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.     

Evaluation of different pretreatment methods for preparing hydrogen-producing seed inocula from waste activated sludge

Waste activated sludge (WAS) is the most favorable inoculum for dark fermentative hydrogen-producing processes, because it can be collected economically. In order to accelerate the start-up process and develop the efficiency and stability of a hydrogen production system, pretreatment of the seed sludge has been examined to enrich hydrogen-producing bacteria. Six pretreatment methods including acid, base, heat-shock, aeration, chloroform and 2-bromoethanesulfonate (BES) were performed on WAS in batch cultures utilizing glucose as the substrate. The results showed that, at 35 °C and initial pH of 7.0, hydrogen yields of the pretreated sludge (except for BES) were higher than the control test. The pretreatment methods resulted in different distributions of soluble metabolites. Acid pretreatment at pH of 3 was the best among all six pretreatment methods, and the maximal hydrogen yield of 1.51 mol/mol-glucose-consumed and the maximal specific hydrogen production of 22.81 mmolH₂/g VSS were observed. The hydrogen yield of the acid treated sludge increased to 1.82 mol/mol-glucose-consumed after five repeated-batch cultivations. It was concluded that acid pretreatment is a simple, economic and effective method for enriching hydrogen-producing bacteria from WAS.     

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.     

Evaluation of alternative methods of preparing hydrogen producing seeds from digested wastewater sludge

Six digested sludges, pre-treated by different methods (heat-shock, aeration, acid and base treatments, 2-bromoethanesulfonic acid (BESA) inhibition and iodopropane inhibition) as well as an untreated sample were compared for their suitability in the preparation of hydrogen producing seeds by cultivations in a sucrose medium. The heat-shock and acid treatment methods completely repressed methanogenic activity; however, they also partially repressed hydrogen production. The base treatment option did not completely repress methanogenic activity and also significantly impacted hydrogen production. The aeration method was unsuccessful at completely repressing methanogenic activity; however, it did not significantly affect the hydrogen production activity. The BESA and iodopropane pre-treatment methods specifically inhibited the methanogens, and there were no significant effects found on hydrogen production. Similar to the aeration pre-treated digested sludge sample, the untreated sludge showed high hydrogen production activity and a small amount of methanogenic activity (lower than the activity detected in the base treatment sample). In the subsequent second-step batch cultivations with the same sucrose medium and the diluted media, methanogenic activity was not detected in any of the test bottles. The microbial seed prepared from base treatment exhibited the highest hydrogen production activity, whereas those prepared from acid treatment did not exhibit any activity. Again, the microbial seed prepared from untreated sludge also exhibited relatively high hydrogen producing activity. A lower pH was detected at the end of the cultivation in all the test bottles. Interestingly, the variations in pH in the different tests bottles indicate that pH is an important parameter in the control of methanogenic activity.     

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.     

The effect of heat pretreatment temperature on fermentative hydrogen production using mixed cultures

The effect of heat treatment at different temperatures on two types of inocula, activated sludge and anaerobically digested sludge, was investigated in batch cultures. Heat treatments were conducted at 65, 80 and 95 °C for 30 min. The untreated inocula produced less amount of hydrogen than the pretreated inocula, with lactic acid as the main metabolite. The maximum yields of 2.3 and 1.6 mol H₂/mol glucose were achieved for the 65 °C pretreated anaerobically digested and activated sludges, respectively. Approximately a 15% decrease in yield was observed with increasing pretreatment temperature from 65 to 95 °C concomitant with an increase in butyrate/acetate ratio from 1.5 to 2.4 for anaerobically digested sludge. The increase of pretreatment temperature of activated sludge to 95 °C suppressed the hydrogen production by lactic acid fermentation. DNA analysis of the microbial community showed that the elevated pretreatment temperatures reduced the species diversity.     

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.     

木糖厌氧发酵产氢特性研究

以木糖作为厌氧发酵产氢底物,热预处理(100℃,处理20 min)的厌氧颗粒污泥作为接种物,研究了中温条件(37℃)下厌氧发酵产氢特性.结果表明,当反应进行至50 h时,累积产氢量最大,为81.11 mL;乙酸、丁酸和乙醇是液相末端产物中的主要物质,其中乙酸和丁酸的浓度分别为1290 mg/L和1225 mg/L,发酵类型是典型的丁酸型发酵;反应体系的pH值开始降低,最后稳定在4.40左右,形成一个稳定的缓冲体系.     

