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Enhancement of anaerobic batch digestion of sisal pulp waste by mesophilic aerobic pre-treatment
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Enhancement of anaerobic batch digestion of sisal pulp waste by mesophilic aerobic pre-treatment Anthony Mshandetea,b, Lovisa Bjo¨rnssona,, Amelia K. Kivaisib, S.T. Rubindamayugib, Bo Mattiassona a
Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-22100, Lund, Sweden b Applied Microbiology Unit, Department of Botany, University of Dar es Salaam, P.O. Box 35060, Dar es Salaam, Tanzania Received 27 May 2004; received in revised form 10 November 2004; accepted 30 November 2004 Available online 7 March 2005
Abstract Pre-treatment of sisal pulp prior to its anaerobic digestion was investigated using an activated sludge mixed culture under aerobic conditions in batch bioreactors at 37 1C. The progression of aerobic pre-treatment of the residue in relation to the activities of some extracellular hydrolytic enzymes in the slurry was monitored. The highest activity of hydrolytic enzymes was obtained at 9 h of pre-treatment. Filter paper cellulase had a maximum activity of 0.90 IU/ml, while carboxymethyl cellulase, amylase and xylanase were produced to a maximum of about 0.40 IU/ml. The methane yield obtained after anaerobic digestion of the pre-treated pulp ranged between 0.12 and 0.24 m3 CH4/kg VS added. The highest and lowest values were obtained for 9 and 72 h of pre-treatment, respectively. Nine hours of pre-treatment of sisal pulp prior to anaerobic digestion demonstrated a 26% higher methane yield when compared to the sisal pulp without pre-treatment. The consortia of microorganisms in activated sludge demonstrated a useful potential in the production of hydrolases acting on major macromolecules of sisal pulp. The fact that a correlation was observed between high enzyme activity and high methane yield at 9 h of aerobic pre-treatment suggests that such a short pretreatment period could be an alternative option for increasing solubilization of sisal pulp and promoting methane productivity. r 2005 Elsevier Ltd. All rights reserved. Keywords: Activated sludge; Aerobic pre-treatment; Anaerobic digestion; Hydrolytic enzymes; Sisal pulp waste
1. Introduction Sisal pulp is amongst the most abundant agroindustry waste in Tanzania. Currently it creates serious environmental pollution problems. It has been estimated Corresponding author. Tel.: +46 46 222 8324;
fax: +46 46 222 4713. E-mail address: [email protected] (L. Bjo¨rnsson).
that about 444,000 tonnes of sisal pulp waste are generated annually from 52 sisal-processing factories. The waste is mainly composed of leaf tissues mixed with small fractions of waste sisal fibres and therefore differs from the traditional cow dung employed in methane production. However, it represents a category of surplus biomass, with great biogas potential. The initial pretreatment of sisal pulp is considered important, as it is not a pre-digested material like cow dung. Pre-treatment of the solid waste has earlier been applied to improve
0043-1354/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2004.11.037
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digester performance and obtain acceleration of the anaerobic process with an increase in biogas yield (Mata-Alvarez et al., 2000). Another important parameter that should be taken into consideration, is that the depletion of degradable material in the biomass should be efficient, thereby leaving very low potential for biogas production, after the bioreactor process is over. Organic matter is converted into methane and carbon dioxide during anaerobic digestion by three bacterial groups, the first of which is a complex of fermentative bacteria that hydrolyses polymers and ferments products to acetic acid and other organic acids, hydrogen and carbon dioxide. The hydrogen-producing bacteria are found in the second group, which converts propionate and higher fatty acids, produced by the first group, into acetate and hydrogen (Verstraete et al., 1996). Methane is generated from acetate and/or hydrogen and carbon dioxide, by species of methanogenic bacteria (Siegrist et al., 1993). However, when treating particulate substrates such as solid waste, both the accessibility of hydrolytic microorganisms to the solid matter and hydrolysis of complex polymeric components constitute rate-limiting steps (Eastman and Ferguson, 1981). Therefore, one way of improving the performance of digesters treating solids is to promote the hydrolysis of organic matter by pre-treatment of the substrate. Several pre-treatment methods have been reported: physical–chemical methods including, mechanical (Hartmann et al., 2000) or thermo-chemical treatment (Patel et al., 1993), and biological methods such as thermophilic bacterial treatment (Mori, 1995). Proteins and polymeric carbohydrates present in particulate organics cannot be taken up by the cells. Therefore, microorganisms produce and excrete hydrolytic enzymes such as cellulases, proteases and lipases to break down and solubilize the macromolecular structures into monomers such as sugars, amino acids and glycerol, and long-chain fatty acids to facilitate transport through the cell membrane (Goel et al., 1998; Lai et al., 2001). The use of a microbial population and its hydrolytic enzymes for pre-treatment of solid waste to increase the yield and rate of solubilization of particulate matter has been demonstrated in many studies. In this field, Del Borghi et al. (1999) tested the ability of activated sludge to solubilize a mixture of sewage sludge and the organic fraction of municipal solid waste. It was observed that during pre-treatment volatile suspended solids significantly decreased while the soluble chemical oxygen demand (COD) increased as a result of the bacterial hydrolysis of polymeric materials. In other studies, Kivaisi and Eliapenda (1994, 1995) observed enhanced anaerobic degradation based on the utilization of rumen microorganisms for the pre-treatment of coconut fibres, bagasse and maize bran. Little information is available in the literature about aerobic pre-treatment of agricultural residues using activated sludge as a seed source.
This study was conducted to investigate the ability of aerobic pre-treatment to enhance the subsequent mesophilic anaerobic digestion of sisal pulp waste using an activated sludge mixed population as a source of inoculum in batch cultures.
2. Materials and methods 2.1. Sources of waste and inoculum Sisal pulp waste (SPW) produced during sisal decortication was obtained from a sisal-processing factory at Ubena Zomozi, Tanzania. The SPW was stored at 20 1C until used. Activated sludge inoculum (ASI) was obtained from an activated sludge process of a municipal wastewater treatment plant at Eslo¨v, Sweden. The characteristics of the sisal pulp and inoculum are summarized in Table 1. 2.2. Chemicals The following reagents were used: filter paper, (Whatman grade 41, ashless filter), carboxymethyl cellulose (CMC), xylan (from oat spelts) (Sigma-Aldrich Co., Ltd., Gillingham-Dorset, UK). Soluble starch (Sigma Chemical Co., St Louis, MO, USA). All other chemicals used were of analytical grade. 2.3. Experimental set-up Screening for optimum pre-treatment time and the effect of second aeration pulse on methane yield were carried out in wide-mouthed 0.5 l Erlenmeyer flasks with Table 1 Characteristics of the sisal pulp waste and activated sludge inoculum Parameters
Sisal pulp waste
Activated sludge inoculum
pH Partial alkalinity (g CaCO3 l1) Total alkalinity (g CaCO3 l1) Total solids (%) Volatile solids (% of TS) Neutral detergent fibresa Acid detergent fibresa Lignina Cellulosea Hemicellulosea
5.6 — — 14.3 82.3 75.7 52.6 5.5 47.1 23.1
7.4 0.48 0.75 0.94 55.3 ND ND ND ND ND
All values are the average of three replicates except for the fibres were obtained from duplicate measurements. ND ¼ not determined. a % TS.
