Improved biohydrogen production by co-fermentation of corn straw and excess sludge: Insights into biochemical process, microbial community and metabolic genes.

The global climate change mainly caused by fossil fuels combustion promotes that zero-carbon hydrogen production through eco-friendly methods has attracted attention in recent years. This investigation explored the biohydrogen production by co-fermentation of corn straw (CS) and excess sludge (ES), as well as comprehensively analyzed the internal mechanism. The results showed that the optimal ratio of CS to ES was 9:1 (TS) with the biohydrogen yield of 101.8 mL/g VS, which was higher than that from the mono-fermentation of CS by 1.0-fold. The pattern of volatile fatty acids (VFAs) indicated that the acetate was the most preponderant by-product in all fermentation systems during the biohydrogen production process, and its yield was improved by adding appropriate dosage of ES. In addition, the content of soluble COD (SCOD) was reduced as increasing ES, while concentration of NH4+-N showed an opposite tendency. Microbial community analysis revealed that the microbial composition in different samples showed a significant divergence. Trichococcus was the most dominant bacterial genus in the optimal ratio of 9:1 (CS/ES) fermentation system and its abundance was as high as 41.8%. The functional genes prediction found that the dominant metabolic genes and hydrogen-producing related genes had not been significantly increased in co-fermentation system (CS/ES = 9:1) compared to that in the mono-fermentation of CS, implying that enhancement of biohydrogen production by adding ES mainly relied on balancing nutrients and adjusting microbial community in this study. Further redundancy analysis (RDA) confirmed that biohydrogen yield was closely correlated with the enrichment of Trichococcus.

Simultaneous production of hydrogen-methane and spatial community succession in an anaerobic baffled reactor treating corn starch processing wastewater.

Corn starch processing wastewater (CSPW) is a high-strength organic wastewater and biological treatment is considered as the dominant process. The present work investigated the effects of pH on the bioenergy production and spatial succession of microbial community in an anaerobic baffled reactor (ABR) treating CSPW. The results showed that above 90.5% of COD removal and above 16.6 L d-1 of methane were achieved at the influent pHs of 8.0 and 7.0 under organic loading rate of 4.0 kg COD·m-3·L-1 condition. Further decreasing the influent pH to 6.0 resulted in the COD removal decreased to 89.7%. Besides, 9.2 L d-1 of hydrogen and 13.0 L d-1 of methane were obtained. There was significant difference in the volatile fatty acids profiles during the variation of pH. Illumina Miseq sequencing showed that Clostridium, Ethanoligenens, Megasphaera, Prevotella and Trichococcus with relative abundance of 2.1%∼28.1% were the dominant hydrogen-producing bacteria in C1. Methanogens (Methanothrix and Methanobacterium) dominated in the last three compartments. Function predicted analysis revealed that the abundance of metabolic-related gene families containing carbohydrate, amino acids and energy in the last three compartments were higher than that in C1. A deduced biodegradation model of CSPW in ABR revealed that the anaerobic sludge in C1 mainly produced hydrogen. Microbial population in C3 was responsible for COD removal and methane production. The redundancy analysis revealed that hydrogen production was highly correlated with some hydrogen-producing bacteria in C1, whereas methane production was positively correlated with microbial group in C2∼ C4.

An enhanced excess sludge fermentation process by anthraquinone-2-sulfonate as electron shuttles for the biorefinery of zero-carbon hydrogen.

Excess sludge (ES) largely produced in municipal wastewater treatment plants is known as a waste biomass and the traditional treatment processes such as landfill and incineration are considered as unsustainable due to the negative environmental impact. Fermentation process of ES for the biorefinery of zero-carbon hydrogen has attracted an increasing interesting and was extensively researched in the last decades. However, the technology is far from commercial application due to the insufficient effectivity. In the present study, anthraquinone-2-sulfonate (AQS) as electron shuttles was introduced into the fermentation process of ES for mediating the composition and activity of bacterial community to get an enhanced biohydrogen production. Inoculated with the same anaerobic activated sludge of 1.12 gVSS/L, a series of batch anaerobic fermentation systems with various dosage of AQS were conducted at the same ES load of 2.75 gVSS/L, initial pH 6.5 and 35 °C. The results showed that the fermentation process was remarkably enhanced by the introduction of 100 mg/L AQS, accompanying the lag phase was shortened to 1.35 h from 7.62. The obtained biohydrogen yield and the specific biohydrogen production rate were also remarkably enhanced to 24.9 mL/gVSS and 0.3 mL/(gVSS·h), respectively. Illumina Miseq sequencing showed that Longilinea and Guggenheimella as the dominant genera had been enriched from 9.2% to 0-12.0% and 4.7%, respectively, in the presence of 100 mg/L AQS. Function predicted analysis suggested that the presence of AQS had increased the abundance of genes involved in the transport and metabolism of carbohydrate, amino acid and energy production. Further redundancy analysis (RDA) revealed that the enhanced hydrogen production was highly positively correlated with the enrichment of genera such as Longilinea and Guggenheimella. The research work presents a novel potential biorefinery of ES for the effective production of zero-carbon hydrogen.