Heat-shock treatment applied to inocula for H2 production decreases microbial diversities,interspecific interactions and performance using cellulose as substrate

The heat-shock pretreatment (HST) is a useful method to select for H₂-producing inocula when soluble substrates are employed. However, the HST has proven to have negative effects on the H₂ production performance from lignocellulosic substrates. We hypothesize that the negative effect of HST on H₂ production from lignocellulosic substrates is due to the loss of species involved in cellulose solubilization. In the present study, we tested this hypothesis by applying a heat-shock pretreatment (105 °C/24 h) on the microbial community for producing hydrogen from microcrystalline cellulose. Specifically, we compared a microbial community treated with 2-bromoethanesulfonate (BES-treated control) versus a heat-shock pretreated microbial community. For both experimental treatments, we determined the major fermentation products (hydrogen, acetic, butyric, propionic, and isovaleric acids), as well as the diversity of bacteria and fungi using Illumina MiSeq of amplicons in five sampling points. We found that HST immediately reduced alpha diversity of microbial communities, being fungi more affected than bacteria. We also found that the bacterial reduction in Comamonas, Ureibacillus, and Aneurinibacillus was related to a low hydrogen production in the heat-shock pretreated community. Strictly anaerobic fungi such as Orpinomyces, Cyllamyces, and Neocallimastix, which are recognized by their role in solubilization of fibrous materials, were unable to survive the HST. The reconstructed bacterial network predicted positive interactions between cellulase-producing and hydrogen-producing families. We conclude that the HST did not promote the high microbial diversity required for hydrogen production from cellulose.     

Effects of various pretreatment methods of anaerobic mixed microflora on biohydrogen production and the fermentation pathway of glucose

The influence of different pretreatment methods on anaerobic mixed inoculum was evaluated for selectively enriching the hydrogen (H₂) producing mixed culture using glucose as the substrate. The efficiency of H₂ yield and the glucose fermentation pathway were found to be dependent on the type of pretreatment procedure adopted on the parent inoculum. The H₂ yield could be increased by appropriate pretreatment methods including the use of heat, alkaline or acidic conditions. Heat pretreatment of the inoculum for 30 min at 80 °C increased the H₂ yield to 53.20% more than the control.When the inoculum was heat-pretreated at 80 °C and 90 °C, the glucose degraded via ethanol (HEt) and butric acid (HBu) fermentation pathways. The degradation pathways shifted to HEt and propionate (HPr) types as the heat pretreatment temperature increased to 100 °C. When the inoculum was alkali- or acid-pretreated, the fermentation pathway shifted from glucose to a combination of the HPr and HBu types. This trend became obvious as the acidity increased. As the fermentation pathway shift from the HEt type to the HPr and HBu types, the H₂ yield decreased.     

Effects of pretreatments of anaerobic sludge and culture conditions on hydrogen productivity in dark anaerobic fermentation

Effects of various pretreatments of the anaerobic sludge on hydrogen production were investigated for enhancing hydrogen production via dark anaerobic fermentation of organic substrates. The pretreatment methods included heat treatment, acid or base treatment, acid/heat treatment and base/heat treatment. After the pretreatment of the anaerobic sludge, productivity of H₂ by the pretreated sludge were evaluated when the culture were grown on glucose-based medium at ambient conditions. The detailed experimental investigation showed highest production of H₂ by the heat-treated culture followed by the heat and acid-treated culture and the heat and base-treated culture.     