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a working volume of 0.3 l. Each bioreactor had a gastight rubber stopper with an outlet equipped with a sampling septum for taking biogas samples and a gastight bag for collecting the biogas. This research was divided into Experiments 1 and 2. 2.3.1. Experiment 1: Screening for optimum pretreatment period During the screening experiments, the untreated and pre-treated sisal pulp waste were compared. To examine the effect of pre-treatment on the subsequent performance of batch anaerobic digestion of sisal pulp waste, experiments were carried out for seven different periods of aerobic conditions: 3, 6, 9, 12, 24, 48 and 72 h. Each digester contained aerated activated sludge as an inoculum and sisal pulp waste as a substrate. The proportions of sisal pulp waste and inoculum appropriate to achieve the 0.3 l working volume mixture were 19 g and 281 ml, respectively. In order to avoid limitation due to digester acidification, 1.5 g NaHCO3 per 0.3 l was added to buffer the system. The presence of dissolved oxygen (DO) was monitored by the addition of resazurin (0.001% w/v). The experimental set-up consisted of a total of 40 digesters. Eight experimental digesters, i.e. 1 for untreated and 7 for treated sisal pulp waste, were used for the following analyses: enzyme activity, total solids (TS), volatile solids (VS), volatile fatty acids (VFAs), total sugar concentration, pH, partial alkalinity (PA), total alkalinity (TA) and COD. The remaining 32 digesters were used for methane yield determinations. The study on the effect of aerobic pre-treatment involved two series of digesters: (i) four digesters without aeration and (ii) 28 digesters with aeration. In the first series, two digesters were charged with untreated sisal pulp waste to simulate the conventional method of treatment, the other two served as controls (inoculum only). Each digester mixture was flushed with a mixture of gases N2 and CO2 (80:20) for 5 min to replace the air (oxygen). The second series consisted of 7 groups consisting of four digesters, each two experimental and two controls. The controls were used to obtain the background biogas production from the inoculum, which was subtracted from that of the test digester at the particular time tested. Surface aeration in the digester mixture was achieved by leaving the digester open and shaking at 135 rpm using a shaking water bath at 37 1C (GFL 1086, Gesellschaft fu¨r Labortechnik mBH, Burgwedel, Germany). The presence of DO was indicated by the pink colour of resazurin. The concentration of DO after the aerobic period was determined with an oximeter (OXi 320, WTW, Germany). The relative concentration of DO recorded was 0.1970.02 mg/l in all experiments. In this study the only sludge used was ASI, in part because this experiment was aimed at evaluating ASI as a seed
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source during aerobic pre-treatment of sisal pulp waste, but also, it has been demonstrated that aerobic activated sludge contain enough anaerobes to become a potential seed source for mesophilic anaerobic digestion (Kim and Speece, 2002). Immediately after the aeration periods of 3, 6, 9, 12, 24, 48 and 72 h respectively, the reaction mixture in each digester was flushed with a mixture of gases N2 and CO2 (80:20) for 5 min to replace the air (oxygen) in order to attain anaerobic conditions more quickly, and also to avoid unfavourable mixtures of oxygen and methane during the process transition. Subsequently, each digester was sealed with a gas-tight rubber stopper which was equipped with an outlet for biogas sampling. To avoid any possibility of leakages, the stoppers were further sealed with parafilm. The digesters were placed in a shaking water bath maintained at 37 1C and shaken at 70 rpm. All the digestion experiments were run in duplicate for 32 days. 2.3.2. Experiment 2: Determination of the effect of a second aeration pulse on methane yield from sisal pulp waste that had previously been pre-treated The experimental arrangements were same as those described in (1) above but only one pre-treatment period was employed (9 h). This experiment was performed to investigate the effect of a second aeration pulse on the methane yield from the previously aerobically pretreated sisal pulp waste. To do this, 12 digesters were used, in three groups of four digesters each, two experimental, and two controls with inoculum only. The first group of four digesters contained untreated sisal pulp waste (conventional) and were designated UT (untreated). The other two groups designated 9T1 and 9T2, were initially given 9 h of aerobic pre-treatment. All three groups were flushed with a mixture of N2 and CO2 (80:20) (for 5 min to replace the air (oxygen), and anaerobic digestion was allowed to proceed. All three groups were initially run for 20 days (this is the period during which the highest amount of biogas production was achieved). Thereafter, at day 24 when the biogas production rate decreased, a second 9 h aeration pulse was introduced into the 9T1 group of digesters. After air exposure the digester conditions were made anaerobic, as described above and anaerobic digestion was allowed to proceed again. All the digestion experiments were run for a total of 85 days. Such a long period was necessary to ascertain whether the oxygen introduced on day 24 had any effect on the process performance of the 9T1 group of digesters. 2.4. Analytical methods The biogas produced in the bioreactors was collected in gas-tight bags. Since small volumes of biogas were produced, the biogas volume was measured using a
