1 1 Effects of heat treatment on hydrogen production potential 2 and microbial community of thermophilic compost 3 enrichment cultures 4 5 Marika E. Nissiläa,*, Hanne P. Tähtib, Jukka A. Rintalab, Jaakko A. Puhakkaa 6 7 a Department of Chemistry and Bioengineering, Tampere University of Technology, 8 Tampere, Finland. 9 b Department of Biological and Environmental Science, University of Jyväskylä, 10 Jyväskylä, Finland 11 * Corresponding author. Address: Tampere University of Technology, Department of 12 Chemistry and Bioengineering, P.O. Box 541, FIN-33101, Tampere, Finland; Tel.: 13 +358 40 198 1140; Fax: +358 3 3115 2869; e- mail: [email protected]. 14 15 Abstract 16 17 Cellulosic plant and waste materials are potential resources for fermentative hydrogen 18 production. In this study, hydrogen producing, cellulolytic cultures were enriched from 19 compost material at 52, 60 and 70ºC. Highest cellulose degradation and highest H2 yield -1 -1 20 were 57 % and 1.4 mol-H2 mol-hexose (2.4 mol-H2 mol-hexose-degraded ), 21 respectively, obtained at 52°C with the heat-treated (80°C for 20 min) enrichment 22 culture. Heat-treatments as well as the sequential enrichment decreased the diversity of 23 microbial communities. The enrichments contained mainly bacteria from families 24 Thermoanaerobacteriaceae and Clostridiaceae, from which a bacterium closely related 2 25 to Thermoanaerobium thermosaccharolyticum was mainly responsible for hydrogen 26 production and bacteria closely related to Clostridium cellulosi and Clostridium 27 stercorarium were responsible for cellulose degradation. 28 29 Keywords: Dark fermentation, cellulose, mixed culture, thermophilic, 30 Thermoanaerobium thermosaccharolyticum 31 32 1 Introduction 33 34 Increasing energy demand and global warming that is associated with the increasing use 35 of fossil fuels increase the demand to produce clean, renewable energy. Hydrogen is 36 considered as a good alternative to conventional fossil fuels because its energy content 37 (122 MJ kg-1) is high (Logan et al., 2002) and because hydrogen results in a cleaner 38 combustion as the only side-product is water (Zhu et al., 2008). The ability to degrade 39 cellulosic material is important since lignocellulosic biomass is the most abundant raw 40 material in nature (Ren et al., 2009) and has low cost. Cellulose is the major constituent 41 of plant biomass and is found in many waste streams, including agricultural and food 42 industry wastes, brewery wastewaters, and waste sludges (Kapdan and Kargi, 2006). In 43 dark fermentation, hydrogen can be produced either by simultaneous cellulose 44 hydrolysis and hydrogen production or by separating these steps into different reactors 45 (Saratale et al., 2008). 46 47 Pure cultures, including species of the genera Enterobacter, Bacillus and Clostridium, 48 have been widely studied for hydrogen production (Hawkes et al., 2002). Simultaneous 49 cellulose degradation and hydrogen production with pure cultures has also been 3 50 reported, for example, with Clostridium acetobutylicum (Wang et al., 2008) and 51 Clostridium thermocellum (Levin et al., 2006). However, use of mixed cultures is 52 preferred because they are easier to operate and control, they can treat non-sterilized 53 feedstocks, their cellulose conversion rates are higher, and they consist of both 54 hydrolytic and fermentative microorganisms (Levin et al., 2004; Hallenbeck and Ghosh, 55 2009; Ren et al., 2009). Hydrogen producing cellulolytic mixed cultures exist in 56 environments such as anaerobic digester sludge and sludge compost (Ueno et al., 1995), 57 forest soil (Ravindran et al., 2010), and hot springs (Koskinen et al., 2008a). Mixed 58 cultures usually contain hydrogen consuming microorganisms, such as methanogens, 59 homoacetogens or sulphate reducers, which have to be inhibited. This can be done with 60 different pretreatments, from which the most common and effective has been heat- 61 treating the cultures at 100°C for 15 min (Li and Fang, 2007). Furthermore, the use of 62 elevated temperatures controls some hydrogen consuming bacteria (Ren et al., 2009). 63 64 In this study, fermentative hydrogen producers were enriched on cellulose at elevated 65 temperatures (52, 60 and 70°C). Microflora for the experiments was derived from 66 thermophilic compost. The effect of different heat treatments on hydrogen production 67 potentials and microbial communities were delineated. To the authors knowledge the 68 effect of heat pretreatment on microbial community composition of compost inoculum 69 has not been studied. 70 71 72 73 74 4 75 2 Materials and Methods 76 77 2.1 Seed Microorganisms 78 79 Compost material from thermophilic (60-70°C) phase of pile compost obtained from a 80 Solid Waste Management Site (Tarastenjärvi, Finland) was used as source material for 81 the enrichments. In the first enrichment step, the ratio of inoculum (28.1 % VS) and 82 substrate (w/w) was set to 1:2. Compost material was weighed prior to the addition. 