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.     

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.     

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。     

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.     

Biohydrogen production from dual digestion pretreatment of poultry slaughterhouse sludge by anaerobic self-fermentation

Poultry slaughterhouse sludge from chicken processing wastewater treatment plant was tested for their suitability as a substrate and inoculum source for fermentation hydrogen production. Dual digestion of poultry slaughterhouse sludge was employed to produce hydrogen by batch anaerobic self-fermentation without any extra-seeds. The sludge (5% TS) was dual digested by aerobic thermophilic digestion at 55 °C with the varying retention time before using as substrate in anaerobic self-fermentation. The best digestion time for enriching hydrogen-producing seeds was 48 h as it completely repressed methanogenic activity and gave the maximum hydrogen yield of 136.9 mL H₂/g TS with a hydrogen production rate of 2.56 mL H₂/L/h. The hydrogen production of treated sludge at 48 h (136.9 mL H₂/g TS) was 15 times higher than that of the raw sludge (8.83 mL H₂/g TS). With this fermentation process, tCOD value in the activated sludge could be reduced up to 30%.     

Effect of acid,heat and combined acid-heat pretreatments of anaerobic sludge on hydrogen production by anaerobic mixed cultures

Activated sludge (AS) from wastewater treatment plant of brewery industry was used as substrate for hydrogen production by anaerobic mixed cultures in batch fermentation process. The AS (10% TS) was pretreated by acid, heat and combined acid and heat. Combined acid- heat treatment (0.5% (w/v) HCl, 110 °C, 60 min) gave the highest soluble COD (sCOD) of 1785.6 ± 27.1 mg/L with the highest soluble protein and carbohydrate of 8.1 ± 0.1 and 38.5 ± 0.8 mg/L, respectively. After the pretreatment, the pretreated sludge was used to produce hydrogen by heat treated upflow anaerobic sludge blanket (UASB) granules. A maximum hydrogen production potential of 481 mL H₂/L was achieved from the AS pretreated with acid (0.5% (w/v) HCl) for 6 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。     

Biohydrogen production with fixed-bed bioreactors

An investigation on anaerobic hydrogen production was conducted in fixed-bed bioreactors containing hydrogen-producing bacteria originated from domestic sewage sludge. Three porous materials, loofah sponge (LS), expanded clay (EC) and activated carbon (AC), were used as the support matrix to allow retention of the hydrogen-producing bacteria within the fixed-bed bioreactors. The carriers were assessed for their effectiveness in biofilm formation and hydrogen production in batch and continuous modes. It was found that LS was inefficient for biomass immobilization, while EC and AC exhibited better biomass yields. The fixed-bed reactors packed with EC or AC (denote as EC or AC reactors) were thus used for continuous hydrogen fermentation at a hydraulic retention time (HRT) of 0.5–5 h. Sucrose was utilized as the major carbon source. With a sucrose concentration of ca. 20 g COD/l in the feed, the EC reactor (workingvolume=300 ml) was able to produce H₂ at an optimal rate of 0.415 l/h/l at HRT=2 h. In contrast, the AC reactor (300 ml in volume) exhibited a better hydrogen production rate of 1.32 l/h/l, which occurred at HRT=1 h. When the AC reactor was scaled up to 3 l, the hydrogen production rate was nearly 0.53–0.68 l/h/l for HRT=1–3 h, but after a short thermal treatment (75°C, 1 h) the rate rose to ca. 1.21 l/h/l at HRT=1 h. The biogas produced with EC and AC reactors typically contained 25–35% of H₂ and the rest was mainly CO₂, while production of methane was negligible (less than 0.1%). During the efficient hydrogen production stage, the major soluble metabolite was butyric acid, followed by propionic acid, acetic acid, and ethanol.     

Effect of Ni2+ concentration on fermentative hydrogen production using waste activated sludge as substrate

The influence of Ni²⁺ concentration on biohydrogen production was investigated using waste activated sludge as substrate. The degradation of substrate, accumulation of volatile fatty acids (VFAs) and distribution of microbial community were analyzed to provide information for influencing mechanisms of Ni²⁺ addition. The experimental results demonstrated that the efficiency of hydrogen fermentation from waste activated sludge could be significantly improved. The optimal Ni²⁺ concentration was 5 mg/L, and under this concentration, the cumulative hydrogen production was 1.29 times of the control group. The degradation of soluble chemical oxygen demand (SCOD) increased from 25.21% to 27.69% when the added Ni²⁺ concentration was 5 mg/L. The analysis of microbial community distribution revealed that Ni²⁺ decreased the microbial diversity, and provided more suitable condition for the microbial growth and activity of hydrogen-producers. Citrobacter was the dominant hydrogen-producers in the control group, they changed into Enterococcus when 5 mg/L Ni²⁺ was added. Besides, the proportion of Clostridium_sensu_stricto_1, which is regarded as the primary hydrogen-producing bacteria under numerous operating conditions, was also significantly increased in the presence of Ni²⁺.     

