Obtainment of lignocellulose degradation microbial community: the effect of acid-base combination after restrictive enrichment.

Microbial communities for bioconversion of lignocellulose have received widespread attention. Many cellulose-degrading microbial communities have been enriched from different sources. Combining two microbial communities with acidic and basic properties (acid-base combination) is a technique used alongside restricted enrichment culturing. Understanding how changes to microbial communities result in community's structure and function is important for mechanistic reconstruction of microbiomes. In this study, we analyzed changes in microbial community structure to elucidate determination of the mechanisms of acid-base combination. We found that after restricted enrichment, the bacteria that were primarily retaining included not only those that decompose and utilize lignocellulose, such as Clostridium and Pseudomonas, but also synergistic microbiota such as Alkalobacillaceae. When the proportion of these two types of bacteria was unbalanced, the degradative ability of the microbial community was low, or pH changes of it did not compound regular changes, which may lead to the failure of restricted enrichment. Microbial communities were re-constituted by acid-base combination, whereby the degrading and synergistic strains were adjusted to a more appropriate proportion. The acid-base combination fixed the instability of microbial communities caused by the randomness of restrictive screening enrichment; it provided an effective method for obtaining high-quality lignocellulose-degrading microbial community.

Bioaugmentation with methanogens cultured in a micro-aerobic microbial community for overloaded anaerobic digestion recovery.

Anaerobic digestion (AD) is widely used for conversion of waste materials into biogas, but inhibition of methane production caused by overloading can be a major problem. The micro-aerobic microbial community MC1 was used to successfully culture methanogens, Methanosarcina acetivorans C2A and Methanosaeta thermophila NBRC 101360. The maximum 16S rRNA gene concentrations of Methanosarcina acetivorans C2A and Methanosaeta thermophila NBRC101360 were 1.06 × 106 and 1.35 × 103 copies/mL, respectively. The five key bacteria in MC1 were quantified to assess the effect of inoculation on the abundances of the bacteria in the mixed culture. The original MC1 total 16S rRNA gene concentration was 1.93 × 108 copies/mL, and the total 16S rRNA gene concentration had increased to 4.79 × 109 copies/mL on day 9 (p < 0.05). The proportions of the key strains in MC1+MST had changed by day 9. Cells were harvested and used to bioaugment and increase the pH values of the high- and medium-temperature anaerobic systems. After bioaugmentation, thermophilic AD recovered well. The cumulative amounts of gas produced were 44.78% and 28.28% higher in the MC1+MST and MC1 groups, respectively, than the sterilized control. The MC1+MST group gave better results than the chemical addition control group (CaCO3). There was no clear effect of bioaugmentation in mesophilic AD. When compared with traditional pure culture of methanogens as inoculants, methanogen cultivation in MC1 was simple and there was no need to separate and purify the target strains. This simplified methanogenic bioaugmentation agent was useful to study the mechanism of bioaugmentation for the recovery from low pH inhibition, showing the potential for practical application.

Efficient phosphorus removal by a novel halotolerant fungus Aureobasidium sp. MSP8 and the application potential in saline industrial wastewater treatment.

Efficient halotolerant phosphorus accumulation microorganisms are of great significance for the treatment of high-salt wastewater. In this study, a halotolerant fungus strain named MSP8 was isolated and identified as Aureobasidium sp. Salinity resistance results showed that strain MSP8 can resist the salinity from 0% to 17%, and 77.2% phosphorus removal was achieved at the optimal salinity of 5%. The strain also showed wide environmental adaptability (pH of 3-7; temperature of 20-30 °C). Batch tests and scanning electron microscope equipped with an energy dispersive spectrometer (SEM-EDS) characterization results verified the key role of extracellular polymeric substance (EPS) secreted by MSP8 in phosphorus removal. The actual brewery and chemical wastewater treatments exhibited that above 53.5% of phosphorus can be removed by MSP8. The excellent adaptation of MSP8 made it a potential candidate for phosphorus removal especially in saline wastewater treatment.

Effect of steel slag in recycling waste activated sludge to produce anaerobic granular sludge.

The amount of waste activated sludge (WAS) has grown dramatically in China. WAS is considered as a problematic and hazardous waste, which should be disposed in a safe and sustainable manner. In order to recycle WAS to an anaerobic granular sludge (AnGS) process for anaerobic digestion, Fe powder and steel slags (rusty and clean slags) were used to enhance the granulation process. The results demonstrated that both rusty and clean slags encouraged the development of granular sludge. Adding 10 g/L clean slags could increase AnGS granulation rate by 37%. In the presence of clean slags, extracellular polymeric substances (EPS) concentration in granules increased noticeably to 715 mg/g mixed liquor suspended solids (MLSS). High throughput sequencing analysis exhibited more diversity and higher abundance of functional microbial communities in the batch bottle with 10 g/L clean slags. This study suggested that adding clean slags at 10 g/L dosage was a sustainable and effective method for the sludge granulation.

A new strategy to recover from volatile fatty acid inhibition in anaerobic digestion by photosynthetic bacteria.

