Enriching hydrogen-producing bacteria from digested sludge by different pretreatment methods

The pretreatment of digested sludge by different methods, including ionizing irradiation, heat-shock, acid and base, was performed for enriching hydrogen-producing bacteria. These methods were evaluated and compared based on their suitability in the enrichment of hydrogen-producing bacteria in dark fermentation with glucose as a substrate in batch tests. The experimental results showed that the seed sludge pretreated by ionizing irradiation achieved the best hydrogen production among the different pretreatment methods, and the maximum hydrogen production potential, maximum hydrogen production rate, hydrogen yield and substrate degradation rate were 525.6 mL, 37.2 mL/h, 267.7 mL/g glucose (2.15 mol/mol glucose) and 98.9%, respectively. Ionizing irradiation can be a good optional pretreatment method for enriching hydrogen-producing bacteria from digested sludge. The effect of ionizing irradiation on the microbial community structure dynamics of the pretreated sludge deserves further study, which will help us to understand the mechanisms leading to the effect of high bio-hydrogen production.     

Enrichment of the hydrogen-producing microbial community from marine intertidal sludge by different pretreatment methods

To determine the effects of pretreatment on hydrogen production and the hydrogen-producing microbial community, we treated the sludge from the intertidal zone of a bathing beach in Tianjin with four different pretreatment methods, including acid treatment, heat-shock, base treatment as well as freezing and thawing. The results showed that acid pretreatment significantly promoted the hydrogen production by sludge and provided the highest efficiency of hydrogen production among the four methods. The efficiency of the hydrogen production of the acid-pretreated sludge was 0.86 ± 0.07 mol H₂/mol glucose (mean ± S.E.), whereas that of the sludge treated with heat-shock, freezing and thawing, base method and control was 0.41 ± 0.03 mol H₂/mol glucose, 0.17 ± 0.01 mol H₂/mol glucose, 0.11 ± 0.01 mol H₂/mol glucose and 0.20 ± 0.04 mol H₂/mol glucose, respectively. The result of denaturing gradient gel electrophoresis (DGGE) showed that pretreatment methods altered the composition of the microbial community that accounts for hydrogen production. Acid and heat pretreatments were favorable to enrich the dominant hydrogen-producing bacterium, i.e. Clostridium sp., Enterococcus sp. and Bacillus sp.. However, besides hydrogen-producing bacteria, much non-hydrogen-producing Lactobacillus sp. was also found in the sludge pretreated with base, freezing and thawing methods. Therefore, based on our results, we concluded that, among the four pretreatment methods using acid, heat-shock, base or freezing and thawing, acid pretreatment was the most effective method for promoting hydrogen production of microbial community.     

Gamma irradiation as a pretreatment method for enriching hydrogen-producing bacteria from digested sludge

Gamma irradiation was used as a pretreatment method for enriching hydrogen-producing bacteria from digested sludge. The experimental results demonstrated that 5.0 kGy was optimal dose among the different doses (0.5–10 kGy) applied in this study. The maximum cumulative hydrogen production, hydrogen yield, hydrogen production rate and substrate degradation efficiency of the sludge irradiated at such dose were 529.4 mL, 267.7 mL/g glucose, 37.25 mL/h and 98.9%, respectively when the fermentation conditions were as follows: at 36 °C, initial pH 7.0 and 10 g/L glucose as substrate. In comparison with the conventional pretreatment methods, such as heat-shock, acid, base, aeration and chloroform, gamma irradiation was more powerful pretreatment method for enriching hydrogen-producing bacteria. The effect of Gamma irradiation on the microbial community structure of the pretreated sludge needs further study.     

Extreme-thermophilic biohydrogen production by an anaerobic heat treated digested sewage sludge culture

Biohydrogen production process from glucose using extreme-thermophilic H₂-producing bacteria enriched from digested sewage sludge was investigated for five cycles of repeated batch experiment at 70 °C. Heat shock pretreatment was used for preparation of hydrogen-producing bacteria comparing to an untreated anaerobic digested sludge for their hydrogen production performance and responsible microbial community structures. The results showed that the heat shock pretreatment completely repressed methanogenic activity and gave the maximum hydrogen production yield of 355-488 ml H₂/g COD in the second cycle of repeated batch cultivation with more stable gas production during the cultivation when compared with control. Hydrogen production was accompanied by production of acetic acid. The average specific hydrogen in five cycles experiment ranged from 150 to 200 ml H₂/g VSS. PCR-DGGE profiling showed that the extreme-thermophilic culture predominant species were closely affiliated to Thermoanaerobacter pseudethanolicus.     

