Bioresource Technology

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1 Bioresource Technology 108 (2012) 8 13 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: Enhancement of starting up anaerobic digestion of lignocellulosic substrate: fique s bagasse as an example Mabel Quintero a, Liliana Castro b, Claudia Ortiz a, Carolina Guzmán a,, Humberto Escalante b, a Escuela de Bacteriología y Laboratorio Clínico Grupo de investigación en Bioquímica y Microbiología, Universidad Industrial de Santander, AA, 678 Bucaramanga, Colombia b Escuela de Ingeniería Química, Grupo de Investigación en Biohidrometalurgia y Medio Ambiente, Universidad Industrial de Santander, AA, 678 Bucaramanga, Colombia article info abstract Article history: Received 24 September 2011 Received in revised form 11 November 2011 Accepted 10 December 2011 Available online 23 December 2011 Keywords: Lignocellulosic residue Ruminal liquid Pig manure sludge Biomethane potential In Colombia there are 20,000 ha of fique fields (Furcraea sp., family Agavaceae), that produce around 93,400 tons of fique s bagasse per year. These residuals are disposed into rivers and soil causing pollution. According to physicochemical characteristics, the lignocellulosic residues from fique crops (fique s bagasse) are appropriate carbon source to biogas production. Anaerobic digestion from fique s Bagasse (FB) requires a specialized microbial consortium capable of degrading its high lignocellulosic concentration. In this study, the capacities of seven microbial consortia for biomethane potential (BMP) from FB were evaluated. Inoculum of ruminal liquid achieved high hydrolytic activity (0.068 g COD/g VSS day), whereas pig waste sludge inoculum showed high methanogenic activity (0.146 g COD/g VSS day). Mixtures of these two inoculums (RL + PWS) showed the best yields for biomethane potential (0.3 m 3 CH4/ Kg VS ad). Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction In Colombia, there are approximately 20,000 ha farmed with fique (Furcraea sp., family Agavaceae). Four percent has been used for commercial applications, while the remaining residues (96%) correspond to agro industrial sub-products (juice and bagasse). These residues are disposed directly to the environment (solid and water) causing serious contamination problems (Finagro, 2011). Physico-chemical composition of FB indicates that these residues are composed by complex polymers such as cellulose, hemicelluloses and lignin. According to this composition, it is possible to treat these residues by anaerobic digestion (AD), because this technology could be an option to upgrade this waste to obtain biogas as an alternative source of renewable energy (Barrera et al., 2009; Castro et al., 2009). AD is carried out in four stages: (a) hydrolytic, (b) acidogenic, (c) acetogenic and (d) methanogenic. These stages are developed by microbial consortia acting in syntrophy (Khanal, 2008). In the hydrolytic stage, mono and poly-carbonated compounds are Abbreviations: BMP, biomethane potential; CM, cow manure; FB, fique s bagasse; HA, hydrolytic activity; PWS, pig waste sludge; SMA, specific methanogenic activity; RL, ruminal liquid; TRS, total reducing sugar; TVFA, total volatile fatty acids; WWS, wastewater sludge. Corresponding authors. addresses: cgluna74@gmail.com (C. Guzmán), escala@uis.edu.co (H. Escalante). generated from biological polymers (cellulose, starches, proteins and fats). Then, volatile organic acids (acetate, propionate, butyrate and valeric acid), carbon dioxide, and hydrogen are produced during the acidogenic stage. Subsequently, acetate, hydrogen and carbon dioxide are synthesized during the acetogenic stage. Finally, during the methanogenic stage, methane and carbon dioxide are formed as main products to the metabolic process (Appels et al., 2008; Yebo et al., 2011). Methane production is developed by different microbial consortia like anaerobic sludge from wastewater treatment plant, ruminal liquid (RL), cow or porcine manures, compost and pure culture of microorganisms (Mshandete et al., 2005; Hu et al., 2004, 2006; Sullivan et al., 2006; Amon et al., 2007; Panichnumsin et al., 