The accumulation of volatile fatty acids (VFAs) can decrease reactor pH and inhibit methane-producing process. For the first time, photosynthetic bacteria (PSB) were used to recover from VFAs inhibition (pH 6.0) of an anaerobic digestion system. After adding PSB for 12 days with and without light condition, the methane content recovered from 33.3% to 60.5% and from 32.1% to 59.3%, respectively; the pH increased to 7.1 and 6.8, respectively, the system alkalinity rapidly increased to 2238 and 1921 mg/L, respectively; the sCOD decreased from 5600 to 995 mg/L and from 5575 to 2025 mg/L, respectively; and the contents of formic acid, acetic acid, propionic acid and total VFA were greatly reduced. Microbial analysis found that PSB bioaugmentation could maintain microbial diversity of the system. PSB bioaugmentation could effectively relieve acids accumulation and stimulate methane production especially under light condition. It is also found that light could accelerate recovery with or without bioaugmentation.

Nitrogen removal bacterial strains, MSNA-1 and MSD4, with wide ranges of salinity and pH resistances.

Nitrogenous wastewater is difficult to treat using conventional microorganisms in high salinity and acidic/alkaline environments. Two halotolerant bacteria, heterotrophic nitrifying Stenotrophomonas sp. MSNA-1 and aerobic denitrifying Pseudomonas sp. MSD4, were isolated, and the amplification of functional genes provided the evidences of nitrogen removal performance. The results regarding salinity and pH resistance showed that strain MSNA-1 is robust at salinities of 0-15% and pH of 3-10. It can remove 51.2% of NH4+-N (180 mg/L) at salinity of 10% (pH: 7) and 49.2% of NH4+-N under pH 4 (salinity: 3%). For strain MSD4, it is robust at salinities of 0-10% and pH of 5-11. It can remove 62.4% of TN (100 mg/L) at salinity of 7% (pH: 7) and 72.2% of TN under pH 9 (salinity: 3%). Their excellent salinity and pH resistances make them promising candidates for treating nitrogenous wastewaters under extreme conditions with low operational cost.

Effect of different initial low pH conditions on biogas production, composition, and shift in the aceticlastic methanogenic population.

Anaerobic digestion (AD) reactors often encounter low pH inhibition during startup and high organic loading periods. The use of a large amount of NaOH in order to raise and buffer the low pH, is reported to be inhibitory to methanogens. In order to address this problem, we acclimatized aceticlastic methanogens to low pH. Methanogens were successfully acclimatized to initial low pH down to 3.5 in a lengthy, five months, acclimatization period. The aceticlastic methanogen, Methanothrix soehngenii which was 96.3% of the total methanogenic population at pH 4.5 and 86.75% at pH 3.5, demonstrated that they were the most tolerant aceticlastic methanogens to low pH. After acclimatization, methane yield at pH 4.5 was comparable to neutral pH. Methanosaeta maintained its dominance over Methanosarcina at an elevated level of acetate (66 mM), and a negative correlation was observed between them. There was a positive correlation between the CH4 content and pH.

Effects of adding trace elements on rice straw anaerobic mono-digestion: Focus on changes in microbial communities using high-throughput sequencing.

Although trace elements are known to aid anaerobic digestion, their mechanism of action is still unclear. High-throughput sequencing was used to reveal the mechanism by which adding trace elements affects microbial communities and their action. The results showed that the highest methane yields, with addition of Fe, Mo, Se and Mn were 289.2, 289.6, 285.3, 293.0mL/g volatile solids (VS), respectively. The addition of Fe, Mo, Se and Mn significantly (P<0.05) reduced the level of volatile fatty acids (VFAs). The dominant bacteria and archaea were Bacteroidetes and Methanosaeta, respectively. Compared with the proportion of Methanosaeta in the control group, treatment with added trace elements increased Methanosaeta by as much as 12.4%. Microbial community analysis indicated that adding trace elements changed the composition and diversity of archaea and bacteria. Methane yield was positively correlated with bacterial diversity and negatively correlated with archaeal diversity for most treatments.

Hydrolysis and acidification of agricultural waste in a non-airtight system: Effect of solid content, temperature, and mixing mode.

A two-phase digestion system for treating agricultural waste is beneficial for methane production. This study explored the effect of solid content, temperature, and mixing mode on the process of hydrolysis and acidification using rice straw and cow dung launched in non-airtight acidogenic system. The results showed that the substrate could be hydrolyzed efficiently in the initial stage, the hydrolysis coefficient (k) of maximum cellulose and hemicellulose can be increased by 217.9% and 290.5%, respectively, compared with those of middle and last stages. High solid content played a leading role in promoting hydrolysis, resulted in hydrolysate content (sCOD) that was significantly higher than in treatments with low solid content (P<0.01), and led to organic acids accumulation up to 5.8 and 6.7g/L at mesophilic and thermophilic temperatures. Thermophilic temperature stimulated the hydrolysis and acidification of low solid content (P<0.05), and improved organic acid accumulation of high solid content only during the middle stage (P<0.01). Mixing mode was not a major factor, but increasing the mixing time was necessary for organic acid accumulation during the last stage (P<0.05). In addition, the study comprehensively analyzed a series of corresponding relationships among each operating parameter during the whole treatment process using canonical correspondence analysis.