Enhanced hydrogen production in a UASB reactor by retaining microbial consortium onto carbon nanotubes (CNTs)

Biohydrogen and subsequent biomethane generation from biomass is a promising strategy for renewable energy supply, because this combination can lead to higher energy recovery efficiency and faster fermentation than single methane fermentation. Microbial consortium control by retaining hydrogen-producers through the addition of microbial carriers is an alternative to constructing hydrogen-producing reactors. Here we report the use of carbon nanotubes (CNTs) as microbial carriers to enhance microbial retention and the production of biohydrogen. Laboratory-scale upflow anaerobic sludge blanket (UASB) reactors with CNTs at 100 mg/L achieved a maximal hydrogen production rate of 5.55 L/L/d and a maximal hydrogen yield of 2.45 mol/mol glucose. Compared to frequently used activated carbon (AC) particles, CNTs resulted in quicker startup and better performance of hydrogen fermentation in UASB reactors. Scanning electron microscopy (SEM) and pyrosequencing results revealed that the reactor with CNTs led to a high proportion of hydrogen-producing bacteria among the microbial consortium, which endowed the microbes with strong flocculation capacity and hydrogen productivity.     

Biohydrogen using leachate from an industrial waste landfill as inoculum

Since hydrogen is a renewable energy source, biohydrogen has been researched in recent years. However, there is little data on hydrogen fermentation by a leachate from a waste landfill as inoculum. We investigated hydrogen production using a leachate from an industrial waste landfill in Kanagawa prefecture. The results showed no methane gas production and the leachate was a suitable inoculum for hydrogen fermentation. The maximum H₂ yield was 2.67 mol of H₂ per mol of carbohydrate added, obtained at 30 °C and initial pH 7. The acetate and butyrate production was significant when the H₂ yield was higher. The oxidation–reduction potential analysis of the culture suggested that hydrogen-producing bacteria in the leachate were facultatively anaerobic. Scanning electron microscope observations revealed hydrogen-producing bacteria comprised bacilli of about 2 μm in length.     

Improvement of hydrogen production with thermophilic mixed culture from rice spent wash of distillery industry

Biohydrogen production processes were investigated using thermophilic bacterial consortia enriched from sludge of the anaerobic digester. A multiple parameter optimization viz. temperature, pH and substrate concentration was performed for maximization of hydrogen production. Heat shock pre-treatment followed by BES (2-bromo ethane sulfonate) treatment was done for the enrichment of hydrogen producing bacteria. Box–Behnken design and response surface methodology were adopted to investigate the mutual interaction among the process parameters. Experimental optimization of process parameters (60 °C, pH 6.5 and 10 g/L) gave the maximum hydrogen production and yield of 3985 mL/L and 2.7 mol/mol glucose respectively in the batch system which is higher than the reported value on UASB. These experimental parameters found concurrent with the values obtained from the theoretical model i.e. 58.4 °C, pH 6.6, 10.8 g/L and yield of 2.71 mol/mol glucose. At optimized conditions, maximum hydrogen production rate (R_m) of 850 mL/h, gas production potential (P) of 4551 mL/L and lag time (λ) of 1.98 h were determined using modified Gompertz equation. Using the optimum conditions, hydrogen production from rice spent wash was conducted in which hydrogen yield of 464 mL/g carbohydrate and hydrogen production rate of 168 mL/L h were obtained. PCR-DGGE profile showed that the thermophilic mixed culture was predominated with species closely affiliated to Thermoanaerobacterium sp.     