83 84 2.2 Batch Enrichment of Thermophilic H2 -Producing Microorganisms 85 86 The microbial enrichments were incubated at 52, 60 and 70ºC. The microbial cultures 87 were manipulated in the following ways: (i) no pretreatment (NHT), (ii) heat treatment 88 at 80ºC for 20 minutes (HT1) and (iii) heat treatment at 100ºC for 10 minutes (HT2). 89 Duplicate samples and two control bottles without substrate were prepared for each 90 temperature. The first enrichments were conducted in 25 mL anaerobic tubes with a 91 working volume of 10 mL. The following two enrichment phases were done in 120 mL 92 serum bottles with 50 mL working volume and with 2 % (v/v) inoculum. The 93 enrichments at 52 and 60°C were incubated on a shaker (150 rpm), while the 94 enrichments at 70°C were done under static conditions. 95 96 The enrichment medium was DSMZ 144 (German Collection of Microorganisms and 97 Cell Cultures, 2008) with the following modifications: 5 g L-cellulose-1 (Sigmacell 98 Cellulose, Type 20) was used instead of glucose, no tryptone was used and yeast extract 99 concentration was decreased to 0.3 g L-1. Headspace in the serum bottles was made 5 100 anaerobic with N2 and sterile cellulose, vitamin solution and Na2S·9 H2O were 101 aseptically added to the medium from stock solutions after sterilization of the medium. 102 Cellulose was sterilized by autoclaving it at 121ºC for 20 min, while vitamin and 103 Na2S·9 H2O solutions were sterile filtered (0.2 μm, Whatman FP 30). Gas production 104 was monitored over time and volatile fatty acids (VFAs) and alcohols were analyzed in 105 the end of the enrichments. 106 107 2.3 Chemical Analyses 108 109 The overpressure from the batch bottles was analyzed according to Owen et al. (1979). 110 The gas composition (H2, CH4 and CO2) in the headspace was analyzed with a 111 Shimadzu gas chromatograph GC-2014 equipped with Porapak N column (80/100 112 mesh) and a thermal conductivity detector (TCD). The temperatures of oven, injector 113 and detector were 80, 110 and 110ºC, respectively. Nitrogen was used as carrier gas at a 114 flow rate of 20 mL min-1. The gas volumes were corrected to a standard pressure (760 115 mm Hg) and temperature (0°C), and cumulative H2 production was calculated according 116 to Logan et al. (2002). 117 118 The concentrations of volatile fatty acids and alcohols were analyzed with Shimadzu 119 High Performance Liquid Chromatography (HPLC) with a Shodex Sugar SH1011 120 column (Showa Denko K.K., Japan) and a refraction index detector (Shimadzu). Mobile -1 121 phase was 5 mM H2SO4 and flow rate 0.7 mL min . Samples for HPLC were pretreated 122 with a solid phase extraction (SPE) method modified from Horspool and McKellar 123 (1991). The cartridge was preconditioned with 2 mL methanol and 2 mL 0.01 M HCl 124 (pH 2), and the VFAs and ethanol were eluted from the sorbent with 1.75 mL 0.05 M 6 125 PBS. Cellulose degradation was calculated based on formed COD in the end of each 126 enrichment phase. COD was calculated as a sum of degradation products and produced 127 hydrogen according to Equation 1 (van Haandel and van der Lubbe, 2007). 128 129 COD = 8 * ( 4x + y – 2z ) / ( 12x + y + 16z ) g COD / g CxHyOz (1) 130 131 2.4 Microbial Community Analyses 132 133 Bacterial communities were characterized using DNA extraction and polymerase chain 134 reaction – denaturing gradient gel electrophoresis (PCR-DGGE) of partial 16S rRNA 135 genes followed by their sequencing. Duplicate samples were taken at the end of each 136 enrichment phase and stored at -20°C. DNA was extracted with a VIOGENE Blood and 137 Tissue Genomic DNA kit (Proteogenix SA, Fegersheim, France). Partial bacterial 16S 138 rRNA genes were amplified using a primer pair GC-BacV3f and 907r as previously 139 described by Koskinen et al. (2007), only exception being that PCR was done with 140 T3000 Thermocycler (Biometra). DGGE was performed with INGENYphorU2 x 2 – 141 system (Ingeny International BV, GP Goes, The Netherlands) as described by Koskinen 142 et al. (2007) with following exceptions: denaturing gradient from 30 % to 70 % was 143 used, gels were run with 100 V for 22.5 h, and dominant bands were eluted in 20 µL of 144 sterile H2O. The re-amplification of bands for sequencing was done as described by 145 Koskinen et al. (2007). Sequence data were analyzed with Bioedit-software (version 146 7.0.5) and compared with sequences in GenBank (http://www.ncb.nlm.nih.gov/blast/). 147 148 149 7 150 151 3 Results and Discussion 152 153 3.1 Enriching Compost Culture for Hydrogen Production 154 155 The original compost culture with or without heat treatment produced negligible 156 amounts of hydrogen when incubated at 70°C and the enrichment of hydrogen 157 producers was unsuccessful.