Pretreatment of non-sterile, rotted silage maize straw by the microbial community MC1 increases biogas production.

Using microbial community MC1 to pretreat lignocellulosic materials increased the yield of biogas production, and the substrate did not need to be sterilized, lowering the cost. Rotted silage maize straw carries many microbes. To determine whether such contamination affects MC1, rotted silage maize straw was pretreated with MC1 prior to biogas production. The decreases in the weights of unsterilized and sterilized rotted silage maize straw were similar, as were their carboxymethyl cellulase activities. After 5d pretreatment, denaturing gradient gel electrophoresis and quantitative polymerase chain reaction results indicated that the proportions of five key strains in MC1 were the same in the unsterilized and sterilized groups; thus, MC1 was resistant to microbial contamination. However, its resistance to contamination decreased as the degradation time increased. Following pretreatment, volatile fatty acids, especially acetic acid, were detected, and MC1 enhanced biogas yields by 74.7% compared with the untreated group.

Influence of cell disruption and elution on cellulase release of Clostridium straminisolvens (CSK1).

Clostridium straminisolvens (CSK1) is a novel cellulolytic bacterium isolated from a cellulose-degrading bacterial community MC1. In this study, the influence of the following cell disruption and elution methods on CSK1cellulase release was investigated: (1) freezing-thawing, (2) ultrasonication, (3) elution, (4) freezing-thawing following elution, (5) ultrasonication following elution, and lastly (6) high-pressure homogenization following elution. The activity of the cellulases CMCase, ß-glucosidase, Avicelase, FPase, and xylanase in crude extracts increased 81.5, 23.8, 87.7, 46.3, and 51.7 %, respectively, with an observed optimal treatment method for each cellulase type. The release of protein from CSK1 cells increased following either cell disruption or elution and was highest at 88.3 % in the homogenization high pressure following elution treatment. A newly observed protein was present following cell elution. The performance of cell elution as determined by real time-PCR indicated that the first time cell elution removed more than 90 % of the CSK1 cells from the substrate. These findings demonstrate that cell disruption and elution are effective methods for inducing cellulase release, and elution is the key step for CSK1. To our knowledge, this study presents the first evidence of optimal treatments for induction of cellulase release of Clostridium straminisolvens. This information will be of great value for use in subsequent efforts to better understand the cellulase characteristics of CSK1 and cellulose degradation mechanisms of the MC1 community.

Dynamic changes in the composite microbial system MC1 during and following its rapid degradation of lignocellulose.

To monitor the dynamics of the composite microbial system MC1 during its degradation of lignocellulose and to improve our understanding of the microbial communities involved in this biomass conversion, MC1 was characterized at eight time points over an 18-day, thermophilic, aerobic, static cultivation. We found the microbial communities to be dynamic, rhythmic consortia capable of changing in response to lignocellulose degradation. The growth curve over 18 days was M-shaped. Based on the quantitative changes in five major components of MC1 (Clostridium straminisolvens CSK-1, Clostridium sp. FG4, Pseudoxanthomonas sp. M1-3, Brevibacillus sp. M1-5, and Bordetella sp. M1-6), reduction in rice straw weight, cellulase (CMCase) activity, xylanase activity, and changes in medium pH, we found that the process comprised two identifiable phases. Rapid degradation occurred from day 0 to day 9, while the post-rapid degradation phase included days 10 to 18. Day 3 and day 12 were two key time points in the rapid degradation phase and post-rapid degradation phase, respectively. Two anaerobes, C. straminisolvens CSK-1 and Clostridium sp. FG4, dominated the MC1 system from day 0 to day 18.

Enhancing the cellulose-degrading activity of cellulolytic bacteria CTL-6 (Clostridium thermocellum) by co-culture with non-cellulolytic bacteria W2-10 (Geobacillus sp.).

The effect of a non-cellulolytic bacterium W2-10 (Geobacillus sp.) on the cellulose-degrading activity of a cellulolytic bacterium CTL-6 (Clostridium thermocellum) was determined using cellulose materials (paper and straw) in peptone cellulose solution (PCS) medium under aerobic conditions. The results indicated that in the co-culture, addition of W2-10 resulted in a balanced medium pH, and may provide the required anaerobic environment for CTL-6. Overall, addition of W2-10 was beneficial to CTL-6 growth in the adverse environment of the PCS medium. In co-culture with W2-10, the CTL-6 cellulose degradation efficiency of filter paper and alkaline-treated wheat straw significantly increased up to 72.45 and 37.79 %, respectively. The CMCase activity and biomass of CTL-6 also increased from 0.23 U ml(-1) and 45.1 µg ml(-1) (DNA content) up to 0.47 U ml(-1) and 112.2 µg ml(-1), respectively. In addition, co-culture resulted in accumulation of acetate and propionate up to 4.26 and 2.76 mg ml(-1). This was a respective increase of 2.58 and 4.45 times, in comparison to the monoculture with CTL-6.