Enhancement of biohydrogen producing using ultrasonication

Ultrasonication was evaluated as a pretreatment for biological hydrogen production from glucose in batch studies, in comparison with heat-shock pretreatment, acid pretreatment, and base pretreatment. The optimized sonication energy for hydrogen production using anaerobic digester sludge was 79 kJ/gTS. Sonication with temperature control (less than 30 °C) increased volumetric hydrogen production by 120% over the untreated sludge, and by 40% over the heat-shock and acid pretreated sludge, with a marginal (∼10%) increase in hydrogen production rate. Upon comparing the molar hydrogen yield in sonicated sludge with and without temperature control, the deleterious effect of heat on some hydrogen producers as reflected by a 30% decrease in yield to 1.03 mol H₂/mol glucose is evident. Sonication with temperature control affected a 45% increase in molar hydrogen yield to 1.55 mol H₂/mol glucose over heat-shock pretreatment at 70 °C for 30 min and acidification to pH 3.0 for 24 h at 4 °C. Sonication with temperature control produced a biomass yield of 0.13 g VSS/g COD, as compared to 0.24 g VSS/g COD for the untreated sludge. The hydrogen yield increased linearly with the molar acetate to butyrate ratio and decreased linearly with the biomass yield.     

Enhanced biohydrogen production from waste activated sludge in combined strategy of chemical pretreatment and microbial electrolysis

Cascade conversion methods are required to treat waste sludge (WAS) targeting at abundant biomass embedded in its cells and extracellular polymer. Two limiting factors have to overcome to obtain efficient conversion: (i) low release of soluble organics in raw WAS; (ii) limited conversion rate from organics to energy. Combined strategy of effective chemical pretreatment and microbial electrolysis was tested. Four kinds of chemicals (SDS, NaOH, peracetic-acid and β-cyclodextrin) were chosen to enhance volatile fatty acids (VFAs) production and following effects on hydrogen production and energy recovery by microbial electrolysis was further studied. The highest VFAs concentration was accumulated to 4712.69 mgCOD/L by β-CD within 3 days, which was increased to 4 times of unpretreated WAS. Other three chemicals respectively achieved ∼2.5-fold increase by SDS and PAA, and ∼2-fold increase by NaOH. However, the highest hydrogen yield was 8.5 mgH₂/gVSS with energy efficiency of 138% ± 8% by SDS pretreatment. The pretreatment substantially affects VFAs components, reflected on cascade changing of current and hydrogen production rate. The cascade conversion indicated that accumulation of acetate and propionate in SDS pretreatment benefited the most hydrogen production in combined strategy.     

Effects of pretreatment methods on cassava wastewater for biohydrogen production optimization

Batch production of biohydrogen from cassava wastewater pretreated with (i) sonication, (ii) OPTIMASH BG^® (enzyme), and (iii) α-amylase (enzyme) were investigated using anaerobic seed sludge subjected to heat pretreatment at 105 °C for 90 min. Hydrogen yield at pH 7.0 for cassava wastewater pretreated with sonication for 45 min using anaerobic seed sludge was 0.913 mol H₂/g COD. Results from pretreatment with OPTIMASH BG^® at 0.20% and pH 7 showed a hydrogen yield of 4.24 mol H₂/g COD. Superior results were obtained when the wastewater was pretreated with α-amylase at 0.20% at pH 7 with a hydrogen yield of 5.02 mol H₂/g COD. In all cases, no methane production was observed when using heat-treated sludge as seed inoculum. Percentage COD removal was found to be highest (60%) using α-amylase as pretreatment followed by OPTIMASH BG^® at 54% and sonication (40% reduction rate). Results further suggested that cassava wastewater is one of the potential sources of renewable biomass to produce hydrogen.     

Improvement of hydrogen production by transposon-mutagenized strain of Pantoea agglomerans BH18

Pantoea agglomerans BH18, isolated from mangrove sludge, could produce hydrogen under marine culture condition. To improve the hydrogen-producing capacity of this strain, we constructed a stable transposon-mutagenized library of P. agglomerans BH18. A Tn7-based transposon was randomly inserted into genomic DNA of P. agglomerans BH18. Mutants were identified by kanamycin resistance and amplification of the inserted transposon sequences. A transposon mutant, named as strain TB212, was screened for the highest hydrogen production ability. The total volume of hydrogen gas evolved by this mutant strain TB212 was 60% higher than that of the wild type. The mutant strain TB212 was able to produce hydrogen over a wide range of initial pH from 5.0 to 10.0, with an optimum initial pH of 7.0, and hydrogen production was 2.52 ± 0.02 mol H₂/mol glucose (mean ± S.E.) under marine culture condition. The mutant strain TB212 could produce hydrogen at the salt concentration from 3 to 6%. It was concluded that the transposon-mutagenized library may be a useful tool for investigation of high efficiency hydrogen-producing bacteria.     