2010). Anaerobic biodegradation of substrates requires microbial consortia with hydrolytic and methanogenic activities. The hydrolytic stage is a rate-limiting step of the anaerobic digestion in lignocelullosic substrates (Yue et al., 2007; Zhao et al., 2009; Hendriks and Zeeman, 2009). Biological conversion of cellulosic materials involved microorganism with high cellulolytic efficiencies (Silva et al., 2004; Khanal, 2008; Valdez-Vazquez and Poggi- Varaldo, 2009). In this research, FB digestion requires a specialized hydrolytic consortium. RL has been studied as a primary inoculum in starting up methanogenic reactors due to its high cellulolytic activity. Additionally, it was reported that microorganisms in RL have also high capacity to decompose cellulose, lignocellulose, and their respective monomers. RL consortium increases the efficiency of anaerobic reactors /$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi: /j.biortech

2 M. Quintero et al. / Bioresource Technology 108 (2012) for degradation of lignocellulosic susbtrates in comparison with the sludge from anaerobic processes (Hu et al., 2006; Sullivan et al., 2006). Moreover, cow and porcine manure have shown high efficiency in methane (CH 4 ) production and high adaptability to different substrates. These characteristics make cow and porcine manure an excellent inoculum for AD of residues. In addition, anaerobic sludge from wastewater treatment plants have high yields in CH 4 production, nevertheless, it has a low affinity for lignocellulosic substrates (Hu and Yu, 2005). Recently, consortia mixtures from different sources and proportions have been proposed as a new alternative to increase substrate biodegradability. This is due to a synergetic action of mixed population of microorganisms promoting a better anaerobic digestion in comparison with processes executed using one consortium alone (Magbanua et al., 2001; McMahon et al., 2001; Stroot et al., 2001; Buendía et al., 2009; Álvarez et al., 2010). Performance of biogas production depends on HA and SMA by the inoculums and BMP potential of the substrate. It is important to consider three conditions for the BMP assay: (1) appropriate microbial inoculum, (2) optimal environmental conditions and (3) appropriate substrate concentration within anaerobic digesters (Carrhá et al., 2006; Monou et al., 2009; Labatut et al., 2011). The specific BMP has been studied in batch fermentation processes using different types of manure like cattle, pig and cow, where methane production yields were 148 ± 41; 356 ± 28 and 275 ± 36 L CH 4 /kg VS, respectively (Moller et al., 2004). Furthermore, the BMP has been evaluated in co-digestion systems using mixtures of cow manure and other agro industrial wastes (Amon et al., 2007; Labatut et al., 2011). Information about microbial anaerobic digestion of fique s bagasse is not available in the literature. The aim of this research was to study the capacities of seven microbial consortia to improve biomethane potential from FB. For this purpose, RL, CM, PWS, WWS inoculums and mixtures of them were evaluated. HA, SMA, and BMP were measured, as response variables to select the best microbial consortia for degradation of FB. Cellulose and acetate were used as model substrates to elucidate the action of the microorganism in the anaerobic process. 2. Methods 2.1. Inoculums Seven microbial consortia, collected in Santander (Colombia), were evaluated: (i) RL; (ii) CM obtained from a municipal meat processing plant; (iii) WWS from a treatment plant and (iv) PWS from a municipal porcine plant. As well as, different combinations of consortia (ratio of 1:1 (v/v)) were used: RL + CM, RL + WWS and RL + PWS. The physicochemical properties of inoculums are shown in Table 1 and were performed according to standard protocols (APHA, 1998). Solid inoculums were diluted to avoid dry anaerobic digestion. Inoculums quantities used for HA and SMA determinations were matched at 1.5 g/l of VSS, according to protocol described by Díaz-Báez et al. (2002) Experimental methodology This research was completed in two steps: Initially HA and SMA were evaluated using model substrates as cellulose