Correlating bacterial and archaeal community with efficiency of a coking wastewater treatment plant employing anaerobic-anoxic-oxic process in coal industry.

Coking wastewater (CWW) contains various complex pollutants, and biological treatment processes are frequently applied in the coking wastewater treatment plants (CWWTPs). The present work is to evaluate the contaminants removal of a full-scale CWWTP with an anaerobic-anoxic-oxic process (A/A/O), to reveal function of bacterial and archaeal community involved in different bioreactors, and to clarify the relationship between the performance and microbial community. Illumina Miseq sequencing of bacteria showed that ß-proteobacteria dominated in three bioreactors with relative abundance of 60.2%~81.7%. 75.2% of sequences were assigned to Petrobacter in the bioreactor A1, while Thiobacillus dominated in A2 and O with relative abundance of 31.8% and 38.7%, respectively. Illumina Miseq sequencing of archaea revealed a high diversity of methanogens existed in A1 and A2 activated sludge. Moreover, Halostagnicola was the dominant archaea in A1 and A2 activated sludge with relative abundance of 41.8% and 66.5%, respectively. Function predicted analysis explored that function of bacteria was similar to that of archaea but the relative abundance differed from each other. A putative biodegradation model of CWW treatment in A/A/O process indicated that A1 and A2 activated sludge mainly reduced carbohydrate, protein, TN, phenol and cyanide, as well as methane production. Bacteria in the bioreactor O were responsible for aerobic biotransformation of residual carbohydrates, refractory organics and nitrification. The redundancy analysis (RDA) further revealed that removal of COD, TN, and NO3--N, phenol and cyanides were highly correlated with some anaerobic bacteria and archaea, whereas the transformation of NH4+-N was positively correlated with some aerobic bacteria.

Functional bacterial and archaeal dynamics dictated by pH stress during sugar refinery wastewater in a UASB.

The operation performance and microbial mechanisms by pH stress were investigated during anaerobic digestion of sugar refinery wastewater in a upflow anaerobic sludge blanket (UASB) reactor to clarify correlations between pH stress, microbial community and process efficiency. Results showed that the COD removal and methane yield were respectively reduced by 24.8% and 25.3% as pH decreased to 5.0. pH decrease resulted in the composition of dominant fermentative acidogenic bacteria was changed to Butyricicoccus, Lactococcus, Brooklawnia, Armatimonadetes_gp2 and Megasphaera from Prevotella, Streptococcus, Acidaminococcus and Megasphaera, causing an increase in propionate production. In addition, the growth of propionate-oxidizing bacteria was also inhibited at pH 5.0, leading the propionate was accumulated, and then reduced the process efficiency. Methane was mainly produced through acetate cleavage by Methanosaeta during the whole operational period of UASB. pH decrease blocked the metabolic balance and community structure between different trophic groups, resulting in the decrease in reactor performance.

Response of Syntrophic Propionate Degradation to pH Decrease and Microbial Community Shifts in an UASB Reactor.

The effect of pH on propionate degradation in an upflow anaerobic sludge blanket (UASB) reactor containing propionate as a sole carbon source was studied. Under influent propionate of 2,000 mg/l and 35ºC, propionate removal at pH 7.5-6.8 was above 93.6%. Propionate conversion was significantly inhibited with stepwise pH decrease from pH 6.8 to 6.5, 6.0, 5.5, 5.0, 4.5, and then to 4.0. After long-term operation, the propionate removal at pH 6.5-4.5 maintained an efficiency of 88.5%-70.1%, whereas propionate was hardly decomposed at pH 4.0. Microbial composition analysis showed that propionate-oxidizing bacteria from the genera Pelotomaculum and Smithella likely existed in this system. They were significantly reduced at pH ≤5.5. The methanogens in this UASB reactor belonged to four genera: Methanobacterium, Methanospirillum, Methanofollis, and Methanosaeta. Most detectable hydrogenotrophic methanogens were able to grow at low pH conditions (pH 6.0-4.0), but the acetotrophic methanogens were reduced as pH decreased. These results indicated that propionate-oxidizing bacteria and acetotrophic methanogens were more sensitive to low pH (5.5-4.0) than hydrogenotrophic methanogens.