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graduated 50 ml gas-tight glass syringe with a sample lock (SGE International Pty Ltd., Ringwood, Australia). Biogas composition was measured using a Varian 3350 gas chromatograph (Walnut Creek, CA, USA) fitted with a Hay sep Q 80/100 mesh column, a molecular sieve column and a thermal conductivity detector. Helium was used as a carrier gas at a flow rate of 12 ml/min. The column temperature was 70 1C and the injector and detector temperatures were 110 and 150 1C, respectively. The compounds detected were methane, carbon dioxide, nitrogen and oxygen. The partial alkalinity, TA, pH and VFA were measured as previously described by Bjo¨rnsson et al. (2001). Samples for the analysis of the total content of sugars and COD were centrifuged at 3000g for 3 min, and the supernatant was analysed as follows. The total concentration of sugars from the hydrolysates was determined by the phenol–sulphuric acid method with glucose standard, according to Dubois et al. (1956). The absorption was measured using an Ultrospec 1000 spectrophotometer (Biochron Ltd., Cambridge, England) at 490 nm. Dr. Lange Cuvette Test COD, LCK 114 and a Lasa 100 spectrophotometer (Dr. Bruno Lange GmbH, Germany) were used for the analysis of COD of the hydrolysates. The contents of neutral detergent fibres, NDF (total fibres in the substrate), acid detergent fibres, ADF (primarily consisting of cellulose and lignin from the substrate), cellulose and lignin (permanganate method) in the sisal pulp waste were analysed in duplicate, according to the method of Goering and van Soest (1970). TS and VS were determined according to standard methods (APHA, AWWA, WPCF, 1985). 2.5. Separation of enzyme and enzyme assays Enzyme activities were determined for each experiment upon termination of the aerobic phase by centrifuging 6 ml of well-mixed slurry at 13,000g for 10 min using a Biofuge 13 (Heraeus Instruments, Germany). The supernatant was collected and used for enzyme assays. Reducing sugars produced in the enzyme-treated samples were assayed using the dinitrosalicylic acid (DNS) method of Miller (1959). Acetate buffer was used in all enzyme assays. The reducing sugar produced after incubation were determined at 540 nm using an Ultrospec 1000 UV/visible spectrophotometer. Amylase was measured using the method of Giraud et al. (1991). A supernatant sample of 0.5 ml was incubated with 0.5 ml of pre-warmed 0.5% (w/v) soluble starch in 1 ml acetate buffer (pH 6.0, 0.05 M) at 50 1C for 30 min. A number of cellulase enzymes were assayed. However, only filter paper cellulase (FPase) and carboxymethyl cellulase (CMCase) showed significant activity. FPase was assayed using the method of Ghose (1987). Supernatant of 0.5 ml was mixed with 1.5 ml pre-warmed acetate buffer (pH 6, 0.05 M), containing 1.0 6.0 mm
(about 50 mg), filter paper and was incubated at 50 1C for 60 min. CMCase was assayed using the method of Ghose (1987). Supernatant 0.5 ml was mixed with 1 ml pre-warmed acetate buffer (pH 6.0, 0.05 M) containing 0.5% (w/v) carboxymethyl cellulose (CMC) and was incubated at 50 1C for 1 h. For the above three enzymes, one enzyme unit was defined in each case as the amount of enzyme that releases 1 mmol of reducing sugars under assay conditions per minute. Xylanase activity was measured by incubating 0.5 ml of the supernatant with 1 ml pre-warmed acetate buffer (pH 6.0, 0.05 M), containing 0.5 ml 0.5% (w/v) xylan (from oat spelts) at 50 1C for 1 h. One unit of enzyme activity was defined as the amount of enzyme, that releases 1 mmol of xylose under assay conditions per minute according to Bailey et al. (1992).
3. Results and discussion 3.1. Synergism in enzymatic degradation of aerobic pretreated sisal pulp waste The production of cellulases and xylanases by pure or mixed microbial cultures usually requires cellulose or xylan as the main carbon source. However, their enzyme activities vary depending on the composition of the substrates and the source of the inoculum employed. In this study, sisal pulp waste was used as the main carbon source and activated sludge was used as the inoculum. The progression of aerobic pre-treatment of the residue in relation to the activities of some extracellular hydrolytic enzymes in the slurry was monitored. FPase had the highest activity, of 0.90 IU/ml, while xylanase, CMCase and amylase were produced to a maximum activity of about 0.40 IU/ml. Irrespective of the maximum enzyme activities, the enzyme production pattern shows that all the enzyme activities increased rapidly within the first 9 h, remained more or less constant until 50 h, and thereafter decreased sharply during the remaining time of the study period (Fig. 1). These results demonstrated that the mixed microbial populations in activated sludge were able to produce extracellular enzymes, and that the mixture of polymers in the sisal pulp waste substrate stimulated enzyme production. The production of extracellular lignocellulosedegrading enzymes is desirable in the hydrolysis of solid sisal pulp waste, which is high in lignocellulosic compounds (Table 1). Similar synergism among these enzymes has been reported earlier in microbial degradation processes of lignocelluloses by Gilbert and Hazlewood (1993). Microbial cellulolytic enzymes, in admixture with xylanase and other hydrolytic enzymes, have high potential in the conversion of surplus biomass to soluble sugars, which can subsequently be used for
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the production of energy carriers such as biogas, organic acids, alcohols and other fermentation products.
methane and carbon dioxide when anaerobic conditions prevail.
3.2. Total sugar contents in sisal pulp waste hydrolysates
3.3. The relationship between concentration of total VFA and soluble COD
The accumulation of sugars resulting from hydrolysis in all the aerobic pre-treatment periods studied ranged between 0.07 and 0.26 g/l (Fig. 2). The lowest and highest values were obtained from untreated sisal pulp waste and sisal pulp waste pre-treated for 48 h, respectively. Large variations in the sugar concentration pattern were observed, and this illustrates the complexity of hydrolytic conditions. Initially, aerobic organisms will use the sugars, later aerobic sugar consumption will slow down due to oxygen deficiency, but the enzymes will still hydrolyse the polymeric structures. In the meantime, fermentative organisms will convert sugars into volatile fatty acids, which will be converted into
Enzyme activity (IU/ml)
FPase
CMCase
Amylase
Xylanase
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75
Time (hours) Fig. 1. Profiles of filter paper cellulase, carboxymethyl cellulase, amylase and xylanase during aerobic pre-treatment of sisal pulp waste in batch bioreactors inoculated with activated sludge prior to anaerobic digestion.
TVFA
COD
Total sugars
3.0
Concentration (g/l)
2.5 2.0 1.5 1.0 0.5 0.0 0
5
10 15 20 25 30 35 40 45 50 55 60 65 70 75
Time (hours)
Fig. 2. Temporary variation of organic matter solubilization in terms of total VFAs, soluble COD and total sugars during aerobic pre-treatment of sisal pulp waste prior to anaerobic digestion.