Photocatalytic pretreatment for the redox conversion of waste activated sludge to enhance biohydrogen production

Photocatalytic pretreatment of waste activated sludge (WAS) using a flat photocatalytic reactor was undertaken. Photocatalytic pretreatment enhanced the release of soluble substances from WAS, in which the soluble protein and soluble carbohydrate concentration increased by about 50% and 80%, respectively. Significant removal of heavy metal ions from the liquid phase of WAS was also achieved after photocatalytic pretreatment. In addition, the highest hydrogen yield and the highest concentration of volatile fatty acids (VFAs) were achieved from the photocatalysis pretreated WAS by batch anaerobic digestion (55 °C). The cumulative hydrogen yield from photocatalysis pretreated WAS was 211.0 ml/l-sludge, much higher than those from UV pretreated WAS (111.0 ml/l-sludge) and from raw WAS (93.0 ml/l-sludge). The results indicate that photocatalysis is a promising WAS pretreatment method for the enhancement of biohydrogen production, probably due to the photo-oxidation of organics and simultaneous photo-reduction of heavy metal ions in WAS.     

Microbial community diversity during fermentative hydrogen production inoculating various pretreated cultures

This study adopted five pretreatment means (base, aeration, γ-radiation, acid and heat-shock) for enriching hydrogen-producing bacteria from anaerobically digested sludge, aiming to investigate the microbial community diversity during fermentative hydrogen production using various pretreatments as inoculum. The experimental results indicated that all five pretreatments could effectively enrich hydrogen-producing bacteria from the seed sludge, while the microbial communities showed a great difference among various pretreated groups. The most three dominant genera were Paraclostridium (28.6%), Clostridium sensu stricto 1 (19.8%) and Terrisporobacter (19.4%) for base pretreated group, Enterococcus (67.2%), Clostridium sensu stricto 1 (10%) and Citrobacter (5.6%) for aeration pretreated group, Clostridium sensu stricto 1 (63.9%), Paeniclostridium (9.3%) and Romboutsia (7%) for γ-radiation pretreated group, Clostridium sensu stricto 1 (51.9%), Romboutsia (22.4%) and Paeniclostridium (8.2%) for acid pretreated group, and Paraclostridium (61.2%), Exiguobacterium (23.1%) and Clostridium sensu stricto 1 (8.1%) for heat-shock pretreated group, respectively. Different microbial communities resulted in diverse process performance and metabolic pathway. Heat-shock pretreatment achieved the maximum hydrogen yield of 1.58 mol/mol-glucose and maximum hydrogen production rate of 37.65 mL/h. The dominance of genus Paraclostridium was supposed to be responsible for the highest hydrogen production.     

Comparison of different pretreatment methods for hydrogen production using environmental microbial consortia on residual glycerol from biodiesel

The pretreatment of environmental microbial consortia by five methods (acid, base, heat shock, dry heat and desiccation, freezing and thawing) was conducted in order to evaluate their applicability for the selection of hydrogen-producing bacteria capable of using residual glycerol from biodiesel synthesis as substrate. Results showed that substrate degradation rates of consortia pretreated with dry heat and desiccation and heat shock were higher compared with controls during the fermentation using glycerol, with degradation rates as high as 65%. The maximal hydrogen and biomass productions were obtained by dry heat and desiccation: 34.19% mol and 4340 mg/L, respectively. Dry heat and desiccation followed by heat shock are simple pretreatments methods that can be used to improve the biotechnological production of hydrogen. DNA sequencing performed to identify the bacteria strains present in the consortium showed that they belonged to the genus Klebsiella and Pantoea.     