Shift of propionate-oxidizing bacteria with HRT decrease in an UASB reactor containing propionate as a sole carbon source.

Propionate is a main intermediate product, and its degradation is crucial for maintaining the efficiency and stability of an anaerobic reactor. However, there was little information about the effects of ecological factor on propionate-oxidizing bacteria. In current research, microbial community composition and quantitative analysis of some identified propionate-oxidizing bacteria with hydraulic retention time (HRT) decrease in an upflow anaerobic sludge blanket (UASB) reactor containing propionate as sole carbon source was investigated. The results showed that propionate-oxidizing bacteria from Syntrophobacter, Pelotomaculum, and Smithella were major functional bacteria in this UASB system. Most propionate-oxidizing bacteria in composition have not changed with HRT decrease. However, the number of previously identified propionate-oxidizing bacteria from these three genera exhibited significant shift. Under HRT 10 h condition, Pelotomaculum schinkii was dominant and its quantity was 1.2 × 10(4) 16S ribosomal RNA (rRNA) gene copies/ng DNA, occupying 56.2 % in total detectable propionate-oxidizing bacteria. HRT decrease from 10 h to 8 and 6 h stepwise resulted in P. schinkii, Syntrophobacter sulfatireducens and Smithella propionica becoming the main population. HRT decrease from 6 to 4 h did not markedly change the amount of propionate-oxidizing bacteria, but S. propionica dominated in the reactor.

Quantitative analysis of previously identified propionate-oxidizing bacteria and methanogens at different temperatures in an UASB reactor containing propionate as a sole carbon source.

Propionate degradation is crucial for maintaining the efficiency and stability of an anaerobic reactor. However, there was little information about the effects of ecological factor on propionate-oxidizing bacteria (POB). In current research, quantitative real-time fluorescence polymerase chain reaction (QPCR) of some identified POB and methanogens with a decrease in temperature in an upflow anaerobic sludge bed (UASB) reactor containing propionate as sole carbon source was investigated. The results showed that there were at least four identified POB, including Pelotomaculum schinkii, Pelotomaculum propionicum, Syntrophobacter fumaroxidans, and Syntrophobacter sulfatireducens, observed in this UASB reactor. Among them, P. schinkii was dominated during the whole operational period. Its quantity was 1.2 × 10(4) 16S rRNA gene copies per nanogram of DNA at 35 °C. A decrease in temperature from 35 to 30 °C led to P. schinkii to be increased by 1.8 times and then it was gradually reduced with a decrease in temperature from 30 to 25, 20, and 18 °C stepwise. A decrease in temperature from 35 to 20 °C did not make the amount of methanogens markedly changed, but hydrogenotrophic methanogens (Methanospirillum) and acetotrophic methanogens (Methanosaeta) at 18 °C were increased by an order of magnitude and 1.0 time, respectively, compared with other experimental conditions.

Syntrophic propionate degradation response to temperature decrease and microbial community shift in an UASB reactor.

Propionate is an important intermediate product during the methane fermentation of organic matter, and its degradation is crucial for maintaining the performance of an anaerobic digester. In order to understand the effect of temperature on propionate degradation, an upflow anaerobic sludge blanket (UASB) reactor with synthetic wastewater containing propionate as a sole carbon source was introduced. Under the hydraulic retention time (HRT) of 10 h and influent propionate of 2,000 mg/l condition, propionate removal was above 94% at 30-35°C, whereas propionate conversion was inhibited when temperature was suddenly decreased stepwise from 30°C to 25°C, to 20°C, and then to 18°C. After a long-term operation, the propionate removal at 25°C resumed to the value at 30- 35°C, whereas that at 20°C and 18°C was still lower than the value at 35°C by 8.1% and 20.7%, respectively. Microbial community composition analysis showed that Syntrophobacter and Pelotomaculum were the major propionate-oxidizing bacteria (POB), and most POB had not changed with temperature decrease in the UASB. However, two POB were enriched at 18°C, indicating they were low temperature tolerant. Methanosaeta and Methanospirillum were the dominant methanogens in this UASB and remained constant during temperature decrease. Although the POB and methanogenic composition hardly changed with temperature decrease, the specific CODPro removal rate of anaerobic sludge (SCRR) was reduced by 21.4%-46.4% compared with the control (35°C) in this system.