and acetate, afterwards, BMP was determined using FB as substrate. To avoid interference with the endogenous metabolism of the inoculums, methane production from each consortium (without substrate), was subtracted from the experiments. All tests were performed in triplicate Determination of HA using cellulose as model substrate HA indicates inherent ability from a microbial population for degrading complex carbon sources (cellulose, starch, etc.), and it is quantified as specific rate of substrate consumption (Miller, 1959; Hu et al., 2004; Mshandete et al., 2005). HA assays were carried out in biodigesters of 500 ml with coiled butyl rubber stoppers and inoculated with microbial consortia. HA of microbial consortia was based on cellulose hydrolysis, as described by Hu et al. (2004). Quantitative filter paper Schleicher & Schuell (589/1, 110 mm) was used as substrate. Anaerobic conditions were maintained by using an anaerobic basal medium composed of cysteine (0.5 g/l), NaHCO 3 (5 g/l) and Na 2 S as a reducing agent, at a ph of (Díaz-Báez et al., 2002). The effective volume of reaction was 400 ml. The experiments were executed during 30 days at 39 ± 2 C of incubation temperature. The experimental configuration had five biodigesters for each inoculum. Three experimental digesters groups were used for analyses of total reducing sugars and others two experimental digesters groups were used as control experiments. TRS produced during cellulose hydrolysis were determined by spectrophotometry at 540 nm according to DNS method (Miller, 1959). Samples were taken at different times from anaerobic reactors and centrifuged at 10,000 rpm during 10 min. The supernatants fractions were recovered and maintained at 4 C for further analysis. HA was determined using the maximum TRS production rate, and defined as g COD/g VSS day. All fermentations were performed in triplicate Determination of SMA using acetate as model substrate SMA is related to microbial biomass capacity for transforming organic matter into methane, and it is expressed in terms of COD that is transformed in methane per biomass and time unit g COD/g VSS day (Le Hyaric et al., 2011). SMA assays were carried out in biodigesters of 500 ml with coiled rubber stoppers and inoculated with the microbial consortium. Methane production was analyzed during 30 days. SMA determination was done using the methodology and the Eq. (A). proposed by Díaz-Báez et al. (2002) with (5 g/l) sodium acetate as substrate BMP determination BMP test refers to the capacity of biodegradability of a substrate. In this study BMP is defined as the amount of methane produced from a given weight of FB as substrate (Angelidaki et al., 2009). FB used was collected in a Fique processing plant in Santander-Colombia and subsequently characterized. FB physicochemical Table 1 Physicochemical characterization of inoculums. Parameter Units CM PWS WWS RL RL + CM RL + PWS RL + WWS ph TS mg/l 107, ,940 35,090 17,470 36,460 43,770 23,640 VS mg/l 80,533 51,660 22, ,840 23,640 29,140 VSS mg/l 58,100 37,900 20, ,920 21,880 28,840 Total alkalinity mgcaco 3 /L TVFA mg/l 20, ,

3 10 M. Quintero et al. / Bioresource Technology 108 (2012) 8 13 properties are shown in Table 2. Elemental composition of FB corresponds to the following empirical formula C 33 H 67 O 33 N 1. BMP test of FB was achieved in batch reactors of 500 ml containing an inoculums/substrate ratio of 1 (g VS of inoculums/g VS of FB). AD was evaluated during 15 days at 39 ± 2 C. TRS and TVFA (Method No. 2310B APHA, 1998) productions were used as parameters for controlling the anaerobic bioprocess. The experimental configuration consisted of 10 digesters by each inoculum, with the respective duplicates. Four digesters were used for methane determinations; five reactors were used for TVFA, TRS, ph, total alkalinity; and one another digester was used as blank. Methane volume production was quantified by the alkaline displacement method (Method No. 2720B APHA, 1998; Angelidaki et al., 2009; Demirer and Chen, 2005). BMP was expressed in terms of specific methane potential at STP conditions (m 3 CH 4 /kg of VS added). Experimental results were analyzed with StatGraphics plus 5.1, StatPoint Inc. (Virginia, EE.UU) Software. Fisher s test (F) was used to verify statistical differences between results. 