. Yang et al. / Bioresource Technology 220 (2016) 609–614
such as acetic, butyric and pyruvic acids, produced by yeast meta-
bolism, may accumulate in the growth medium. This has been con-
sidered to enhance ethanol toxicity, resulting in inhibition of yeast
growth and of fermentation (Gibson et al., 2007). However, our
previous data showed that S. cerevisiae cells exposed simultane-
ously to toxic concentrations of ethanol and low concentrations
of acetic acid displayed higher survival than cells treated only with
ethanol. These results indicate that acetic acid induces a cellular
response which provides protection against the cytotoxic effect
of ethanol.
4. Conclusions
In this paper, we studied ethanol fermentation characteristics of
reusing mixed water composed of anaerobic digestion and thin
stillage by Saccharomyces cerevisiae. The advantages of the inte-
grated ethanol-methane fermentation process are the reduction
of energy consumption and water consumption and expected to
achieve the goal of ‘‘zero wastewater”. In addition, fermentation
time was shortened by reusing mixed water. Moreover, the reusing
of mixed water process provides reference for other submerged
fermentation industries to manage the wastewater.
Acknowledgements
This study was financially supported by the following projects:
1. Shandong Province Natural Science Foundation of University
Joint Special-Project – China granted No. ZR2013CL007. 2. Indepen-
dent Research Program of Jiangnan University Youth Fund – China
granted No. 1012050205150710. 3. Suzhou Science and Technol-
ogy Support Project – China granted No. SS201412. 4. National Nat-
ural Science Foundation of China – China granted No. 21506075.
5. The Fundamental Research Funds for the Central Universities –
China granted No. JUSRP51504.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2016.08.
040.
References
Abbott, D.A., Ingledew, W.M., 2004. Buffering capacity of whole corn mash alters
concentrations of organic acids required to inhibit growth of Saccharomyces
cerevisiae and ethanol production. Biotechnol. Lett. 26, 1313–1316.
Alkan-Ozkaynak, A., Karthikeyan, K.G., 2011. Anaerobic digestion of thin stillage for
energy recovery and water reuse in corn-ethanol plants. Bioresour. Technol.
102, 9891–9896.
Bothast, R.J., Schlicher, M.A., 2004. Biotechnological processes for conversion of corn
into ethanol. Appl. Microbiol. Biotechnol. 67, 19–25.
Devantier, R., Pedersen, S., Olsson, L., 2005. Characterization of very high gravity
ethanol fermentation of corn mash. Effect of glucoamylase dosage, pre-
saccharification and yeast strain. Appl. Microbiol. Biotechnol. 68, 622–629.
Eleutherio, E., Panek, A., Mesquita, J.F., Trevisol, E., Magalhães, R., 2014. Revisiting
yeast trehalose metabolism. Curr. Genet. 61, 263–274.
Foxon, K.M., Brouckaert, C.J., Buckley, C.A., 2010. Anaerobic digestion of domestic
wastewater: the role of alkalinity in the rate and extent of digestion. Water
Pract. Technol. 5, 1–21.
Gibney, P.A., Schieler, A., Chen, J.C., Rabinowitz, J.D., Botstein, D., 2015.
Characterizing the in vivo role of trehalose in Saccharomyces cerevisiae using
the AGT1 transporter. Proc. Natl. Acad. Sci. U.S.A. 112, 6116–6121.
Gibson, B.R., Lawrence, S.J., Leclaire, J.P., Powell, C.D., Smart, K.A., 2007. Yeast
responses to stresses associated with industrial brewery handling. FEMS
Microbiol. Rev. 31 (5), 535–569.
Guo, Z.P., Zhang, L., Ding, Z.Y., Wang, Z.X., Shi, G.Y., 2009. Interruption of glycerol
pathway in industrial alcoholic yeasts to improve the ethanol production. Appl.
Microbiol. Biotechnol. 82, 287–292.
Jain, N.K., Roy, I., 2009. Effect of trehalose on protein structure. Protein Sci. 18, 24–
36.
Jördening, H.J., Winter, J., 2005. Environmental Biotechnology: Concept and
Application. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany.
Kim, J.S., Kim, B.G., Lee, C.H., Kim, S.W., 1997. Development of clean technology in
alcohol fermentation industry. J. Clean. Prod. 5, 263–267.
Lee, P.-H., Bae, J., Kim, J., Chen, W.-H., 2011. Mesophilic anaerobic digestion of corn
thin stillage: a technical and energetic assessment of the corn-to-ethanol
industry integrated with anaerobic digestion. J. Chem. Technol. Biotechnol. 86,
1514–1520.
Mohammad, J., Taherzadeh, C.N.A.G.L., 1997. Acetic acid – friend or foe in anaerobic
batch conversion of glucose to ethanol by Saccharomyces cerevisiae? Chem.
