Anaerobic digestion of distillery spent wash: Inuence of enzymatic

pre-treatment of intact yeast cells

P. Mallick, J.C. Akunna
*
, G.M. Walker
School of Contemporary Sciences, University of Abertay Dundee, Dundee DD1 1HG, Scotland, UK
a r t i c l e i n f o
Article history:
Received 24 July 2009
Received in revised form 26 September
2009
Accepted 30 September 2009
Available online 1 November 2009
Keywords:
Anaerobic digestion
Distillery spent wash centrate
Distillery pot ale
Enzymatic hydrolysis
Yeast cell lysis
a b s t r a c t
The potential benets of enzymatic digestion of intact yeast cells on anaerobic digestion of Scotch whisky
distillery spent wash and pot ale were investigated. Various yeast cell wall hydrolytic enzymes were
studied based on their effect on dissolution of cell wall glucan and mannoprotein. The synergistic activity
of beta-glucanase and protease showed greater than 90% yeast cell digestion at 37 C in 24 h. The widely-
used industrial enzyme papain showed 95% yeast cell digestion in spent wash at 1% enzyme concentra-
tion within 22 h at 50 C. Anaerobic digestion of pot ale residues containing intact yeast cells pre-treated
with lytic enzymes showed COD reductions of 87%, compared with only 13% without enzymes. Similar
results were observed with distillery spent wash centrate. The hydrolysis of intact yeast cells in distillery
liquid residues was found to be a rate-limiting step in anaerobic treatment of such residues.
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Typical liquid residues remaining after Scotch whisky distilla-
tion include pot ale and spent lees from wash and spirit stills,
respectively (Goodwin and Stuart, 1994). It is estimated that 8.5
11.5 l of pot ale is discharged per 1 l of pure alcohol in the case
of malt whisky and 1621 l of spent wash per 1 l of grain whisky
(Tokuda et al., 1998). Hence, disposing the liquid waste appropri-
ately is a major concern for distilleries. Distillery residues tend to
have a very strong inuence on receiving waters, due to their high
chemical oxygen demand (COD). In addition to this, the presence of
putrefying organics and sulphur compounds causes the formation
of obnoxious odour. The estimated LD
50
dose for spent wash was
found to be 0.5% using bio-toxicity tests on fresh water sh Cypri-
nus carpio var, communis (Pant and Adholeya, 2007). It is therefore
apparent that appropriate treatment and disposal methods for dis-
tillery residues are urgently required.
Several procedures for treating distillery liquid residues have
been examined including chemical and/or biological processes
and their common feature is relatively high cost of operation and
creation of other hazardous by-products and pollutants. Aerobic
treatment of pot ale is associated with problems such as high acid-
ity, high oxygen demand, sludge bulking and high cost of operation
in terms of energy consumption (Uzal et al., 2003). Although anaer-
obic treatment processes are relatively slow, they have several
advantages such as high degree of solids breakdown, high organic
loading rate and efcient recovery of biogas which represents util-
isable renewable energy for other distillery operations. Pot ale and
spent lees consist mainly of readily biodegradable organic com-
pounds, and possess chemical oxygen demand (COD) concentra-
tions ranging from 30,000 to 50,000 and 1000 to 2000 mg/l,
respectively (Goodwin et al., 2001). Due to the high COD of these
residues, anaerobic digestion is benecial not only in reducing this,
but also in producing signicant amounts of useful methane gas.
Anaerobic digestion with methane fermentation is therefore con-
sidered the most suitable approach to treat distillery liquid resi-
dues (Vlissidis and Zouboulis, 1993) and a number of reactors
like the up-ow anaerobic sludge blanket (UASB) reactor, up-ow
anaerobic lter process (UAFP) and anaerobic bafed reactor (ABR)
have already been optimised and commercialized (Tokuda et al.,
1999; Akunna and Clark, 2000; Baloch et al., 2006).
In addition to the organic composition of typical pot ale and
spent wash which contributes to about two thirds of the composi-
tion, the remainder includes inorganics like potassium, calcium,
magnesium silicates, sulphates and phosphates (Koziorowski and
Kucharski, 1972). Undiluted pot ale is a light brownish turbid li-
quid with a pH below 4 and consists of particulate matter largely
comprising of intact yeast cells which are generally allowed to set-
tle to the bottom of the reactors (Goodwin and Stuart, 1994). Yeast
cells are enveloped by a thick cell wall comprising of complex ma-
trix of phosphomannans, glucans, chitin and protein (Walker,
0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.09.089