3. Results and discussion 3.1. Physicochemical characterization of fique s bagasse According to Table 2, FB is composed mainly by two fractions: one soluble fraction, formed by non-structural carbohydrates and TVFA; another particulate fraction, formed by cellulose, hemicellulose, proteins and lipids. The composition of FB indicates that this residue is appropriate for the start-up of AD process. However, the acidity of FB substrate could inhibit methanogenic bacteria performance reducing the methane production (Carrhá et al., 2006; Monou et al., 2009). This problem can be resolved by the buffer capacity of inoculums (see Table 1). In addition, low sulfur content in FB allows better development of anaerobic digestion processes because it reduces the hydrogen sulfide production (Khanal, 2008) due to FB heating power, this raw material is appropriate for renewable energy production TRS production from different inoculums using cellulose as substrate Fig. 1 shows the performance of each inoculum to produce TRS. During the first eight days, TRS values were not increased probably due to microbial lag phase. Since this moment, a significant increase in TRS production was observed in all inoculums with the Table 2 Physicochemical characteristics of FB. Parameter Units Fique s bagasse ph 4 TS g TS kg VS g VS kg COD g O 2 kg 1 TS 1230 Total alkalinity mgcaco 3 /L 3300 TVFA g VFA kg 1 TS 48.1 Proteins g kg 1 TS 43.8 Lipids g kg 1 TS 41.3 Holocellulose g kg 1 TS Non atructural aarbohydrates g kg 1 TS 69.4 Lignin g kg 1 TS C % H % 6.02 O % N % 1.32 C/N 26.9 Sulfur % Heating power kcal kg highest specific production mg/ml by RL, this value represents an increase on TRS production of 34% with respect to RL + PWS. By contrast, inoculums WWS and RL + WWS performed the worst with values of mg/ml and mg/ml, respectively. From day 13th, all inoculums maintained a steady production of TRS. There were three levels of TRS production in accordance with the consortium used. RL consortium produced the highest TRS values. These results are associated with the native characteristics of the RL matrix. Most Probable Number of RL bacteria was very high comparing with the others inoculums. Russell and Rychlik (2001) confirm that bacterial numbers in rumen play a dominant role in the fermentation of fibers. High yields values of TRS in RL represent an efficient conversion from cellulose (slowly hydrolysable fraction) into sugar (soluble fraction) favoring the hydrolytic step in anaerobic degradation of lignocellulosic waste. Lower concentrations were found by Hu et al. (2004); mg/ml and Mshandete et al. (2005); 0.26 mg/ ml when using sisal-wws and cellulose-lr, respectively, as substrate-consortium systems for biogas production Methane production by different inoculums using acetate as a model substrate Methane production by different inoculums using acetate as substrate is shown in Fig 2. PWS and admixed RL + PWS (660 and 572 ml, respectively), exhibit better substrate metabolizing capacities and start into reactor comparing to the others inoculums. Taking as reference the estimated theoretical methane value, PWS and RL + PWS percentage conversion was higher (41% and 47%, respectively) compare with the other inoculums, between 12% and 26% (RL, CM, WWS and their combination) Methane production by different inoculums using FB In Fig. 3, results for methane production of the inoculums using FB are presented. Compare with previous results, all the consortia showed a similar behavior. After the day eight, PWS and RL + PWS showed the best methane production favoring the biodegradability process. The other inoculums maintained a stable production during the assay. The methane production from FB using RL + PWS improves 38% with respect to the yields obtained using WWS as microbial consortia. This demonstrated that the degradation of lignocellulosic materials by conventional anaerobic microorganisms is not efficient. Similar results are available in the literature for substrates such as