Diversity and distribution of methanogenic archaea in an anaerobic baffled reactor (ABR) treating sugar refinery wastewater.

The diversity and distribution of methanogenic archaea in a four-compartment anaerobic baffled reactor (ABR) treating sugar refinery wastewater were investigated by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). At an organic loading rate of 5.33 kg COD/m3·day, the ABR could perform steadily with the mean chemical oxygen demand (COD) removal of 94.8% and the specific CH4 yield of 0.21 l/g COD(removed). The CH4 content in the biogas was increased along the compartments, whereas the percentage of H2 was decreased, indicating the distribution characteristics of the methanogens occurred longitudinally down the ABR. A high phylogenetic and ecological diversity of methanogens was found in the ABR, and all the detected methanogens were classified into six groups, including Methanomicrobiales, Methanosarcinales, Methanobacteriales, Crenarchaeota, Arc I, and Unidentified. Among the methanogenic population, the acid-tolerant hydrogenotrophic methanogens including Methanoregula and Methanosphaerula dominated the first two compartments. In the last two compartments, the dominant methanogenic population was Methanosaeta, which was the major acetate oxidizer under methanogenic conditions and could promote the formation of granular sludge. The distribution of the hydrogenotrophic (acid-tolerant) and acetotrophic methanogens in sequence along the compartments allowed the ABR to perform more efficiently and steadily.

Linking performance with microbial community characteristics in an anaerobic baffled reactor.

The performance and microbial community characteristics of a laboratory scale anaerobic baffled reactor (ABR) with four compartments (C1-C4) treating sugar refinery wastewater were investigated. The COD removal was 94.8 % with a CH4 yield of 0.21 L g(-1) CODremoved at total organic loading rate (OLR) of 5.33 kg COD/m(-3) day(-1). Fermentative bacteria were dominant in C1 and C2, while syntrophic acetogens and methanogens were dominant in C3 and C4. Some acid-tolerant methanogens were enriched in acidogenic phase. The present of the acid-tolerant methanogens could improve the efficiency and stability of the ABR as the most of the methanogens are vulnerable to low pH. In addition, high functional redundancy of the fermentative bacteria implicated that the microbial communities in acidogenic phase were stable functionally and allowed the ABR to balance perturbation. It was also found that syntrophic acetogenesis might be a weakness in the ABR as syntrophic acetogens were poor as compared with fermentative bacteria and methanogens.

Metabolic pathways of hydrogen production in fermentative acidogenic microflora.

Biohydrogen production from organic wastewater by anaerobically activated sludge fermentation has already been extensively investigated, and it is known that hydrogen can be produced by glucose fermentation through three metabolic pathways, including the oxidative decarboxylation of pyruvic acid to acetyl-CoA, oxidation of NADH to NAD+, and acetogenesis by hydrogen-producing acetogens. However, the exact or dominant pathways of hydrogen production in the anaerobically activated sludge fermentation process have not yet been identified. Thus, a continuous stirred-tank reactor (CSTR) was introduced and a specifically acclimated acidogenic fermentative microflora obtained under certain operation conditions. The hydrogen production activity and potential hydrogen-producing pathways in the acidogenic fermentative microflora were then investigated using batch cultures in Erlenmeyer flasks with a working volume of 500 ml. Based on an initial glucose concentration of 10 g/l, pH 6.0, and a biomass of 1.01 g/l of a mixed liquid volatile suspended solid (MLVSS), 247.7 ml of hydrogen was obtained after a 68 h cultivation period at 35 +/- 1 degrees C. Further tests indicated that 69% of the hydrogen was produced from the oxidative decarboxylation of pyruvic acid, whereas the remaining 31% was from the oxidation of NADH to NAD+. There were no hydrogen-producing acetogens or they were unable to work effectively in the anaerobically activated sludge with a hydraulic retention time (HRT) of less than 8 h.

Dry anaerobic digestion of cow dung for methane production: effect of mixing.

