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Bioresource Technology 101 (2010) 1681–1685

Bioresource Technology 101 (2010) 1681–1685 Contents lists available at ScienceDirect Bioresource Technology

Contents lists available at ScienceDirect

Bioresource Technology

journal homepage: www.elsevi er.com/locate/biortech Anaerobic digestion of distillery spent wash: Influence of

Anaerobic digestion of distillery spent wash: Influence 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

article info

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

abstract

The potential benefits 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 16–21 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 influence 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 fish 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-

* Corresponding author. Tel.: +44 1382 308141; fax: +44 1382 308117. E-mail address: j.akunna@abertay.ac.uk (J.C. Akunna).

0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi: 10.1016/j.biortech.2009.09.089

obic treatment processes are relatively slow, they have several advantages such as high degree of solids breakdown, high organic loading rate and efficient 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 beneficial not only in reducing this, but also in producing significant 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-flow anaerobic sludge blanket (UASB) reactor, up-flow anaerobic filter process (UAFP) and anaerobic baffled 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,

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1998 ). Due to the presence of thick cell walls, yeast cells are diffi- 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 significant 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 benefits for anaerobic digestion, and this was the focus of the present investigation.

2. Methods

2.1. Distillery liquid residues

Pot ale samples were obtained from Blair 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 firstly conducted

using the control distiller’s yeast (DCLM) with 500 l l 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 flasks 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 filtered through a Whatman Type 70 mm glass microfiber filter (1.2 l m retention) and the filtrate 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 4.0–4.2; centrate pH 3.6–4.1; pot ale (Blair Athol) 3.5–4.5. Pot ale samples contained intact yeast cells, which stained with methy- lene blue dye, confirming 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 fibrous 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.

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100 pH 5 90 pH 6 80 pH-7 70 60 50 40 30 20 10
100
pH 5
90
pH 6
80
pH-7
70
60
50
40
30
20
10
0
Percentage lysis of cell

123456789

Enzymes

Fig. 1a. Percentage of distiller’s 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].

100 pH 5 90 pH 6 80 pH 7 70 60 50 40 30 20
100
pH 5
90
pH 6
80
pH 7
70
60
50
40
30
20
10
0
Percentage lysis of cell

123456789

Enzymes

Fig. 1b. Percentage of distiller’s 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].

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 distiller’s 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 first 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 distiller’s 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 efflu- 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 pH and later recovery are indicative of the acido- genic and methanogenic dominant stages, respectively. The distillery centrate typically consisted of high levels of protein, hence

pH

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P. Mallick et al. / Bioresource Technology 101 (2010) 1681–1685

Table 1 Characteristics of distillery residues.

8

7.5

7

6.5

6

5.5

5

untreated pot ale pre-treated pot ale + papain pre-treated pot ale with beta-glucanase + protease
untreated pot ale
pre-treated pot ale +
papain
pre-treated pot ale
with beta-glucanase
+ protease
control
0 2
4
6
8
10
12

Parameter

Spent wash

Pot ale

 

Total solids (g/l) Total suspended solids (g/l) Volatile suspended solids (g/l) Total Kjeldahl nitrogen (mg/l) Total COD (g/l) pH

23

17.0

9.6

8.3

9.4

8.1

37.0

92.0

46.3

61.5

3.8

4.1

 

pH

9.5

9

8.5

8

7.5

7

6.5

6

5.5

5

4.5

untreated centrate pretreated centrate with papain pretreated centrate with beta- glucanase + protease control 0
untreated centrate
pretreated centrate with
papain
pretreated centrate with beta-
glucanase + protease
control
0
2
4
6
8
10
12

Time (days)

Fig. 2a. pH variation during digestion of distillery spent wash.

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 filtered 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.

Time (days)

Fig. 3a. pH variation during digestion of distillery pot ale.

10000 untreated pot ale pretreated pot ale 9000 + papain 8000 pre-pot ale +beta glu+potease
10000
untreated pot ale
pretreated pot ale
9000
+ papain
8000
pre-pot ale +beta
glu+potease
7000
6000
5000
4000
3000
2000
1000
0
0 2
4
6
8
10
12
COD (mg/l)

Time (days)

Fig. 3b. COD variation during digestion of pot ale.

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 .

untreated centrate 18000 pretreated centrate with papain 16000 pretreated centrate with beta-glucanase + protease
untreated centrate
18000
pretreated centrate with
papain
16000
pretreated centrate with
beta-glucanase + protease
14000
12000
10000
8000
6000
4000
2000
0
0
2
4
6
8
10
12
COD (mg/l)

Time (days)

Fig. 2b. COD variation during digestion of the centrate.

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 significantly greater than in the centrate; hence, the post digestion ammonia concentration in the former was cor- respondingly greater. Fig. 3b illustrates filtered COD reduction from samples with pot ale with beta-glucanase plus protease having a significantly 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

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temperature conditions before their large-scale application. The study also showed that significant 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 efficiency.

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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