As can be seen in Fig. 2, the trend in the variation of total VFAs (TVFAs) with pre-treatment time was similar to that of the soluble COD concentration. VFAs are among the main products of the acidogenesis of organic matter. The highest concentrations of VFAs were attained after 9 h of aerobic pre-treatment. The aerobic conditions (aeration with low DO concentrations) and the mixing of the reaction mixture in shakebioreactors provided suitable growth conditions for acidogenic and hydrolytic bacteria. VFA formation indicates fermentative conditions also during the aeration phase. Accumulation of VFAs under microaerobic conditions during solubilization of organic sludge when using aerobic bacteria as a pre-treatment method has been reported by Hagesawa et al. (2000). However, in their case, pre-treatment was carried out under thermophilic conditions. The combined action of microbial consortia in ASI on sisal pulp waste resulted in hydrolysis and fermentation of polymeric compounds, thus resulting in the solubilization of the organic content. Soluble COD is a parameter that represents the extent of solubilization. Solubilization of organic matter, in terms of soluble COD increased rapidly to a maximum of 2.5 COD g/l obtained at 9 h of pretreatment. This represents a conversion of about 28% in reference to 9 g TS/l of sisal pulp waste added, which represents a very good conversion rate to soluble COD. However, during aerobic pre-treatment it appears that aerobic/fermentative organisms degraded soluble COD. Also, some of the volatile compounds, such as VFAs, could have been lost through evaporation, since the bioreactors were left open and shaken during pretreatment. 3.4. Total biogas production and methane yield The main results of batch anaerobic digestion of sisal pulp waste after different aerobic pre-treatment time periods, in terms of total biogas production and methane yield are given in Table 2. Total biogas production ranged between 0.46 and 0.97 l, which were obtained for 72, and 9 h of pre-treatment, respectively, from 19 g of sisal pulp waste after 32 days of digestion. The methane yield ranged between 0.12 and 0.24 m3 CH4/kg VS added. The highest methane yield was recorded for 9 h of pre-treatment. This represents an increase of 26% compared with using sisal pulp waste without pre-treatment. In the literature, little can be found on the use of microbes from activated sludge as an inoculum for pre-treating agricultural residues under
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Table 2 Total biogas production and methane yield during anaerobic digestion of aerobic pre-treated sisal pulp waste in batch bioreactors after 32 days of digestion Pre-treatment time (h)
Total biogas production (l)
Methane yield (m3 CH4/kg VS added)
0 3 6 9 12 24 48 72
0.79 0.79 0.92 0.97 0.93 0.91 0.64 0.46
0.19 0.19 0.22 0.24 0.23 0.22 0.14 0.12
aerobic conditions prior to anaerobic digestion. Hagesawa et al. (2000) reported that 1.5 times as much biogas was generated when sewage sludge was solubilized under thermophilic microaerobic conditions for 24 h compared with non-pre-treated sludge. When degrading organic matter for the purpose of producing biogas, it is important to convert the accessible fraction of the matter as efficiently as possible, otherwise, biomethanation may proceed when the process is terminated and the bioreactor contents evacuated. Such biomethanation may lead to dissipation of methane to the atmosphere, thereby contributing to the greenhouse effect. During aerobic pre-treatment of sisal pulp waste, some of the large molecules were efficiently hydrolysed, making them more easily available for anaerobic digestion. However, at the same time, prolonged pre-treatment periods were disadvantageous to the subsequent anaerobic digestion. A major drawback, was a loss of the potential methane yield of 26% and 37% recorded following pre-treatment for 48 and 72 h, respectively, compared with the situation with sisal pulp waste without pre-treatment. For such pre-treatment times, significant amounts of organic material were aerobically degraded, causing lower methane yields. Thus, it is inferred that, based on the results presented in Table 2, 9 h of aerobic pre-treatment of sisal pulp waste is promising for enhanced methane yield under mesophilic conditions amongst the time periods investigated. 3.5. The effect of treatment with a second aeration pulse on methane yield The hypothesis behind these experiments was that introducing air (oxygen) might stimulate the microorganisms to produce more enzymes for further degradation of the sisal pulp waste, so that more biogas could be extracted. The enzymes were assumed to be active also after anaerobic conditions had been restored. The sisal pulp waste given a second aerobic pulse gave a
slightly lower methane yield (94%) than the one with only 9 h pre-treatment. Therefore, an aeration pulse introduced into the sisal pulp waste after the first aerobic pre-treatment period did not improve the subsequent anaerobic digestion. A second aerobic pulse coupled with reinoculation could be more advantageous to the process, however, this remains to be studied.
4. Conclusions The overall conclusions from the results presented in this study are summarized below.
Aerobic
pre-treatment of sisal pulp waste, prior to anaerobic digestion, had an effect on the subsequent digestion of the substrate in batch cultures using a microbial consortium originating from activated sludge as an inoculum. Twenty-six percent more methane was obtained after 9 h of pre-treatment than with untreated sisal pulp waste. However, neither prolonged pre-treatment periods of 48 and 72 h, nor a second aeration pulse introduced to the sisal pulp waste after the first 9 h of pre-treatment increased the amount of biogas that could be extracted from sisal pulp waste. The method of using a consortium of microbes from activated sludge under mesophilic aerobic conditions offers rapid pre-treatment (9 h of pre-treatment). It has great potential to promote hydrolysis and solubilization of organic matter and improve the subsequent anaerobic digestion of sisal pulp waste. Also, the low level of oxygen employed is an added advantage since aeration is expensive. Aerobic pre-treatment (9 h) of sisal pulp waste using activated sludge inoculum has been applied during hydrolysis/acidification stage of a two-stage anaerobic digestion. It is shown to be a promising pretreatment option for selective production of VFAs suitable for methane production from sisal pulp waste.
Acknowledgements The authors express their gratitude to The Swedish International Development Cooperation Agency (SIDA), via the BIO-EARN project and the SAREC Project SWE-2003-021, for their financial support.
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ARTICLE IN PRESS A. Mshandete et al. / Water Research 39 (2005) 1569–1575 Bailey, M.J., Biely, P., Poutanen, K., 1992. Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 23, 257–270. Bjo¨rnsson, L., Murto, M., Jantsch, T.G., Mattiasson, B., 2001. Evaluation of new methods for the monitoring of alkalinity, dissolved hydrogen and the microbial community in anaerobic digestion. Water Res. 35 (12), 2833–2840. Del Borghi, A., Converti, A., Pallazi, E., Del Borghi, M., 1999. Hydrolysis and thermophilic anaerobic digestion of sewage sludge and organic fraction of municipal solid waste. Bioprocess Eng. 20, 553–560. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Calorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Eastman, J.A., Ferguson, J.F., 1981. Solubility of particulate organic carbon during the acid phase of anaerobic digestion. J. Water Pollut. Control Fed. 53, 352–366. Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257–268. Gilbert, H.J., Hazlewood, G.P., 1993. Bacterial cellulases and xylanases J. Gen. Microbiol. 139, 187–194. Giraud, E., Brauman, A., Keleke, S., Lelong, B., Raimbault, M., 1991. Isolation and physiological study of an amylolytic strain of Lactobacillus plantarum. Appl. Microbiol. Biotechnol. 36, 379–383. Goel, R., Mino, T., Satoh, H., Matsuo, T., 1998. Enzyme activities under anaerobic and aerobic conditions in activated sludge sequencing batch reactor. Water Res. 32 (7), 2081–2088. Goering, H.K., van Soest, P.J., 1970. Forage fibre analysis (apparatus, reagents, procedures and some applications). Agricultural Handbook, vol. 379. Agricultural Research Service, United States Department of Agriculture, Washington DC, USA. Hagesawa, S., Shiota, N., Katsura, K., Akashi, A., 2000. Solubilization of organic sludge by thermophilic aerobic
