Aerobic thermophilic bacteria enhance biogas production

J Mater Cycles Waste Manag (2005) 7:48–54 DOI 10.1007/s10163-004-0125-y

© Springer-Verlag 2005

ORIGINAL ARTICLE M.S. Miah · Chika Tada · Yingnan Yang Shigeki Sawayama

Aerobic thermophilic bacteria enhance biogas production

Received: December 3, 2003 / Accepted: July 29, 2004

Abstract The enhancing effect of aerobic thermophilic (AT) bacteria on the production of biogas from anaerobically digested sewage sludge (methanogenic sludge) was investigated. Sewage sludge (5%, w/w) was incubated at 65°C with shaking for a few months to prepare the AT seed sludge. AT sludge was prepared by incubation of the AT seed sludge (5%, v/v) and sewage sludge (5%, w/w) at 65°C with shaking. The addition of this AT sludge (1.2% 0.5% of total volatile solids) to methanogenic sludge enhanced the production of biogas. The optimum volume of the addition and the pretreatment temperature of the AT sludge for optimum biogas production were 5% (v/v) and 65°C. Batchfed anaerobic digestion was covered with the addition of various AT sludges. The AT sludge prepared with the AT seed sludge improved the biogas production by 2.2 times relative to that from the sewage sludge addition. The addition of sludge without AT seed sludge weakly enhanced biogas production. An aerobic thermophilic bacterium (strain AT1) was isolated from the AT seed sludge. Strain AT1 grew well in a synthetic medium. The production of biogas from the anaerobic digestion of sewage sludge was improved by the addition of 5% (v/v) AT1 bacterial culture compared with that from the sewage sludge addition. The addition of AT1 culture reduced the volatile solids by 21%, which was higher than the 12.6% achieved with the sewage sludge addition. The AT1 bacterial culture enhanced the biogas production more than the AT seed sludge. The phylogenetic analysis of the 16 S rRNA gene revealed that strain AT1 is closely related to Geobacillus thermodenitrificans (100% sequence similarity). The improvement in the production of biogas with the AT sludge could be caused by thermophilic bacterial activity in the AT sludge.

M.S. Miah · C. Tada · Y. Yang · S. Sawayama (*) Biomass Group, Energy Technology Research Institute, National Institute of Advanced Industrial Science and Technology, 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan Tel./Fax 81-29-861-8184 e-mail: [email protected]

Key words Aerobic thermophilic bacteria · Anaerobic digestion · Sewage sludge · Bioenergy · 16 S rRNA

Introduction The vast amounts of municipal, industrial, and agricultural wastes that are released every day create serious environmental problems. Water pollution by solid waste disposal is also becoming a severe problem. To protect the environment, landfilling and ocean dumping are now discouraged. Because of its high content of organic matter, sewage sludge is used as a substrate for anaerobic digestion to recover biogas.1 Anaerobic digestion of organic wastes is an effective technology for both treatment and energy conversion. Pretreatment processes have been developed to improve the sludge degradation rate and handling. Different pretreatment processes have been studied; in particular heat treatment at 40°–180°C,2–4 chemical treatment,5 and mechanical disintegration and biological hydrolysis with or without enzyme addition.6 Hasegawa et al.7 reported that aerobic thermophilic (AT) bacteria have great potential as a cost-effective pretreatment for biological waste for anaerobic digestion, and allowing easy management of the sludge. An AT pretreatment system could be useful for the anaerobic digestion of sewage sludge.8,9 Thermal pretreatment and excreted enzymes were found to influence hydrolysis in anaerobic conditions.10–12 The treatment of municipal and industrial waste by AT bacteria was also studied.13 Several researchers have reported that thermal bacteria hydrolyze sludge, making it easily degraded in anaerobic conditions.14 Several researchers have been studying the aerobic thermophilic pretreatment of whole sewage sludge,7–9 and a fullscale aerobic and anaerobic thermophilic digestion plant for sewage sludge is operated in Tacoma, WA, USA. We investigated the effect of the addition of a small amount of AT bacterial sludge to methanogenic sludge to accelerate anaerobic digestion. We isolated AT bacteria and studied their enhancement of anaerobic digestion.