* Corresponding author. Tel.: +44 1382 308141; fax: +44 1382 308117.
E-mail address: j.akunna@abertay.ac.uk (J.C. Akunna).
Bioresource Technology 101 (2010) 16811685
Contents lists available at ScienceDirect
Bioresource Technology
j our nal homepage: www. el sevi er . com/ l ocat e/ bi or t ech
1998). Due to the presence of thick cell walls, yeast cells are dif-
cult to digest and their presence may deleteriously affect anaerobic
digestion processes. There is therefore a need to pre-treat or hydro-
lyse intact yeast cells before distillery residues are subject to
anaerobic digestion.
To improve the digestion of slurry-like wastewaters it is some-
times necessary to enhance the hydrolysis phase by pre-treating
the wastewater using various processes such as: heating, ultra-
sound treatment, ozonation, alkaline treatment or biological enzy-
molysis. Apart from enzyme pre-treatment, most of these
processes require signicant amounts of energy. Enzymatic hydro-
lysis has been successfully used in treating wastewater from dair-
ies, distilleries and slaughterhouses (Cammarota et al., 2001;
Masse et al., 2001, 2003; Jung et al., 2002; Bochmann et al.,
2007). Enzymolysis can enhance the degradation of the complex
organic fractions to simple products and this has led to the com-
mercial production of enzymes which can be used as aids to anaer-
obic digestion systems. Consequently, the application of
appropriate enzymes to pre-treat yeast cells in distillery residues
may have potential benets for anaerobic digestion, and this was
the focus of the present investigation.
2. Methods
2.1. Distillery liquid residues
Pot ale samples were obtained fromBlair Athol malt whisky dis-
tillery (Perthshire, Scotland, UK). Spent wash was obtained from
Cameronbridge grain distillery (Fife, Scotland, UK). Since the spent
wash contained grain particles, it was centrifuged to allow the
grains to settle and the supernatant, known as centrate, was
recovered for experimentation. All distillery samples were stored
at 4 C to minimize microbial contamination.
2.2. Control yeast strain
Saccharomyces cerevisiae M-type distillers yeast strain (DCLM)
obtained from Kerry Biosciences, Menstrie, Scotland was used as a
control organism in order to study the effects of various enzymes.
The yeast strain was maintained on malt extract agar slopes and
sub-cultured once every three weeks. Malt extract broth (Oxoid)
was used as a growth medium dispensed as 100 ml volumes. To
evaluate the effect of enzyme digestion on dead yeast cells, cul-
tures were heat killed by boiling the slurry of live cells at 100 C
for 60 min. Yeast viability was assessed using methylene blue
staining (Mills, 1941).
2.3. Commercial enzymes
The following commercially available enzymes were used for
enzymatic hydrolysis of the control yeast strain and distillery sam-
ples at various temperatures, pH values and enzyme dosages: lyt-
icase, alpha amylase, cellulase, beta-glucosidase, beta-glucanase,
lipase, protease, enzyme complex and papain. Cultures, and appro-
priate blanks, were prepared in incubation bottles, placed in a
shaking incubator at 130 rpm for 24 h. The following buffers were
used to control pH: 10 mM MES buffer (from Sigma, UK) was used
to adjust cell suspensions to pH 5 and 6; 10 mM PIPES buffer (also
from Sigma, UK) was used to adjust pH to 7.
2.4. Effect of enzymatic pre-treatment of control intact and distillery
residues
A comparative enzyme pre-treatment was rstly conducted
using the control distillers yeast (DCLM) with 500 ll of different
commercial enzymes (except papain) but at various pH values
and temperatures. The enzymes that produced relatively high rates
of hydrolysis were noted and used for repeat set of experiments,
replacing the DCLM with distillery liquid residues.
Using the optimum pH and temperature values obtained from
the above experiments, further experiments were carried out to
obtain the optimum dosage of the enzyme (or combination) which
produced the best hydrolytic effect on the distillery samples. Three
dosages were tested, viz: 5, 2.5 and 0.5 ml/g (of dry weight of the
solids in the culture).
2.5. Effect of enzyme pre-treatment on anaerobic digestion of distillery
residues
Separate anaerobic digestion trials were conducted for distillery
spent wash centrate and pot ale in order to investigate the effects
of pre-treatment with enzymes (combined beta-glucanase plus
protease and papain), on the digestion process. The samples were
pre-treated with enzyme dosages of 2.5 ml/g (of dry weight of
the solids in the culture) for beta-glucanase plus protease for
24 h, 1% by volume for papain with centrate and 10% by volume