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bacteria as a pre-treatment for anaerobic digestion. Water Sci. Technol. 41 (3), 163–169. Hartmann, H., Angelidaki, I., Ahring, B.K., 2000. Increase of anaerobic degradation of particulate organic matter in fullscale biogas plants by mechanical maceration. Water Sci. Technol. 41 (3), 145–153. Kim, M., Speece, R.E., 2002. Aerobic waste activated sludge (WAS) for start-up seed of mesophilic and thermophilic anaerobic digestion. Water Res. 36 (15), 3860–3866. Kivaisi, A.K., Eliapenda, S., 1994. Pretreatment of bagasse and coconut fibres for enhanced anaerobic degradation by rumen microorganisms. Renew. Energy 5, 791–795. Kivaisi, A.K., Eliapenda, S., 1995. Application of rumen microorganisms for enhanced anaerobic degradation of bagasse and rice bran. Biomass Bioenergy 8, 45–50. Lai, T.E., Nopharatana, A., Pullammanappallil, P.C., Clarke, W.P., 2001. Cellulolytic activity in leachate during leach-bed anaerobic digestion of municipal solid waste. Biores. Technol. 80, 205–210. Mata-Alvarez, J., Mace´, S., Llabre´s, P., 2000. Anaerobic digestion of organic wastes. An overview of research achievements and perspectives. Biores. Technol. 74, 3–16. Miller, G., 1959. Use of the dinitrosalicylic acid reagent for the determination of reducing sugars. Anal. Chem. 31, 426–428. Mori, T., 1995. Treatment of highly concentrated organic wastewaters by thermophilic aerobic digestion. J. Water Waste 37, 40–44. Patel, V., Desai, M., Madamwar, D., 1993. Thermochemical pretreatment of water hyacinth for improved biomethanation. Appl. Biochem. Biotechnol. 42, 67–74. Siegrist, H., Renggli, D., Gujer, W., 1993. Mathematicalmodelling of anaerobic mesophilic sewage-sludge treatment. Water Sci. Technol. 27 (2), 25–36. Verstraete, W., de Beer, D., Pena, M., Lettinga, G., Lens, P., 1996. Anaerobic bioprocessing of organic wastes. World J Microb. Biotechnol. 12, 221–238.
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Enhancement of anaerobic batch digestion of sisal pulp waste by mesophilic aerobic pre-treatment Anthony Mshandetea,b, Lovisa Bjo¨rnssona,, Amelia K. Kivaisib, S.T. Rubindamayugib, Bo Mattiassona a
Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-22100, Lund, Sweden b Applied Microbiology Unit, Department of Botany, University of Dar es Salaam, P.O. Box 35060, Dar es Salaam, Tanzania Received 27 May 2004; received in revised form 10 November 2004; accepted 30 November 2004 Available online 7 March 2005
Abstract Pre-treatment of sisal pulp prior to its anaerobic digestion was investigated using an activated sludge mixed culture under aerobic conditions in batch bioreactors at 37 1C. The progression of aerobic pre-treatment of the residue in relation to the activities of some extracellular hydrolytic enzymes in the slurry was monitored. The highest activity of hydrolytic enzymes was obtained at 9 h of pre-treatment. Filter paper cellulase had a maximum activity of 0.90 IU/ml, while carboxymethyl cellulase, amylase and xylanase were produced to a maximum of about 0.40 IU/ml. The methane yield obtained after anaerobic digestion of the pre-treated pulp ranged between 0.12 and 0.24 m3 CH4/kg VS added. The highest and lowest values were obtained for 9 and 72 h of pre-treatment, respectively. Nine hours of pre-treatment of sisal pulp prior to anaerobic digestion demonstrated a 26% higher methane yield when compared to the sisal pulp without pre-treatment. The consortia of microorganisms in activated sludge demonstrated a useful potential in the production of hydrolases acting on major macromolecules of sisal pulp. The fact that a correlation was observed between high enzyme activity and high methane yield at 9 h of aerobic pre-treatment suggests that such a short pretreatment period could be an alternative option for increasing solubilization of sisal pulp and promoting methane productivity. r 2005 Elsevier Ltd. All rights reserved. Keywords: Activated sludge; Aerobic pre-treatment; Anaerobic digestion; Hydrolytic enzymes; Sisal pulp waste
1. Introduction Sisal pulp is amongst the most abundant agroindustry waste in Tanzania. Currently it creates serious environmental pollution problems. It has been estimated Corresponding author. Tel.: +46 46 222 8324;
fax: +46 46 222 4713. E-mail address: [email protected] (L. Bjo¨rnsson).
that about 444,000 tonnes of sisal pulp waste are generated annually from 52 sisal-processing factories. The waste is mainly composed of leaf tissues mixed with small fractions of waste sisal fibres and therefore differs from the traditional cow dung employed in methane production. However, it represents a category of surplus biomass, with great biogas potential. The initial pretreatment of sisal pulp is considered important, as it is not a pre-digested material like cow dung. Pre-treatment of the solid waste has earlier been applied to improve
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digester performance and obtain acceleration of the anaerobic process with an increase in biogas yield (Mata-Alvarez et al., 2000). Another important parameter that should be taken into consideration, is that the depletion of degradable material in the biomass should be efficient, thereby leaving very low potential for biogas production, after the bioreactor process is over. Organic matter is converted into methane and carbon dioxide during anaerobic digestion by three bacterial groups, the first of which is a complex of fermentative bacteria that hydrolyses polymers and ferments products to acetic acid and other organic acids, hydrogen and carbon dioxide. The hydrogen-producing bacteria are found in the second group, which converts propionate and higher fatty acids, produced by the first group, into acetate and hydrogen (Verstraete et al., 1996). Methane is generated from acetate and/or hydrogen and carbon dioxide, by species of methanogenic bacteria (Siegrist et al., 1993). However, when treating particulate substrates such as solid waste, both the accessibility of hydrolytic microorganisms to the solid matter and hydrolysis of complex polymeric components constitute rate-limiting steps (Eastman and Ferguson, 1981). Therefore, one way of improving the performance of digesters treating solids is to promote the hydrolysis of organic matter by pre-treatment of the substrate. Several pre-treatment methods have been reported: physical–chemical methods including, mechanical (Hartmann et al., 2000) or thermo-chemical treatment (Patel et al., 1993), and biological methods such as thermophilic bacterial treatment (Mori, 1995). Proteins and polymeric carbohydrates present in particulate organics cannot be taken up by the cells. Therefore, microorganisms produce and excrete hydrolytic enzymes such as cellulases, proteases and lipases to break down and solubilize the macromolecular structures into monomers such as sugars, amino acids and glycerol, and long-chain fatty acids to facilitate transport through the cell membrane (Goel et al., 1998; Lai et al., 2001). The use of a microbial population and its hydrolytic enzymes for pre-treatment of solid waste to increase the yield and rate of solubilization of particulate matter has been demonstrated in many studies. In this field, Del Borghi et al. (1999) tested the ability of activated sludge to solubilize a mixture of sewage sludge and the organic fraction of municipal solid waste. It was observed that during pre-treatment volatile suspended solids significantly decreased while the soluble chemical oxygen demand (COD) increased as a result of the bacterial hydrolysis of polymeric materials. In other studies, Kivaisi and Eliapenda (1994, 1995) observed enhanced anaerobic degradation based on the utilization of rumen microorganisms for the pre-treatment of coconut fibres, bagasse and maize bran. Little information is available in the literature about aerobic pre-treatment of agricultural residues using activated sludge as a seed source.