Enhanced biohydrogen production from tofu residue by acid/base pretreatment and sewage sludge addition

Anaerobic dark fermentation is considered a promising technology for clean energy production and waste reduction. In the present work, tofu residue and sewage sludge were utilized as substrates for fermentative hydrogen production. To increase the biodegradability, tofu residue was pretreated for 30 min in the presence of HCl and NaOH at various concentrations (0, 0.5, 1.0, and 2.0%), and then fermented by a thermophilic (60 °C) mixed culture. The solubility (SCOD/TCOD ratio) of the tofu residue increased from 4 to 30–40% after pretreatment, and the increased soluble constituents were mainly protein rather than carbohydrate compounds. However, in spite of a slight increase of carbohydrate solubility, the H₂ production performance was significantly enhanced by pretreatment, owing to the degradability of thermophilic cultures used on insoluble tofu residues. The limited H₂ yield of 0.30 mol H₂/mol hexose_added achieved in the raw tofu residue was increased 1.6- to 4-fold with the highest H₂ yield of 1.25 mol H₂/mol hexose_added at 1.0% HCl concentration. Carbohydrate degradation and the H₂ production rate also increased from 39 to 50–65% and 27 to 50–120 mL H₂/L/h, respectively. The role of pretreatment was not only to increase the biodegradability but also to suppress the activity of indigenous non H₂-producers such as lactic acid bacteria and propionic acid bacteria. When sewage sludge was added to acid pretreated (1.0% HCl) tofu residue as a co-substrate, the H₂ yield and H₂ production rate increased to 1.48 mol H₂/mol hexose_added and 161 mL H₂/L/h, respectively, which was attributed to the abundant minerals, vitamins, and metals contained in sewage sludge.     

Impact of operational conditions on development of the hydrogen-producing microbial consortium in an AnSBBR from cassava wastewater rich in lactic acid

Biohydrogen production from cassava starch wastewater was evaluated in anaerobic sequencing batch biofilm reactor (AnSBBR) using different inoculum (mixed cultures from naturally fermented wastewater and anaerobic sludge thermally treated) and feeding strategies (batch and fed-batch). The highest hydrogen productivity (2.4 LH₂ L⁻¹ d⁻¹) and yield (11.7 molH₂ kg⁻¹Carbohydrates) were verified in low and intermediate organic load rates (12 and 14 g L⁻¹ d⁻¹) and longer cycle time (4 h), respectively. The productivity was favored by fed-batch strategy, and yield by batch. The hydrogen production was verified in both inoculum sources. However, in the assays inoculated from naturally fermented wastewater, with higher organic load rate (18 g L⁻¹ d⁻¹) and intermediate cycle time (3 h) no hydrogen was observed, regardless the feeding strategy, indicating that the inhibitory effects of the indigenous microorganisms present in cassava starch wastewater were more expressive in these conditions. The operational conditions applied to hydrogen production in AnSBBR from cassava starch wastewater may influence the microflora development in the reactor. In this study three possible scenarios were verified: hydrogen-producing bacteria (HPB) growth; hydrogen-producing bacteria inhibition or coexistence between ones and lactic acid bacteria (LAB), which are autochthones of this wastewater.     

Enhanced methane production using pretreated sludge in MEC-AD system: Performance,microbial activity,and implications at different applied voltages

Although various pretreatment methods are employed to promote sludge hydrolysis and thereby promoting methane production in the subsequent microbial electrolysis cell assisted anaerobic digestion (MEC-AD) system, the questions arise are, “which pretreatment method on waste activated sludge (WAS) maximises the sludge hydrolysis and what is the optimal applied voltage on anaerobic digestion (AD) to stimulates the direct interspecies electron transfer (DIET) performance and thereby accelerating the methane production fed with pretreated WAS?” was still unanswered. Herein, firstly, a series of pretreatment methods to hydrolyse and mineralise the organic matter of WAS was performed to evaluate solubilization efficiency and thereafter, the influence of different applied voltages (0.3 V, 0.6 V, and 0.9 V) on coupled MEC-AD reactors fed with pretreated WAS was investigated to apprehend the DIET promotion for methane production. The results indicated that in MEC-AD reactors, the methane yield increased by 27.2%, 44.8%, and 37.3% when the applied voltages were 0.3 V, 0.6 V, and 0.9 V, respectively. Therefore, the alkaline-thermal pretreatment (ATP) enhanced the sludge hydrolysis in WAS, followed by an applied voltage of 0.6 V in the MEC-AD reactor fed with pretreated WAS, enhanced methane production under DIET stimulation induced by the increased abundance of electroactive microorganisms (EAM) and the advanced electron transfer. Besides, the energy balance estimation validates that with an applied voltage of 0.6 V in MEC-AD could achieve higher net energy input.     