corn stover and cattail, among others (Thanakoses et al., 2003; Hu et al., 2006). Furthermore, the BMP of FB was reduced drastically when RL was used as inoculum. In fermentation assays, with an inoculum substrate ratio of: 1:1, it was observed a VFA accumulation during the process with RL (7440 mg/l) comparing with a consumption behavior with RL + PWS. Despite of the high capacity of RL to metabolize complex substrate into soluble compound, VFA accumulation affect biogas yield values. Yebo et al. (2011) reported that VFA excess may inhibit the methanogenesis reaction Evaluation of HA, SMA and BMP parameters Using the results obtained in Figs. 1 and 2, it was calculated the HA, SMA values of inoculums. For BMP determination, acetate was replaced by FB as substrate carbon source (Table 3). RL and PWS showed high hydrolytic activities, attain values of and g COD/g VSS day, respectively; the high enzymatic activity of these inoculums showing the ability to degrade complex carbohydrates, such as cellulose, has been reported (Thanakoses et al., 2003; Hu et al., 2004; Mshandete et al., 2005). When FB

4 M. Quintero et al. / Bioresource Technology 108 (2012) Fig. 1. TRS production by inoculums using cellulose as substrate (ph: 7.0, 39 ± 2 C). Fig. 2. Methane volume production (STP condition) by inoculums using acetate as substrate (ph: 7.0, 39 ± 2 C). Fig. 3. Production of methane (STP condition) by different inoculums using FB as substrate (ph: 7.0, 39 ± 2 C).

5 12 M. Quintero et al. / Bioresource Technology 108 (2012) 8 13 Table 3 HA and SMA of inoculums and BMP of FB. Inoculums HA (g COD/g VSS day) SMA (g COD/g VSS day) BPM (m 3 CH 4 /kg VS added) CM PWS WWS RL RL + CM RL + PWS RL + WWS (Grant ), Ministerio de Agricultura y Desarrollo Rural MADR from Colombia (Grant 2008J ) and VIE from the Universidad Industrial de Santander (UIS). Appendix A P SMA ¼ FC V SSV where, BMP = specific methanogenic activity (g COD/g VSS day); P = maximum slop of methane production kinetics for each experiment (ml/day); CF = conversion factor of g COD (430 ml of humid CH 4 /g CDO at 39 C); V = volume of inoculum (L); SSV = volatile suspended solids in the inoculum (g/l). ðaþ was evaluated as substrate, instead of cellulose, HA results using RL were significantly higher than the others inoculums (HA of g COD/g VSS day). RL was corroborated as the best inoculums for hydrolysis of cellulose. On the other hand, SMA is strongly related to the capacity of microbial consortia to effectively transform VFA in acetate and subsequently converted into methane per time unit, at the next stage (Díaz-Báez et al., 2002; Madigan et al., 2004). In this study, PWS and RL + PWS show the best results with values of and g COD/g VSS day, respectively. Maximum values of BMP of FB were obtained with RL + PWS and PWS consortia (0.299 and m 3 CH 4 /kg VS added, respectively). Given that there are significant differences of RL + PWS compared with the other consortia (P = 0.000), the mixed consortium had the best affinity to FB among the analyzed consortia (Fig. 4). Besides of the high hydrolytic activity, the mixture RL + PWS was selected as the best choice. In the mixture, each consortium is acting synergistically for cellulose degradation, transforming these sugars in VFA and finally into methane. It was demonstrated that admixed system of RL + PWS constitutes appropriate inoculum for degradation of agro-residues substrates such as FB. This finding is very important during the start-up of anaerobic reactors for lignocellulosic waste treatment. 4. Conclusions This is the first time that anaerobic degradation of FB using different consortia was evaluated. RL + PWS inoculum guarantees the best biogas production from FB. Its hydrolytic and specific metanogenic activities (0.05 g COD glucose/g VSS day and 0.14 g COD CH 4 / g VSS day, respectively) determined the start up of biogas production from lignocelullosic residue. The BMP of FB (0.3 m 3 CH 4 /kg VS added) demonstrated the high bioconversion potential by the admixed inoculum RL + PWS. Acknowledgements Fig. 4. BMP mean numbers (±standard error) of FB. 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