This study was conducted to investigate the ability of aerobic pre-treatment to enhance the subsequent mesophilic anaerobic digestion of sisal pulp waste using an activated sludge mixed population as a source of inoculum in batch cultures.
2. Materials and methods 2.1. Sources of waste and inoculum Sisal pulp waste (SPW) produced during sisal decortication was obtained from a sisal-processing factory at Ubena Zomozi, Tanzania. The SPW was stored at 20 1C until used. Activated sludge inoculum (ASI) was obtained from an activated sludge process of a municipal wastewater treatment plant at Eslo¨v, Sweden. The characteristics of the sisal pulp and inoculum are summarized in Table 1. 2.2. Chemicals The following reagents were used: filter paper, (Whatman grade 41, ashless filter), carboxymethyl cellulose (CMC), xylan (from oat spelts) (Sigma-Aldrich Co., Ltd., Gillingham-Dorset, UK). Soluble starch (Sigma Chemical Co., St Louis, MO, USA). All other chemicals used were of analytical grade. 2.3. Experimental set-up Screening for optimum pre-treatment time and the effect of second aeration pulse on methane yield were carried out in wide-mouthed 0.5 l Erlenmeyer flasks with Table 1 Characteristics of the sisal pulp waste and activated sludge inoculum Parameters
Sisal pulp waste
Activated sludge inoculum
pH Partial alkalinity (g CaCO3 l1) Total alkalinity (g CaCO3 l1) Total solids (%) Volatile solids (% of TS) Neutral detergent fibresa Acid detergent fibresa Lignina Cellulosea Hemicellulosea
5.6 — — 14.3 82.3 75.7 52.6 5.5 47.1 23.1
7.4 0.48 0.75 0.94 55.3 ND ND ND ND ND
All values are the average of three replicates except for the fibres were obtained from duplicate measurements. ND ¼ not determined. a % TS.
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a working volume of 0.3 l. Each bioreactor had a gastight rubber stopper with an outlet equipped with a sampling septum for taking biogas samples and a gastight bag for collecting the biogas. This research was divided into Experiments 1 and 2. 2.3.1. Experiment 1: Screening for optimum pretreatment period During the screening experiments, the untreated and pre-treated sisal pulp waste were compared. To examine the effect of pre-treatment on the subsequent performance of batch anaerobic digestion of sisal pulp waste, experiments were carried out for seven different periods of aerobic conditions: 3, 6, 9, 12, 24, 48 and 72 h. Each digester contained aerated activated sludge as an inoculum and sisal pulp waste as a substrate. The proportions of sisal pulp waste and inoculum appropriate to achieve the 0.3 l working volume mixture were 19 g and 281 ml, respectively. In order to avoid limitation due to digester acidification, 1.5 g NaHCO3 per 0.3 l was added to buffer the system. The presence of dissolved oxygen (DO) was monitored by the addition of resazurin (0.001% w/v). The experimental set-up consisted of a total of 40 digesters. Eight experimental digesters, i.e. 1 for untreated and 7 for treated sisal pulp waste, were used for the following analyses: enzyme activity, total solids (TS), volatile solids (VS), volatile fatty acids (VFAs), total sugar concentration, pH, partial alkalinity (PA), total alkalinity (TA) and COD. The remaining 32 digesters were used for methane yield determinations. The study on the effect of aerobic pre-treatment involved two series of digesters: (i) four digesters without aeration and (ii) 28 digesters with aeration. In the first series, two digesters were charged with untreated sisal pulp waste to simulate the conventional method of treatment, the other two served as controls (inoculum only). Each digester mixture was flushed with a mixture of gases N2 and CO2 (80:20) for 5 min to replace the air (oxygen). The second series consisted of 7 groups consisting of four digesters, each two experimental and two controls. The controls were used to obtain the background biogas production from the inoculum, which was subtracted from that of the test digester at the particular time tested. Surface aeration in the digester mixture was achieved by leaving the digester open and shaking at 135 rpm using a shaking water bath at 37 1C (GFL 1086, Gesellschaft fu¨r Labortechnik mBH, Burgwedel, Germany). The presence of DO was indicated by the pink colour of resazurin. The concentration of DO after the aerobic period was determined with an oximeter (OXi 320, WTW, Germany). The relative concentration of DO recorded was 0.1970.02 mg/l in all experiments. In this study the only sludge used was ASI, in part because this experiment was aimed at evaluating ASI as a seed
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source during aerobic pre-treatment of sisal pulp waste, but also, it has been demonstrated that aerobic activated sludge contain enough anaerobes to become a potential seed source for mesophilic anaerobic digestion (Kim and Speece, 2002). Immediately after the aeration periods of 3, 6, 9, 12, 24, 48 and 72 h respectively, the reaction mixture in each digester was flushed with a mixture of gases N2 and CO2 (80:20) for 5 min to replace the air (oxygen) in order to attain anaerobic conditions more quickly, and also to avoid unfavourable mixtures of oxygen and methane during the process transition. Subsequently, each digester was sealed with a gas-tight rubber stopper which was equipped with an outlet for biogas sampling. To avoid any possibility of leakages, the stoppers were further sealed with parafilm. The digesters were placed in a shaking water bath maintained at 37 1C and shaken at 70 rpm. All the digestion experiments were run in duplicate for 32 days. 2.3.2. Experiment 2: Determination of the effect of a second aeration pulse on methane yield from sisal pulp waste that had previously been pre-treated The experimental arrangements were same as those described in (1) above but only one pre-treatment period was employed (9 h). This experiment was performed to investigate the effect of a second aeration pulse on the methane yield from the previously aerobically pretreated sisal pulp waste. To do this, 12 digesters were used, in three groups of four digesters each, two experimental, and two controls with inoculum only. The first group of four digesters contained untreated sisal pulp waste (conventional) and were designated UT (untreated). The other two groups designated 9T1 and 9T2, were initially given 9 h of aerobic pre-treatment. All three groups were flushed with a mixture of N2 and CO2 (80:20) (for 5 min to replace the air (oxygen), and anaerobic digestion was allowed to proceed. All three groups were initially run for 20 days (this is the period during which the highest amount of biogas production was achieved). Thereafter, at day 24 when the biogas production rate decreased, a second 9 h aeration pulse was introduced into the 9T1 group of digesters. After air exposure the digester conditions were made anaerobic, as described above and anaerobic digestion was allowed to proceed again. All the digestion experiments were run for a total of 85 days. Such a long period was necessary to ascertain whether the oxygen introduced on day 24 had any effect on the process performance of the 9T1 group of digesters. 2.4. Analytical methods The biogas produced in the bioreactors was collected in gas-tight bags. Since small volumes of biogas were produced, the biogas volume was measured using a