Optimization of high-solid waste activated sludge concentration for hydrogen production in microbial electrolysis cells and microbial community diversity analysis

To enhance hydrogen recovery from high-solid waste activated sludge (WAS), microbial electrolysis cells (MECs) were used as an efficient device. The effects of WAS concentrations were firstly investigated. Optimal concentration for hydrogen production was 7.6 g VSS/L. Maximum hydrogen yields reached to 4.66 ± 1.90 mg-H₂/g VSS and 11.42 ± 2.43 mg-H₂/g VSS for MECs fed with raw WAS (R-WAS) and alkaline-pretreated WAS (A-WAS) respectively, which was much higher than that obtained traditional anaerobic digestion. Moreover, no propionic acid accumulation was achieved at the optimal concentration. Effective sludge reduction was also achieved in MECs feeding with A-WAS. 52.9 ± 1.3% TCOD were removed in A-WAS MECs, meanwhile, protein degradation were 50.4 ± 0.8%. The 454 pyrosequencing analysis of 16S rRNA gene revealed the syntrophic interactions were existed between exoelectrogen Geobacter and fermentative bacteria Petrimonas, which apparently drove the efficient performance of MECs fed with WAS.     

Biohydrogen production by anaerobic co-digestion of municipal food waste and sewage sludges

Food waste (FW), primary sludge (PS) and waste activated sludge (WAS) were characterized and found to be complementary in the concentrations of carbohydrates, total Kjeldahl nitrogen (TKN), PO₄–P and some metal for biological hydrogen production. Moreover, FW was found to have low pH buffering capacity while the values for PS and WAS were relatively higher. An anaerobic toxicity analysis (ATA) derived from a methanogenic ATA protocol showed that these waste materials had no toxicity to hydrogen production. Adding phosphate buffer to the FW significantly improved hydrogen production while initial pH was 7.0. Co-digestion of FW and sewage sludge was studied using a batch respirometric cultivation system. All combinations of the feedstocks (FW+PS, FW+WAS and FW+PS+WAS) showed enhanced hydrogen production potential as compared with the individual wastes. A mixing ratio of 1:1 was found to be the best among the ratios tested for all three co-digestion groups. A hydrogen yield of 112 mL/g volatile solid (VS) added was obtained from a combination of FW, PS and WAS. This yield was equivalent to 250 mL/g VS added if only FW contributed to hydrogen production. The reason for the enhancement of hydrogen production was postulated to be multifold in which the increase in buffer capacity in the co-digestion mixture was verified.     

Isolation and characterization of a novel hydrogen-producing strain Clostridium sp. suitable for immobilization

A hydrogen-producing strain of bacteria suitable for immobilization was isolated from anaerobic sludge obtained from a methane fermentation plant. The isolated strain, CFPA-20 was identified as a novel species of the genus Clostridium by phylogenetic analysis of the 16S rRNA sequence. The changes in free energy of interaction of adhesion to polymer resin and self-aggregation were both negative. This indicated that CFPA-20 was thermodynamically favored for immobilization. CFPA-20 grew at a temperature range of 25–37 °C and at a pH range of 4.5–9.0. Immobilization of CFPA-20 on block copolymer polyethylene glycol-b-polypropylene glycol gave a radically improved hydrogen production yield (2.91 mol/mol-glucose) and a maximum hydrogen production rate (568 mL/L-culture/h) compared to the non-immobilized isolate. In addition, the biofilm of CFPA-20 acquired tolerance for volatile fatty acids. Further investigation into this mechanism may ultimately improve the hydrogen production capacity of CFPA-20.