49 Fig. 1. Flow chart of the sewage treatment and sludges used in this experiment

Materials and methods Effect of AT sludge addition Sewage sludge and anaerobically digested and dewatered sludge (methanogenic sludge) were collected from a sewage treatment plant in Ibaraki, Japan (Fig. 1). The sewage sludge contained 11.2% of volatile solids (VS). The AT seed sludge (aerobic thermophilic bacteria) was prepared from the sewage sludge, which was diluted to 5% (w/w) with distilled water and kept at 65°C with shaking at 140 r.p.m. in a thermostat with a shaker for a few months. The AT sludge (aerobic thermophilic bacterial culture) was prepared from the AT seed sludge (5%, v/v) and the sewage sludge (5%, w/w) at 65°C with shaking for 5 days. The sewage sludge was used as a substrate for aerobic thermophilic bacteria. The anaerobic reactors (100 ml glass vials) received 32–39.6 ml methanogenic sludge. Various volumes (1%, 3%, 5%, 7%, 10%, and 20%) of AT sludge were added to the methanogenic sludge. The initial VS content in each reactor ranged from 0.85% to 0.87%. The reactors were then placed in a thermostat at 35°C. All the activity tests were carried out in triplicate. Biogas production and VS reduction were measured.

Effect of temperature on AT sludge incubation The AT sludge was prepared as previously described. To determine the optimal temperature for the AT bacteria, the AT sludge was maintained at 60, 65, and 70°C for 5 days with shaking at 140 r.p.m. Anaerobic reactors received 2 ml AT sludge and 38 ml methanogenic sludge. The volume of inoculum in AT sludge was 5% (v/v) of the total active volume in all cases. Biogas production and VS reduction were measured.

Batch-fed anaerobic digestion The AT sludge, sludge incubated at 65°C for 5 days without AT seed sludge, and sewage sludge (all 5%, v/v) were added as samples to the methanogenic sludge. Anaerobic reactors (500 ml glass vials) received a 200-ml mixture of methanogenic sludge and sample. The initial VS content in each reactor was 0.85%–0.87%. The air in the anaerobic reactors was replaced with nitrogen.The reactor was capped with a screw-cover cape, fitted with two inlets and outlets, and made air-tight for the anaerobic conditions. The contents of all reactors were stirred continuously. The reactors were kept in a thermostat at 35°C. In the batch-fed culture, 100 ml of the reactor contents was withdrawn, and 95 ml of methanogenic sludge and 5 ml of sample were added every 15 days. Biogas production was periodically measured by monitoring its volume on the measuring scale of a plastic syringe with a polyethylene gas container. The experiments were stopped after 45 days (i.e., after two batch–batch feeds).

Isolation of AT bacteria from AT seed sludge The AT seed sludge was diluted to 1 : 104 with distilled water. The diluted sample was spread on an F–JX agar plate. The F–JX agar contained yeast extract 0.1% (w/v), trypticase peptone 0.1% (w/v), and 3% (w/v) of a special type of agar (Taiyo-agar, GP-700, Shimizu Shokuhin, Japan).15 The pH of the F–JX medium was adjusted to 7.0. After the growth of bacterial colonies on the agar plate, an isolated colony was picked up from the plate and streaked onto another agar plate. This procedure was repeated at least five times to ensure a pure culture. The isolated pure strain of AT bacteria was designated strain AT1. Autoclaved culture medium (100 ml) was added to a 250-ml flask. The isolated colony was transferred from the plate into the flask. The flask culture was incubated at 65°C with shaking at

50

140 r.p.m. The growth of AT1 bacteria was measured every 12 h with a spectrophotometer at 660 nm.