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graduated 50 ml gas-tight glass syringe with a sample lock (SGE International Pty Ltd., Ringwood, Australia). Biogas composition was measured using a Varian 3350 gas chromatograph (Walnut Creek, CA, USA) fitted with a Hay sep Q 80/100 mesh column, a molecular sieve column and a thermal conductivity detector. Helium was used as a carrier gas at a flow rate of 12 ml/min. The column temperature was 70 1C and the injector and detector temperatures were 110 and 150 1C, respectively. The compounds detected were methane, carbon dioxide, nitrogen and oxygen. The partial alkalinity, TA, pH and VFA were measured as previously described by Bjo¨rnsson et al. (2001). Samples for the analysis of the total content of sugars and COD were centrifuged at 3000g for 3 min, and the supernatant was analysed as follows. The total concentration of sugars from the hydrolysates was determined by the phenol–sulphuric acid method with glucose standard, according to Dubois et al. (1956). The absorption was measured using an Ultrospec 1000 spectrophotometer (Biochron Ltd., Cambridge, England) at 490 nm. Dr. Lange Cuvette Test COD, LCK 114 and a Lasa 100 spectrophotometer (Dr. Bruno Lange GmbH, Germany) were used for the analysis of COD of the hydrolysates. The contents of neutral detergent fibres, NDF (total fibres in the substrate), acid detergent fibres, ADF (primarily consisting of cellulose and lignin from the substrate), cellulose and lignin (permanganate method) in the sisal pulp waste were analysed in duplicate, according to the method of Goering and van Soest (1970). TS and VS were determined according to standard methods (APHA, AWWA, WPCF, 1985). 2.5. Separation of enzyme and enzyme assays Enzyme activities were determined for each experiment upon termination of the aerobic phase by centrifuging 6 ml of well-mixed slurry at 13,000g for 10 min using a Biofuge 13 (Heraeus Instruments, Germany). The supernatant was collected and used for enzyme assays. Reducing sugars produced in the enzyme-treated samples were assayed using the dinitrosalicylic acid (DNS) method of Miller (1959). Acetate buffer was used in all enzyme assays. The reducing sugar produced after incubation were determined at 540 nm using an Ultrospec 1000 UV/visible spectrophotometer. Amylase was measured using the method of Giraud et al. (1991). A supernatant sample of 0.5 ml was incubated with 0.5 ml of pre-warmed 0.5% (w/v) soluble starch in 1 ml acetate buffer (pH 6.0, 0.05 M) at 50 1C for 30 min. A number of cellulase enzymes were assayed. However, only filter paper cellulase (FPase) and carboxymethyl cellulase (CMCase) showed significant activity. FPase was assayed using the method of Ghose (1987). Supernatant of 0.5 ml was mixed with 1.5 ml pre-warmed acetate buffer (pH 6, 0.05 M), containing 1.0 6.0 mm
(about 50 mg), filter paper and was incubated at 50 1C for 60 min. CMCase was assayed using the method of Ghose (1987). Supernatant 0.5 ml was mixed with 1 ml pre-warmed acetate buffer (pH 6.0, 0.05 M) containing 0.5% (w/v) carboxymethyl cellulose (CMC) and was incubated at 50 1C for 1 h. For the above three enzymes, one enzyme unit was defined in each case as the amount of enzyme that releases 1 mmol of reducing sugars under assay conditions per minute. Xylanase activity was measured by incubating 0.5 ml of the supernatant with 1 ml pre-warmed acetate buffer (pH 6.0, 0.05 M), containing 0.5 ml 0.5% (w/v) xylan (from oat spelts) at 50 1C for 1 h. One unit of enzyme activity was defined as the amount of enzyme, that releases 1 mmol of xylose under assay conditions per minute according to Bailey et al. (1992).
3. Results and discussion 3.1. Synergism in enzymatic degradation of aerobic pretreated sisal pulp waste The production of cellulases and xylanases by pure or mixed microbial cultures usually requires cellulose or xylan as the main carbon source. However, their enzyme activities vary depending on the composition of the substrates and the source of the inoculum employed. In this study, sisal pulp waste was used as the main carbon source and activated sludge was used as the inoculum. The progression of aerobic pre-treatment of the residue in relation to the activities of some extracellular hydrolytic enzymes in the slurry was monitored. FPase had the highest activity, of 0.90 IU/ml, while xylanase, CMCase and amylase were produced to a maximum activity of about 0.40 IU/ml. Irrespective of the maximum enzyme activities, the enzyme production pattern shows that all the enzyme activities increased rapidly within the first 9 h, remained more or less constant until 50 h, and thereafter decreased sharply during the remaining time of the study period (Fig. 1). These results demonstrated that the mixed microbial populations in activated sludge were able to produce extracellular enzymes, and that the mixture of polymers in the sisal pulp waste substrate stimulated enzyme production. The production of extracellular lignocellulosedegrading enzymes is desirable in the hydrolysis of solid sisal pulp waste, which is high in lignocellulosic compounds (Table 1). Similar synergism among these enzymes has been reported earlier in microbial degradation processes of lignocelluloses by Gilbert and Hazlewood (1993). Microbial cellulolytic enzymes, in admixture with xylanase and other hydrolytic enzymes, have high potential in the conversion of surplus biomass to soluble sugars, which can subsequently be used for
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the production of energy carriers such as biogas, organic acids, alcohols and other fermentation products.
methane and carbon dioxide when anaerobic conditions prevail.
3.2. Total sugar contents in sisal pulp waste hydrolysates
3.3. The relationship between concentration of total VFA and soluble COD
The accumulation of sugars resulting from hydrolysis in all the aerobic pre-treatment periods studied ranged between 0.07 and 0.26 g/l (Fig. 2). The lowest and highest values were obtained from untreated sisal pulp waste and sisal pulp waste pre-treated for 48 h, respectively. Large variations in the sugar concentration pattern were observed, and this illustrates the complexity of hydrolytic conditions. Initially, aerobic organisms will use the sugars, later aerobic sugar consumption will slow down due to oxygen deficiency, but the enzymes will still hydrolyse the polymeric structures. In the meantime, fermentative organisms will convert sugars into volatile fatty acids, which will be converted into
Enzyme activity (IU/ml)
FPase
CMCase
Amylase
Xylanase
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75
Time (hours) Fig. 1. Profiles of filter paper cellulase, carboxymethyl cellulase, amylase and xylanase during aerobic pre-treatment of sisal pulp waste in batch bioreactors inoculated with activated sludge prior to anaerobic digestion.