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.     

Pretreatment of methanogenic granules for immobilized hydrogen fermentation

Hydrogen can be produced through fermenting sugars in a mixed bacterial culture under anaerobic conditions. Anaerobic granular sludge was proposed as immobilized hydrogen producing bacteria to be used in hydrogen fermentation after methanogenic activity of the granule was eliminated in the pretreatment process. This paper reports an innovative treatment method to directly convert methanogenic granules to hydrogen producing granules using chloroform. Chloroform treatment was compared against acid and heat treatments of sewage sludge and methanogenic granules in terms of effectiveness to eliminate methanogenic activity. The results showed that chloroform treatment was the most effective among the three methods tested. Acid and heat treatments that were effective for sewage sludge treatment were observed not highly effective for application to granules because of the protection from the granular structure. In contrast, methanogens were very sensitive to the chloroform even at very low level. Methane production was almost completely inhibited in both sewage sludge and granules with the treatment with only 0.05% chloroform addition into the culture medium. If the chloroform concentration was controlled at low levels, chloroform selectively inhibited methanogenic activity while did not affect the hydrogen production. At high concentration range, chloroform also inhibited hydrogen production. Chloroform caused irreversible methanogenic activity elimination but hydrogen production recovered to normal after chloroform addition stopped. Chloroform showed desirable selectivity on inhibition of methanogens from hydrogen producing bacteria, nearly permanently eliminated the methane production, postponed the hydrogen consumption to acetic acid, while allowed recovery for normal hydrogen production. Chloroform treated granules were repeatedly cultured for eight time without noticeable damage. Continuous culture with chloroform treated granules showed that the granule structure could be kept for over 15 days and new granules started to form after 10 days operation. The hydrogen productivity reached 11.6 L/L/day at HRT 5.3 h, which all showed potential application of chloroform treatment of methanogenic granules in the immobilized hydrogen fermentation.     

Enhanced anaerobic digestion of sewage sludge by thermal or alkaline-thermal pretreatments: Influence of hydraulic retention time reduction

The influence of hydraulic retention times (HRTs) reduction from 25 days to 15 days on the enhancement effects of two pretreatments (thermal pretreatment and alkaline-thermal pretreatment) on the continuous anaerobic digestion (AD) of sewage sludge was studied in a long-term experiment (196 days). The operation of the semi-continuous AD fed with raw sludge or pretreated sludge was stable at the three HRTs. The methane production increased from 70.6 to 165.8 ml/L·d to 75.2–172.6 ml/L·d and the methane yield decreased from 98.9 to 234.9 ml/g added volatile solid to 65.6–144.9 ml/g added volatile solid when the HRT reduced from 25 days to 15 days. The two pretreatments reduced the HRT of raw sludge AD by over 40%, and the effects of the alkaline-thermal pretreatment were greater than those of the thermal pretreatment. The reduction of HRT from 25 days to 15 days increased the enhancement effects of the two pretreatments on the removal of organic matter (4.7–15.9% for volatile solid), average hydrolysis ratio (36.9–116.4%), and specific hydrolysis rate (44.1–155.6%) but decreased the enhancement effects of the pretreatments on the methane production (0.9–4.6%) and yield (4.0–15.8%), average reaction ratios (0.4–8.2%), and specific rates of the last three AD steps (0.1–13.9%). The influence of HRT reduction on the enhancement effects of the alkaline-thermal pretreatment for sludge AD was slightly greater than on the enhancement effects of the thermal pretreatment.     

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.     