Phylogenetic analysis DNA was extracted from strain AT1 with a FastPrep kit (Bio 101, Vista, CA, USA). A polymerase chain reaction (PCR) was run with a primer set of 530F (5-GTGCCA GCMGCCGCGG) and 1100R (5-TCTCGCTCGTTGCCTGACT-3).16 The PCR program consisted of 15 cycles, each of 1 min at 95°C, 1 min at 50°C, and 2 min at 72°C. The PCR amplification and cloning analysis were carried out according to Sekiguchi et al.17 The PCR products were then used for cloning according to the manufacturer’s protocol (TA cloning kit, Invitrogen, Carlsbad, CA, USA). The cloned DNA was sequenced with a rhodamin dye terminator cycle sequencing FS ready reaction kit (Applied Biosystems, Foster City, CA, USA) and an automated sequence analyzer (model 3700, Applied Biosystems). Sequences were compared with reported 16 S rRNA gene sequences by a nucleotide–nucleotide BLAST search of the GenBank DNA database.18 The best matching sequences were noted. A phylogenetic tree was constructed by the neighbor joining method using the MEGA V2.1 package.19,20

Accession numbers The organisms whose 16 S rRNA sequences were used for the phylogenic analysis (and their accession numbers) were Geobacillus thermodenitrificans (AB116114), G. subterraneus (AF276307), G. anatolicus (AF411064), G. stearothermophilus (AF478064), G. uralicus (AY079151), G. uzenensis (AF276304), G. gargaensis (AY193888), G. lituanicus (AY044055), Bacillus caldotenax (Z26922), G. sacchari (AY074879), G. thermoleovorans (AJ489329), G. kaustophilus (AF478063), G. thermoglucosidasius (X60641), G. toebii (AB116116), G. caldoxylosilyticus (AJ489326), Bacillus sp. TS-3 (AB063312), B. pallidus (Z26930), and B. smithii (Z26935).

Effect of AT1 bacterial culture on anaerobic digestion The isolated strain AT1 bacterium was grown in the F–JX medium at 65°C with shaking. The growth of AT1 was measured as its optical density at 660 nm. The incubation period was 36 h. To find out the effect of strain AT1 on the anaerobic digestion of sewage sludge, the AT1 bacterial culture, AT seed sludge, or sewage sludge were added at 5% (v/v) to the methanogenic sludge. Biogas production and VS reduction were measured.

Analytical methods The VS content was determined by heating at 105°C for 24 h, and by heating at 600°C for 1 h. The methane concentra-

Fig. 2. Cumulative biogas production from the anaerobic digestion of sewage sludge with various volumes of aerobic thermophilic (AT) sludge added. Open triangles, 1% AT sludge; closed circles, 3% AT sludge; diamonds, 5% AT sludge; open circles, 7% AT sludge; stars, 10% AT sludge; squares, 20% AT sludge; closed triangles, DW

tion of biogas was determined with a gas chromatograph (model GC-12A, Shimadzu, Japan) with a Porapak Q column (Shinwakakou, Japan) at 90°C. The volume of methane produced was given by multiplying the volume of the biogas produced by the methane concentration.

Results Effect of AT sludge addition on the anaerobic digestion of sewage sludge Figure 2 shows the cumulative biogas production from the anaerobic digestion of sewage sludge with various volumes of AT sludge added. The addition of 5% (v/v) AT sludge (1.2% 0.5% of total VS) to the methanogenic sludge produced the most biogas (22 ml/reactor). The addition of more AT sludge reduced the biogas production. The lowest volume of biogas (9 ml/ reactor) was produced when distilled water (DW) was added. Therefore, the addition of the AT sludge in the range tested to the methanogenic sludge enhanced biogas production. The methane concentration in the biogas was 50%–67%. A methane yield of 44 ml/g-VS was observed with the addition of 5% AT sludge, and a yield of 15 ml/g-VS was observed with the addition of 5% DW (Table 1). The addition of AT sludge with 1.2% 0.5% of total VS reduced VS by 17.2% (Table 1). The addition of DW only

51 Table 1. Methane production and volatile solids (VS) reduction with the addition of various volumes of aerobic thermophilic (AT) sludge to the methanogenic sludge Inoculum volume of AT sludge (v/v)

Methane production (ml/reactor)

Methane yield (ml/g-VS)

CH4 (%)

VS reduction (%)

1% 3% 5% 7% 10% 20% DW 5%

10 2.5 12 3.3 15 2.5 11 1.5 8 2.2 6 1.1 5 1.0

29 35 44 32 23 17 15

61 2 66 3 67 2 62 1 56 2 55 1 50 3

9.4 14.2 17.2 13 13 11 9.5

The VS content of the added AT sludge was 1.2% 0.5% of total VS in all cases DW, distilled water