TVFA
COD
Total sugars
3.0
Concentration (g/l)
2.5 2.0 1.5 1.0 0.5 0.0 0
5
10 15 20 25 30 35 40 45 50 55 60 65 70 75
Time (hours)
Fig. 2. Temporary variation of organic matter solubilization in terms of total VFAs, soluble COD and total sugars during aerobic pre-treatment of sisal pulp waste prior to anaerobic digestion.
As can be seen in Fig. 2, the trend in the variation of total VFAs (TVFAs) with pre-treatment time was similar to that of the soluble COD concentration. VFAs are among the main products of the acidogenesis of organic matter. The highest concentrations of VFAs were attained after 9 h of aerobic pre-treatment. The aerobic conditions (aeration with low DO concentrations) and the mixing of the reaction mixture in shakebioreactors provided suitable growth conditions for acidogenic and hydrolytic bacteria. VFA formation indicates fermentative conditions also during the aeration phase. Accumulation of VFAs under microaerobic conditions during solubilization of organic sludge when using aerobic bacteria as a pre-treatment method has been reported by Hagesawa et al. (2000). However, in their case, pre-treatment was carried out under thermophilic conditions. The combined action of microbial consortia in ASI on sisal pulp waste resulted in hydrolysis and fermentation of polymeric compounds, thus resulting in the solubilization of the organic content. Soluble COD is a parameter that represents the extent of solubilization. Solubilization of organic matter, in terms of soluble COD increased rapidly to a maximum of 2.5 COD g/l obtained at 9 h of pretreatment. This represents a conversion of about 28% in reference to 9 g TS/l of sisal pulp waste added, which represents a very good conversion rate to soluble COD. However, during aerobic pre-treatment it appears that aerobic/fermentative organisms degraded soluble COD. Also, some of the volatile compounds, such as VFAs, could have been lost through evaporation, since the bioreactors were left open and shaken during pretreatment. 3.4. Total biogas production and methane yield The main results of batch anaerobic digestion of sisal pulp waste after different aerobic pre-treatment time periods, in terms of total biogas production and methane yield are given in Table 2. Total biogas production ranged between 0.46 and 0.97 l, which were obtained for 72, and 9 h of pre-treatment, respectively, from 19 g of sisal pulp waste after 32 days of digestion. The methane yield ranged between 0.12 and 0.24 m3 CH4/kg VS added. The highest methane yield was recorded for 9 h of pre-treatment. This represents an increase of 26% compared with using sisal pulp waste without pre-treatment. In the literature, little can be found on the use of microbes from activated sludge as an inoculum for pre-treating agricultural residues under
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Table 2 Total biogas production and methane yield during anaerobic digestion of aerobic pre-treated sisal pulp waste in batch bioreactors after 32 days of digestion Pre-treatment time (h)
Total biogas production (l)
Methane yield (m3 CH4/kg VS added)
0 3 6 9 12 24 48 72
0.79 0.79 0.92 0.97 0.93 0.91 0.64 0.46
0.19 0.19 0.22 0.24 0.23 0.22 0.14 0.12
aerobic conditions prior to anaerobic digestion. Hagesawa et al. (2000) reported that 1.5 times as much biogas was generated when sewage sludge was solubilized under thermophilic microaerobic conditions for 24 h compared with non-pre-treated sludge. When degrading organic matter for the purpose of producing biogas, it is important to convert the accessible fraction of the matter as efficiently as possible, otherwise, biomethanation may proceed when the process is terminated and the bioreactor contents evacuated. Such biomethanation may lead to dissipation of methane to the atmosphere, thereby contributing to the greenhouse effect. During aerobic pre-treatment of sisal pulp waste, some of the large molecules were efficiently hydrolysed, making them more easily available for anaerobic digestion. However, at the same time, prolonged pre-treatment periods were disadvantageous to the subsequent anaerobic digestion. A major drawback, was a loss of the potential methane yield of 26% and 37% recorded following pre-treatment for 48 and 72 h, respectively, compared with the situation with sisal pulp waste without pre-treatment. For such pre-treatment times, significant amounts of organic material were aerobically degraded, causing lower methane yields. Thus, it is inferred that, based on the results presented in Table 2, 9 h of aerobic pre-treatment of sisal pulp waste is promising for enhanced methane yield under mesophilic conditions amongst the time periods investigated. 3.5. The effect of treatment with a second aeration pulse on methane yield The hypothesis behind these experiments was that introducing air (oxygen) might stimulate the microorganisms to produce more enzymes for further degradation of the sisal pulp waste, so that more biogas could be extracted. The enzymes were assumed to be active also after anaerobic conditions had been restored. The sisal pulp waste given a second aerobic pulse gave a
slightly lower methane yield (94%) than the one with only 9 h pre-treatment. Therefore, an aeration pulse introduced into the sisal pulp waste after the first aerobic pre-treatment period did not improve the subsequent anaerobic digestion. A second aerobic pulse coupled with reinoculation could be more advantageous to the process, however, this remains to be studied.
4. Conclusions The overall conclusions from the results presented in this study are summarized below.
Aerobic
pre-treatment of sisal pulp waste, prior to anaerobic digestion, had an effect on the subsequent digestion of the substrate in batch cultures using a microbial consortium originating from activated sludge as an inoculum. Twenty-six percent more methane was obtained after 9 h of pre-treatment than with untreated sisal pulp waste. However, neither prolonged pre-treatment periods of 48 and 72 h, nor a second aeration pulse introduced to the sisal pulp waste after the first 9 h of pre-treatment increased the amount of biogas that could be extracted from sisal pulp waste. The method of using a consortium of microbes from activated sludge under mesophilic aerobic conditions offers rapid pre-treatment (9 h of pre-treatment). It has great potential to promote hydrolysis and solubilization of organic matter and improve the subsequent anaerobic digestion of sisal pulp waste. Also, the low level of oxygen employed is an added advantage since aeration is expensive. Aerobic pre-treatment (9 h) of sisal pulp waste using activated sludge inoculum has been applied during hydrolysis/acidification stage of a two-stage anaerobic digestion. It is shown to be a promising pretreatment option for selective production of VFAs suitable for methane production from sisal pulp waste.
Acknowledgements The authors express their gratitude to The Swedish International Development Cooperation Agency (SIDA), via the BIO-EARN project and the SAREC Project SWE-2003-021, for their financial support.
References APHA, AWWA, WPCF, 1985. Standard Methods for Examination of Water and Wastewater, sixteenth ed. American Public Health Association, Washington DC, USA.
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