Hydrogenase activity monitoring in the fermentative hydrogen production using heat pretreated sludge: A useful approach to evaluate bacterial communities performance

The conversion of organic compounds into H₂ has received increasing attention. Enrichment of inocula by heat pretreatment eliminates non-spore forming hydrogen consuming microorganisms and promotes spore germination in genus Clostridium, which is known as one of the key hydrogen producers. Useful information about metabolic pathway is provided by some intermediate metabolites, such as: acetic, propionic, butyric and formic acids. The increase of acetic/butyric acids ratio indicates H₂ production in heat pretreated inoculum when compared to untreated inoculum in the same cultivation conditions. The effect of heat pretreatment on inocula and consequently on the performance of bacterial communities responsible for H₂ production was monitored through the measurement of the level of hydrogenase gene expression, as well as through the content and distribution of volatile fatty acids. The acetic acid type fermentation was followed by the microorganisms presented in untreated and heat pretreated sludge. The medium containing untreated sludge presented a ratio of acetic/butyric acid of approximately 4, the same parameter was 7 when heat pretreated sludge was employed. The level of hydrogenase gene expression tripled when heat pretreated inoculum was used, indicating a higher production of H₂.     

预处理温度对活性污泥发酵产氢特性的影响

为寻求适宜的种泥热处理方法,利用摇瓶发酵实验,考察了城市污水处理厂好氧活性污泥分别经65、80、95、110℃热处理30min后,其利用葡萄糖发酵产氢的特性。结果表明:在初始pH=7.0、葡萄糖浓度10g/L、接种量2gMLVSS/L条件下,35℃培养72h,经65℃和95℃处理的种泥表现出较好的发酵产氢性能,其葡萄糖的氢气转化率分别达到1.08和1.11mol/mol,污泥的比产氢率分别为8.36和9.05mmol/gMLVSS;经65℃预处理的种泥发酵体系,表现为丁酸型发酵,其葡萄糖降解率和最大产氢速率分别高达82%和11.29mL/h,而经95℃预处理的种泥发酵体系则呈现混合酸发酵特征,其葡萄糖转化率和最大产氢速率分别仅为76%和4.45mL/h。     

厨余和污泥不同混合比例碱处理产氢特性研究

以厨余垃圾和污泥为反应底物,加热预处理的污泥为发酵接种物,考察了碱处理下厨余与污泥不同混合比例的发酵产氢特性。结果表明:不同pH碱液对厨余垃圾进行预处理后,其效果以pH=13时最佳,预处理3h后SCOD和还原糖含量分别为31316.8mg/L和5.54mg/mL;碱预处理后的污泥与厨余联合发酵能够改善物料的营养平衡,缩短反应延迟时间到1h内;当厨余与污泥混和比例为5:1时为本试验最佳的试验条件,其氢气含量、比产氢速率峰值和氢产率分别为52.69%,1.73mL H_2/(h·gVS)和50.27mL H_2/gVS。     

Influence of thermal and chemical pretreatment on structural stability of granular sludge for high-rate hydrogen production in an UASB bioreactor

Fermentation of organic waste materials presents an alternate route instead of photosynthetic and chemical routes for hydrogen production. Low yield of biohydrogen production is the major challenge in the fermentative hydrogen-producing technology. Improvement of fermentation process by various sludge pretreatment methods is one of the ways that have been applied to boost hydrogen productivity. This study sheds new light on the impact of thermal and chemical pretreatments on the hydrogen-producing granular sludge morphology and strength as well as up-flow anaerobic sludge blanket (UASB) reactor performance treating palm oil mill effluent (POME). Thermal pretreatment showed devastating effects on the morphological and structural characteristics of the granules. However, the chemically pretreated granules remained structurally stable and relatively undamaged. The thermal pretreatment increased the cumulative hydrogen production by 40% and 76% over chemical pretreatment and control test (untreated), respectively.     

Regulation of biohydrogen production by heat-shock pretreatment facilitates selective enrichment of Clostridium sp.

The effect of heat-shock treatment to selectively enrich acidogenic, H₂ producing consortia was investigated for inoculum preparation and to control the process operation. Long term operation (520 days) in suspended-batch mode bioreactors illustrated relative efficiency and feasibility of heat-shock treated consortia (15.78 mol/kg COD_R) in enhancing H₂ production (3.31 mol/kg COD_R) when compared to parent (control) consortia. On the contrary, substrate degradation was higher in the control operation (ξ_COD, 62.86%; substrate degradation rate (SDR), 1.34 kg COD_R/m³-day) compared to heat-shock operation (ξ_COD, 52.6%; SDR, 1.10 kg COD_R/m³-day). Heat-shock pretreatment has resulted in a marked fermentation pathway shift towards acetic-butyric acid type production. The microbial diversity illustrated dominance in the Clostridia class after applying heat-shock pretreatment. The redox catalytic currents and Tafel analysis strongly support the conclusion of an improved biocatalyst performance after pretreatment with regards to H₂ production.     