Co-fermentation of sewage sludge and algae and Fe2+ addition for enhancing hydrogen production

Co-fermentation of sewage sludge and algae was performed for enhancing the hydrogen production, and the effect of Fe²⁺ on co-fermentation process was examined. Results showed that both co-fermentation process and Fe²⁺ addition promoted hydrogen production. Highest hydrogen production of 28 mL/100 mL (14.8 mL H₂/g VS_added) was obtained from the co-fermentation group with 600 mg/L Fe²⁺ addition, which was 2.15 times, 2.00 times and 1.87 times of mono-fermentation of sludge, mono-fermentation of algae, and the co-fermentation group without Fe²⁺ addition. Both volatile solids and protein degradation were stimulated by co-fermentation process. Microbial analysis showed that co-fermentation groups with Fe²⁺ addition enriched Clostridium sensu stricto 13, Clostridium tertium and Terrisporobacter, which were positively correlated with cumulative hydrogen production. This study suggested that the co-fermentation of sludge and algae in the presence of Fe²⁺ could significantly improve the hydrogen production by stimulating the hydrogen-producing metabolism.     

Influence of chemical nature of organic wastes on their conversion to hydrogen by heat-shock digested sludge

The influence of the chemical nature of high-solid organic wastes (HSOW) on their biohydrogen generation was investigated using simulated high-solid bioreactors under mesophilic conditions. The bioreactors were filled with 10% total solid of rice, potato, fat meat, chicken skin, egg, and lean meat. Experimental results indicate that hydrogen-producing potential of carbohydrate-rich HSOW (rice and potato) was approximately 20 times larger than that of fat-rich HSOW (fat meat and chicken skin) and of protein-rich HSOW (egg and lean meat). According to development trends of pH and hydrogen, pH around 6.0 might be threshold for heat-shock digested sludge; that is Clostridium-rich sludge, converting fat- and protein-rich HSOW to hydrogen; but pH threshold for Clostridium-rich sludge consuming carbohydrates-rich HSOW occurred at around 5.0. In bulk solution, volatile fatty acids (VFA) and alcohols occurred concurrently and the trends of carbohydrate-rich HSOW were similar to those of protein-rich HSOW. Considering developments of carbohydrates and VFAs together with that of hydrogen one infers that lipids would be hydrolyzed to carbohydrates and the carbon flow would proceed through acetate/H₂+CO₂ cleavage. Indications from cluster analysis of pH development trends are that a cometabolism would be obtained in wastes rich in carbohydrate and protein.     

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 hydrogen production in co-gasification of sewage sludge and industrial wastewater sludge by a pilot-scale fluidized bed gasifier

This research provides a perspective on sludge-to-energy using sewage sludge (SS) and industrial wastewater sludge (IS) co-gasification in a pilot-scale fluidized bed gasifier with temperature controlled at (600–800 °C) using IS addition ratio (0%–60%), and steam-to-biomass ratio (S/B) (0–1.0). The experimental results show that the increase in thermal reaction activity occurred in concordance with the increase in the IS addition. The explanation for such phenomena is that relatively high catalytic Fe/Mn content in industrial wastewater sludge could lower the activation energy. Hydrogen production was increased from 9.1% to 11.94% with an increase in industrial wastewater sludge ratios from 0% to 60%. The produced gas heating value ranged from 4.84 MJ/Nm³ to 5.11 MJ/Nm³, which was coupled with the cold gas efficiency (CGE) ranging from 33.91% to 36.15%. Enhanced hydrogen production in sewage sludge and industrial wastewater sludge co-gasification is investigated in this study.     

Hydrogen production from sludge of poultry slaughterhouse wastewater treatment plant pretreated with microwave

This study aims to produce hydrogen from sludge of poultry slaughterhouse wastewater treatment plant (5% total solid) by anaerobic batch fermentation with Enterobactor aerogenes or mixed cultures from hot spring sediment as the inoculums. Sludge was heated in microwave at 850 W for 3 min. Results indicated that a soluble chemical oxygen demand (sCOD) of pretreated sludge was higher than that of raw sludge. Pretreated sludge inoculated with E. aerogenes and supplemented with the Endo nutrient had a higher hydrogen yield (12.77 mL H₂/g tCOD) than the raw sludge (0.18 mL H₂/g tCOD). When considered the hydrogen yield, the optimum initial pH for hydrogen production from microwave pretreated sludge was 5.5 giving the maximum value of 12.77 mL H₂/g tCOD. However, when considered the hydrogen production rate (R_m), the optimum pH for hydrogen production would be 9.0 with the maximum R_m of 22.80 mL H₂/L sludge·h.