Eng. Sci. 52, 2653–2659.
Moraes, B.S., Zaiat, M., Bonomi, A., 2015. Anaerobic digestion of vinasse from
sugarcane ethanol production in Brazil: challenges and perspectives. Renewable
Sustainable Energy Rev. 44, 888–903.
Narendranath, N.V., Thomas, K.C., Ingledew, M.W., 2001. Effect of acetic acid and
lactic acid on growth of Saccharomyces cerevisiae in a minimal medium. J. Ind.
Microbiol. Biotechnol. 26, 171–177.
Rasmussen, M.L., Khanal, S.K., Pometto 3rd, A.L., van Leeuwen, J.H., 2014. Water
reclamation and value-added animal feed from corn-ethanol stillage by fungal
processing. Bioresour. Technol. 151, 284–290.
Saha, N., Balakrishnan, M., Batra, V., 2005. Improving industrial water use: case
study for an Indian distillery. Resour. Conserv. Recycl. 43, 163–174.
Shojaosadati, S.A., Sanaei, H.R., Fatemi, S.M., 1996. The use of biomass and stillage
recycle in conventional ethanol fermentation. J. Chem. Technol. Biotechnol. 67,
362–366.
Sun, F., Mao, Z., Tang, L., Zhang, H., Zhang, C., Zhai, F., Zhang, J., 2013. Exploration of
water-recycled cassava bioethanol production integrated with anaerobic
digestion treatment. Afr. J. Biotechnol. 9, 6182–6190.
Thomas, K.C., Hynes, S.H., Ingledew, W.M., 2002. Influence of medium buffering
capacity on inhibition of Saccharomyces cerevisiae growth by acetic and lactic
acids. Appl. Environ. Microbiol. 68, 1616–1623.
van Leeuwen, J., Rasmussen, M.L., Sankaran, S., Koza, C.R., Erickson, D.T., Mitra, D.,
Jin, B., 2012. Fungal Treatment of Crop Processing Wastewaters with Value-
Added Co-Products, pp. 13–44.
Wang, K., Mao, Z., Zhang, C., Zhang, J., Zhang, H., Tang, L., 2012. Influence of nitrogen
sources on ethanol fermentation in an integrated ethanol-methane
fermentation system. Bioresour. Technol. 120, 206–211.
Wang, K., Zhang, J., Tang, L., Zhang, H., Zhang, G., Yang, X., Liu, P., Mao, Z., 2013.
Establishment and assessment of a novel cleaner production process of corn
grain fuel ethanol. Bioresour. Technol. 148, 453–460.
Wang, K., Zhang, J.-H., Liu, P., Cao, H.-S., Mao, Z.-G., 2014a. Reusing a mixture of
anaerobic digestion effluent and thin stillage for cassava ethanol production. J.
Clean. Prod. 75, 57–63.
Wang, P.M., Zheng, D.Q., Chi, X.Q., Li, O., Qian, C.D., Liu, T.Z., Zhang, X.Y., Du, F.G.,
Sun, P.Y., Qu, A.M., Wu, X.C., 2014b. Relationship of trehalose accumulation with
ethanol fermentation in industrial Saccharomyces cerevisiae yeast strains.
Bioresour. Technol. 152, 371–376.
Wilkie, A.C., Riedesel, K.J., Owens, J.M., 2000. Stillage characterization and anaerobic
treatment of ethanol stillage from conventional and cellulosic feedstocks.
Biomass Bioenergy 19, 63–102.
Zhang, C.M., Mao, Z.G., Wang, X., Zhang, J.H., Sun, F.B., Tang, L., Zhang, H.J., 2010a.
Effective ethanol production by reutilizing waste distillage anaerobic digestion
effluent in an integrated fermentation process coupled with both ethanol and
methane fermentations. Bioprocess Biosyst. Eng. 33, 1067–1075.
Zhang, Q.H., Lu, X., Tang, L., Mao, Z.G., Zhang, J.H., Zhang, H.J., Sun, F.B., 2010b. A
novel full recycling process through two-stage anaerobic treatment of distillery
wastewater for bioethanol production from cassava. J. Hazard. Mater. 179, 635–
641.
Zhao, X.Q., Bai, F.W., 2009. Mechanisms of yeast stress tolerance and its
manipulation for efficient fuel ethanol production. J. Biotechnol. 144 (1), 23–30.
Zhao, R., Bean, S.R., Crozier-Dodson, B.A., Fung, D.Y., Wang, D., 2009. Application of
acetate buffer in pH adjustment of sorghum mash and its influence on fuel
ethanol fermentation. J. Ind. Microbiol. Biotechnol. 36, 75–85.