Table 2. Methane production and VS reduction with the addition of AT sludge treated at 60–70°C Samples added to the methanogenic sludge



CH4 (%)

VS reduction (%)

AT sludge incubated at 60°C AT sludge incubated at 65°C AT sludge incubated at 70°C DW

17 1.3 22 1.1 19 1.5 10 1.2

49 64 55 29

70 1 72 2 71 1 65 2

14.7 17.6 16.2 10.4

The VS content of AT sludge was added at 1.1% 0.2% of total VS in all cases

reduced VS by 9.5%. This difference in VS reduction was greater than the VS with AT sludge added.

Optimum temperature for AT sludge Figure 3 shows the cumulative biogas production from the anaerobic digestion of sewage sludge containing 5% AT sludge (1.1% 0.2% of total VS) that had been incubated at 60°–70°C. The highest volume of biogas (30 ml/reactor) was produced with the addition of AT sludge that had been aerobically incubated at 65°C. The methane yield of 64 ml/gVS was observed with the addition of AT sludge incubated at 65°C, but a yield of 29 ml/g/VS was observed with the addition of DW (Table 2). The methane gas composition was 65%–72%. The VS reduction with the AT sludge incubated at 65°C was 17.6% (Table 2).

Effect of AT sludge addition to batch-fed anaerobic digestion Figure 4 shows the cumulative biogas production from a batch-fed anaerobic reactor with the addition of AT sludge. The addition of AT sludge to the methanogenic sludge produced a relatively high amount of biogas (136 ml/reactor). On the other hand, neither sewage sludge nor sludge without AT seed sludge enhanced biogas production. Table 3 shows the methane production and percentage of VS reduction under batch-fed anaerobic digestion. The methane gas concentration in the biogas was 80%–90%. The highest methane yield of 70 ml/g-VS was observed with the addition of AT sludge. The lowest methane yield of

Fig. 3. Cumulative biogas production from the anaerobic digestion of sewage sludge with additions of AT sludge incubated at 60°–70°C. Triangles, AT incubation at 60°C; closed circles, AT incubation at 65°C; diamonds, AT incubation at 70°C; open circles, DW

52 Table 3. Methane production and VS reduction under batch-fed anaerobic digestion Samples added to the methanogenic sludge



CH4 (%)

VS reduction (%)

AT sludge Sludge incubated at 65°C for 5 days without AT seed sludge Sewage sludge

121 3.1 69 1.5

70 40

90 1 86 2

18.8 13.6

56 1.6

33

80 2

13.1

The VS content of added AT sludge was 1.3% 0.2 % of total VS in all cases. The VS of the added sewage sludge was 2.2% 0.2% of the total VS

Table 4. Methane production and VS reduction with the addition of AT seed sludge and AT1 bacterial culture to the anaerobic digestion of sewage sludge Samples added to the methanogenic sludge



CH4 (%)

VS reduction (%)

AT seed sludge AT1 bacterial culture Sewage sludge DW

28 1.2 43 2.3 24 1.1 22 1.6

81 125 70 64

74 1 78 2 74 2 74 1

15.1 21 12.6 12.9

The VS of the added AT seed sludge was 0.5% 0.1% of the total VS. The VS of the added sewage sludge was 2.2% 0.2% of the total VS

showed that AT1 is related to Geobacillus thermodenitrificans (100% sequence similarity). The 16 S rRNA gene sequence of AT1 has been deposited in the DDBJ/EMBL/GenBank database under the accession number AB112914.

Effect of the addition of isolated strain AT1 bacterial culture

Fig. 4. Cumulative biogas production from a batch-fed anaerobic reactor with the addition of various AT sludges. Triangles, AT sludges; circles, sludge without AT seed sludge; diamonds, sewage sludge

33 ml/g-VS was observed with the addition of sewage sludge to the methanogenic sludge.