for papain with pot ale for 22 h, at pH of 7.5, 6.0 and 37 and
50 C for combined beta-glucanase plus protease and papain,
respectively. Four different digesters were then operated for each
of the distillery samples, in 1 l capacity digesters connected to
500 ml conical asks for gas collection, with and without enzyme
pre-treatment. Seed inoculum was obtained from a mesophilic
anaerobic digester treating domestic wastewater sludge at the Uni-
ted Utilities Wastewater Treatment Plant, at Hatton, Angus, Scot-
land. Initial pH was adjusted to 7.5 for all digesters. The digesters
were placed in a shaking incubator at 37 C at 80 rpm for a period
of 10 days. Samples were collected periodically for analysis.
2.6. Analytical methods
Aliquots from anaerobic digesters were ltered through a
Whatman Type 70 mm glass microber lter (1.2 lm retention)
and the ltrate analysed for COD, ammonia, and total suspended
solids (TSS) by Standard Methods (APHA, 1992). Detailed analytical
methods can be obtained from Mallick (2007).
3. Results and discussion
3.1. Characteristics of distillery residues
Distillery samples were light brown to brown turbid liquids
wherein the solid content settled down upon standing. Dry
weights for pot ale and spent wash centrate samples were, respec-
tively, 72.7 and 59.2 g/l. pH values of samples were; spent wash pH
4.04.2; centrate pH 3.64.1; pot ale (Blair Athol) 3.54.5. Pot ale
samples contained intact yeast cells, which stained with methy-
lene blue dye, conrming that the cells were dead but with rela-
tively intact cell walls. In addition to yeast cells, a lot of solid
matter was observed in the samples which consisted of brous
particles (primarily originating from spent grains) and rod shaped
bacteria at approximately 1 10
8
cells/ml that stained Gram posi-
tive (presumptive Lactobacilli).
3.2. Effects of enzymatic pre-treatment of control intact yeast cells
(DCLM)
The results are shown in Figs. 1a and 1b and are presented as
percentage lysis of cells, determined by microscopic analysis of
the treated cultures after 24 h incubation, using a haemocytometer.
1682 P. Mallick et al. / Bioresource Technology 101 (2010) 16811685
It was apparent from these results that treatment with beta-
glucanase, beta-glucanase plus protease, lyticase and the pool of
enzymes led to greater than 50% lysis of intact distillers yeast cells.
In particular, beta-glucanase plus protease and lyticase all showed
approximately 90% digestion at 37 C pH 7. Protease on its own did
not digest yeast very well.
3.3. Effect of enzymatic pre-treatment of distillery wastes
The results of a repeat of the rst series of experiments using
only beta-glucanase, beta-glucanase plus protease and lyticase en-
zymes (which showed high rates of hydrolysis), with distillery li-
quid residues at the optimised conditions of 37 C and pH 7.5
showed that beta-glucanase was an effective yeast lytic enzyme
on its own, but worked synergistically with protease producing a
90% yeast cell digestion in spent wash centrate and 80% cell diges-
tion in pot ale. Although lyticase effectively digested control cells
of distillers yeast, it did not show good results when tested on dis-
tillery samples. In addition, whilst lyticase action was very pH
dependent, beta-glucanase and protease were effective over a
range from pH 6.5 to 7.5.
The optimum dosage of the enzyme combination, beta-glucan-
ase plus protease (which produced the best hydrolytic effect on the
distillery samples) at the optimum pH and temperature of 7.5 and
37 C, respectively was found to be 2.5 ml/g of solids.
3.4. Effect of pre-treatment of distillery wastes with papain
Papain, which is a commonly used industrial proteolytic en-
zyme, was tested at pH 6.0 and 50 C at concentrations of 1% and
10% (by volume). After 24 h incubation the samples were micro-
scopically analysed and the results show that papain was ineffec-
tive in the hydrolysis of the intact control cells of distillers yeast,
but effective for the centrate and pot ale resulting in more than
90% cell digestion. It was also found that papain dosages of 1%
and 10% respectively for centrate and pot ale wastes brought about
effective hydrolysis.
3.5. Anaerobic digestion of distillery spent wash centrate
The physical and chemical characteristics of the distillery efu-
ent used (namely spent wash centrate) are presented in Table 1.
Fig. 2a shows the variation of pH values over the digestion peri-
od. The early fall in pHand later recovery are indicative of the acido-
genic and methanogenic dominant stages, respectively. The
distillery centrate typically consisted of high levels of protein, hence
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Enzymes
P
e
r
c
e
n
t
a
g
e