The performance characteristics of a dry batch reactor with a blender treating cow dung has been evaluated for 35 days in a single-stage batch reactor of 3 L effective volume at 35 +/- 1 degree C to investigate the effect of continuous-mixing on biogas production and organic materials removal. The results showed that the performance of unmixed and mixed digesters was quite different and the dry digester with mixing system produced methane of 0.358 LCH4/gVS(r) which was 7.50% higher than that for unmixed digester. Moreover, the organic material removal efficiency was increased by 9.73% in term of VS. The wide diversity of prominent bacteria and methanogenic archaea affiliated with all steps along the anaerobic degradation pathway made the process stable. But the dry digester with mixing system during start up was not beneficial, as it resulted in relatively higher volatile fatty acids, higher volatile fatty acid to alkalinity ratio, lower pH and consequently prolonged start up time.

A DREB gene from Limonium bicolor mediates molecular and physiological responses to copper stress in transgenic tobacco.

The DRE-binding (DREB) transcription factors play an important role in regulating stress-related genes. In the present study, a novel DREB gene (LbDREB) from Limonium bicolor was cloned. To characterize the function of DREB in heavy metal stress tolerance, LbDREB-transformed tobacco plants were generated and subjected to CuSO(4) stress. Analysis of the role of LbDREB in tolerance to copper stress in transgenic tobacco showed that overexpression of LbDREB increased the contents of soluble protein and proline, and elevated the ratio of K to Na under CuSO(4) stress. Moreover, overexpression of LbDREB can up-regulate some stress-related genes, including Cu/Zn superoxide dismutase (Cu/Zn SOD), peroxidases (PODs), late embryogenesis abundant (LEA), and lipid transfer proteins (LTP). These results suggest that LbDREB can enhance plant copper tolerance by up-regulating a series of stress-related genes, thereby mediating physiological processes associated with stress tolerance in plants.

[Cloning and expression of a novel metallothionein gene LbMT2 from Limonium bicolor].

The full length cDNA of a novel metallothionein (LbMT2) gene was cloned from a cDNA library of Limonium bicolor. The LbMT2 gene cloned is 518 bp in length, which includes a 64 bp of 5' untranslated region (UTR) and a 205 bp of 3' untranslated region. This gene has an open reading frame (ORF) of 249 bp in length, encoding a protein of 82 amino acid residues with the molecular mass of 8.1 kDa and theoretical pI of 4.71. The expression of LbMT2 gene in L. bicolor in response to CuSO4, CdCl2, NaCl, cold, and PEG was further investigated using real time quantitative PCR. In both leaf and root of L. bicolor, the expression of LbMT2 was induced by CuSO4, CdCl2, NaCl, and cold, but inhibited by PEG stress. LbMT2 gene was inserted into a prokaryotic expression vector (pGEX-4T-2) to produce the recombinant expression vector pGEX-LbMT2. The expression of LbMT2 in E. coli BL21 was induced with IPTG, which produced a protein band with expected size of 35 kDa on SDS-PAGE.

Generation and analysis of expressed sequence tags from a NaHCO3-treated Limonium bicolor cDNA library.

Limonium bicolor, a halophytic species of Plumbaginaceae, can thrive in saline or saline-alkali (sodic) soil, demonstrating that it has developed an efficient saline-alkali resistance system, and is an ideal material for the study of saline-alkali tolerance. In order to identify and characterize the complexity of this adaptation, expressed sequence tags (ESTs) analysis and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were conducted. We constructed a cDNA library of L. bicolor exposed to 0.4M NaHCO3 for 48h, and obtained 2358 ESTs, representing 1735 unique genes. A BLASTX search revealed that 1393 ESTs, representing 873 unique genes, showed significant similarity (E-values <10(-4)) to protein sequences in the non-redundant database. These ESTs were further grouped into 12 functional categories according to their functional annotation. The most abundant categories were metabolism (18.74%), photosynthesis (14.86%), unknown classification (12.20%), defense (12.20%), and transport facilitation (10.19%). In total, 286 putative abiotic stress related transcripts, representing 121 unique genes, were identified. Among them, the two most abundant genes encoded metallothionein (EH794553) and lipid transfer protein (EH794695), each of which accounted for 1.4% of the total ESTs. The expression of 18 putative stress-related genes were further analyzed in roots and leaves of L. bicolor using real-time RT-PCR, and 14 genes were differentially expressed by more than 2-fold as a result of the NaHCO3 stress. The results of this study may contribute to our understanding of the molecular mechanism of saline-alkali tolerance in L. bicolor.