Phylogenetic analysis of isolated bacterial strain AT1 Strain AT1 was isolated from the AT seed sludge. A phylogenetic analysis showed a similarity to aerobic thermophilic bacteria (Fig. 5). Analysis of the 16 S rRNA gene sequence

Figure 6 shows biogas production from the anaerobic digestion of sewage sludge with the addition of AT seed sludge (0.5% 0.1% of total VS), AT1 bacterial culture, sewage sludge (2.2% 0.2% of total VS), and DW. Biogas was produced at 55 ml/reactor with the addition of AT1 bacterial culture. In contrast, only 32 ml/reactor of biogas was produced when the sewage sludge was added to the methanogenic sludge. The methane gas concentration in the biogas was 74%–78%. The highest methane yield of 125 ml/g-VS was observed with the addition of the AT1 bacterial culture (Table 4). The VS reduction was 21% when the AT1 bacterial culture was added to the methanogenic sludge (Table 4).

Discussion To maximize biogas production, the optimum inoculum volume of the AT sludge was 5% (1.2% VS). Production decreased as the inoculation volume of the AT sludge increased. Therefore, a higher volume of AT sludge inhibited methanogenesis. Hasegawa et al.7 reported that during sludge solubilization, AT bacteria excreted enzymes which actively enhanced biogas production. Schieder et al.11

53 Fig. 5. Phylogenetic tree of the isolated AT1 strain based on 16 S rRNA gene analysis. The tree was constructed by the neighbor-joining method. Numbers at nodes represent bootstrap values (100 replicates)

Fig. 6. Cumulative biogas production from the anaerobic digestion of sewage sludge with the addition of AT1 bacterial culture. Triangles, AT seed sludge; closed circles, AT1 culture; diamonds, sewage sludge; open circles, DW

reported that enzymes from thermal bacteria influenced the hydrolysis of the sludge during anaerobic digestion. These reports suggested that this enhancement of biogas production by the AT sludge is caused by the enzymatic effect of the AT bacteria. Biogas production was enhanced when the AT sludge was incubated at 65°C. Kume and Fujio14 reported that thermophilic bacteria excreted enzymes at high temperatures. Aerobic thermophilic bacteria are able to hydrolyze the sludge by producing extracellular enzymes. Hasegawa et al.7 reported that AT bacteria grew best at a temperature range of 60°–70°C. Blonskaji and Vaalu12 reported that the growth rate of thermal bacteria was high in the same temperature range. The incubation of the AT sludge at 65°C is therefore appropriate for the growth of AT bacteria in sludge. The VS reductions in the methanogenic sludge did not show any differences as a result of AT sludge incubation temperatures (60, 65, and 70°C). The addition of AT sludge and AT seed sludge enhanced biogas production. The addition of sludge without AT seed sludge to the methanogenic sludge weakly enhanced biogas production.The AT bacteria in the AT seed sludge appeared to play an important role in the enhancement of biogas production. Relatively high methane concentrations could be caused by relatively high pH (data not shown) in the liquid phase of the reactors. Biogas production with the addition of the AT1 bacterial culture was higher than that with the addition of the sewage sludge. The enhancement of biogas production could be caused by the AT1 bacteria in the AT sludge. Kume and Fujio14 reported that aerobic thermophilic bacteria, especially Bacillus sp., produce extracellular enzymes which

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act as a catalyst in improving the degradation efficiency of the organic matter in the sludge. The 16 S rRNA gene sequence analysis of strain AT1 showed that this isolate is likely Geobacillus thermodinitrificans. Therefore, our results indicate that G. thermodinitrificans could play an important role in the enhancement of biogas production by the addition of AT sludge. Further studies of AT1 are necessary to find the optimum conditions for the anaerobic digestion of the sewage sludge, and to understand how AT1 enhances the digestion.

Conclusions Our results allow us to draw the following conclusions. 1. The improvement of biogas production and VS reduction by the addition of AT sludge could be caused by aerobic thermophilic bacterial activity in the AT sludge. 2. The optimum additional volume and incubation temperature of AT sludge were 5% (v/v, 1.2% VS) and 65°C. 3. Geobacillus spp. could play an important role in the enhancement of biogas production by methanogenic sludge. Acknowledgments This work was partially supported by the Japan Society for the Promotion of Science (JSPS), Japan. We also thank Dr. T. Ogi, Dr. T. Yagishita, and Mr. Tsukahara for useful discussions.

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