l
y
s
i
s

o
f

c
e
l
l
pH 5
pH 6
pH-7
Fig. 1a. Percentage of distillers yeast cell lysis with various enzymes at 30 C at different pH values [1: cellulase; 2: beta-glucosidase; 3: lipolase; 4: alpha amylase; 5:
protease (subtilisin); 6: beta-glucanase; 7: protease + beta-glucanase; 8: lyticase and 9: pool of enzymes].
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Enzymes
P
e
r
c
e
n
t
a
g
e

l
y
s
i
s

o
f

c
e
l
l
pH 5
pH 6
pH 7
Fig. 1b. Percentage of distillers yeast cell lysis with various enzymes at 37 C at different pH values [1: cellulase; 2: beta-glucosidase; 3: lipolase; 4: alpha amylase; 5:
protease (subtilisin); 6: beta-glucanase; 7: protease + beta-glucanase; 8: lyticase and 9: pool of enzymes].
P. Mallick et al. / Bioresource Technology 101 (2010) 16811685 1683
increased ammonia concentration (to a peak value of 300 mg N/l)
due to protein degradation was observed in all samples.
Fig. 2b shows the overall ltered COD reduction in each diges-
ter. Centrate pre-treated with beta-glucanase plus protease
showed COD reduction of 45% within two days of digestion while
the centrate (with intact yeast cells) without enzyme addition
showed negligible reduction in COD within the same time period.
Fig. 2b clearly shows that there was considerable COD reduction
when the centrate was exposed to hydrolytic enzymes. It is thus
evident that hydrolysis of intact yeast cells is a limiting step in
their effective anaerobic digestion.
3.6. Anaerobic digestion of distillery pot ale
The physical and chemical characteristics of the distillery pot
ale sample are also presented in Table 1.
Fig. 3a suggests that the acidogenic activities were dominant
during the initial 24 h marked by a drop in pH. From then on the
pH gradually increased, indicating increased methanogenic activ-
ity. Ammonia production also increased with time (with a peak va-
lue of 600 mg N/l), and this was attributed to the breakdown of
protein contained in the pot ale. The nitrogen content of pot ale
was generally signicantly greater than in the centrate; hence,
the post digestion ammonia concentration in the former was cor-
respondingly greater.
Fig. 3b illustrates ltered COD reduction from samples with pot
ale with beta-glucanase plus protease having a signicantly better
COD reduction (of up to 87%) compared with pot ale without en-
zyme pre-treatment. When the pot ale was pre-treated with 10%
papain, 50% COD reduction was achieved. In contrast, pot ale with-
out enzyme pre-treatment showed only 13% COD removal. As with
previous experiments with spent wash centrate, enzymatic hydro-
lysis greatly improved anaerobic digestion of pot ale.
4. Conclusions
This study has shown that some commercially available en-
zymes can be effective in digesting intact yeast cells in distillery li-
quid residues. However, in-depth preliminary studies are required
to identify the most appropriate enzymes, optimal dosage, pH and
Table 1
Characteristics of distillery residues.
Parameter Spent wash Pot ale
Total solids (g/l) 23 17.0
Total suspended solids (g/l) 9.6 8.3
Volatile suspended solids (g/l) 9.4 8.1
Total Kjeldahl nitrogen (mg/l) 37.0 92.0
Total COD (g/l) 46.3 61.5
pH 3.8 4.1
4.5
5
5.5
6
6.5
7
7.5
8
8.5
9
9.5
0 2 4 6 8 10 12
Time (days)
p
H
untreated centrate
pretreated centrate with
papain
pretreated centrate with beta-
glucanase + protease
control
Fig. 2a. pH variation during digestion of distillery spent wash.
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
0 2 4 6 8 10 12
Time (days)
C
O
D

(
m
g
/
l
)
untreated centrate
pretreated centrate with
papain
pretreated centrate with
beta-glucanase + protease
Fig. 2b. COD variation during digestion of the centrate.
5
5.5
6
6.5
7
7.5
8
0 2 4 6 8 10 12
Time (days)
p
H
untreated pot ale
pre-treated pot ale +
papain
pre-treated pot ale
with beta-glucanase
+ protease
control
Fig. 3a. pH variation during digestion of distillery pot ale.
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
0 2 4 6 8 10 12
Time (days)
C
O
D

(
m
g
/
l
)
untreated pot ale
pretreated pot ale
+ papain
pre-pot ale +beta
glu+potease
Fig. 3b. COD variation during digestion of pot ale.
1684 P. Mallick et al. / Bioresource Technology 101 (2010) 16811685
temperature conditions before their large-scale application. The
study also showed that signicant improvement in COD reduction
of more than 50% can be achieved following enzymatic pre-treat-
ment of distillery residues prior to anaerobic digestion. It is thus
apparent that the hydrolysis of intact yeast cells in pot ale and
spent wash from Scotch whisky distilleries is a rate-limiting step
in the bioconversion of these residues by anaerobic processes.
Enzymatic pre-treatment of these waste streams can therefore be
an effective pre-treatment for increased process efciency.
Acknowledgements
We thank colleagues at Diageo Ltd. for useful discussion (espe-
cially D. Murray and K. Law) and for provision of distillery samples.
The provision of inoculum for anaerobic digesters from United
Utilities Wastewater Treatment Plant (Hatton, Angus, Scotland) is
gratefully acknowledged.
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