Novel dark fermentation involving bioaugmentation with constructed bacterial consortium for enhanced biohydrogen production from pretreated sewage sludge

The present study summarizes the observations on various nutrient and seed formulation methods using sewage sludge that have been aimed at ameliorating the biohydrogen production potential. Pretreatment methods viz., acid/base treatment, heat treatment, sterilization, freezing–thawing, microwave, ultrasonication and chemical supplementation were attempted on sludge. It was observed that pretreatment was essential not only to reduce the needless, competitive microbial load but also to improve the nutrient solublization of sludge. Heat treatment at 121 °C for 20 min was found to be most effective in reducing the microbial load by 98% and hydrolyzing the organic fraction of sludge. However, this pretreatment alone was either not sufficient or inconsistent in developing a suitable microbial consortium for hydrogen production. Hydrogen yield was found to improve 1.5–4 times upon inoculation with H₂-producing microorganisms. A defined microbial consortium was developed consisting of three established bacteria viz., Enterobacter cloacae IIT-BT 08, Citrobacter freundii IIT-BT L139 and Bacillus coagulans IIT-BT S1. Following pretreatments soluble proteins and lipids (the major component of the sludge) were also found to be consumed besides carbohydrates. This laid out the concurrent proteolytic/lipolytic ability of the developed H₂-producing consortium. 1:1:1 v/v ratio of these bacteria in consortium was found to give the maximum yield of H₂ from sludge, 39.15 ml H₂/g COD_reduced. 15%v/v dilution and supplementation with 0.5%w/v cane molasses prior to heat treatment was found to further improve the yield to 41.23 ml H₂/g COD_reduced.     

Influence of seed sludge and pretreatment method on hydrogen production in packed-bed anaerobic reactors

Several studies have reported on the effects of inoculums source and pretreatment on biological hydrogen production. However, there have been few studies on continuous reactors. This paper investigated the influence of different seed sludge sources and pretreatment methods on biohydrogen production in up-flow anaerobic fixed-bed reactors fed with sucrose. The following inoculum sources were included in the study: (1) anaerobic sludge from an up-flow anaerobic sludge blanket (UASB) reactor used to treat poultry slaughterhouse wastewater (Sl), (2) anaerobic sludge from a UASB reactor used to treat swine wastewater (Sw) and (3) autofermentation (A). Heat (He) and acid (Ac) shock were used to increase hydrogen production and suppress hydrogen consumption. The average hydrogen yields (HY) in the experiment were 2.1 (A), 2.0 (SlHe), 2.0 (SlAc), 1.0 (Sl), 1.0 (SwAc), 0.7 (SwHe) and 0.7 (Sw) mol H₂ mol⁻¹ sucrose. Although heat shock produced the maximum HY value (SlHe), acid pretreatment (SlAc) resulted in more stable hydrogen production with the largest average value, which could be an advantage of using pH shock. The autofermentation process presented HY values similar to those produced with SlAc and SlHe, making it a suitable seed sludge for biohydrogen production because pretreatment was not required.     

Fermentative hydrogen production by conventionally and unconventionally heat pretreated seed cultures: A comparative assessment

In this study, the effects of pretreatment temperature and time during conventional and unconventional, microwave-assisted heat shock on the hydrogen producing capability of anaerobic seed sludge from soluble starch was focused. It was found that the different heat transfer techniques resulted in seed cultures with comparable hydrogen production potentials, with the highest obtainable values of approximately 0.9 L H₂/L-d. A comprehensive, statistical analysis revealed that both treatment temperature and time could be designated as significant process variables, however, in distinguishable extents for the two alternative methods. The results indicated that microwave-based sludge pretreatment needed remarkably shorter curing times (2 min) to eliminate H₂-consuming, methanogenic activity in comparison to the conventional heat shock method (30 min). It was also demonstrated that microwave irradiation increased the soluble organic matter content in the seed sludge.