Novel Gas Diffusion Cloth Bioanodes for High-Performance Methane-Powered Microbial Fuel Cells.

Microbial fuel cells (MFCs) are a promising technology that converts chemical energy into electricity. However, up to now only few MFCs have been powered by gas fuels, such as methane, and their limited performance is still challenged by the low solubility and bioavailability of gases. Here, we developed a gas diffusion cloth (GDC) anode to significantly enhance the performance of methane-powered MFCs. The GDC anode was constructed by simply coating waterproof GORE-TEX cloth with conductive carbon cloth in one step. After biofilm enrichment, the GDC anodes obtained a methane-dependent current up to 1130.2 mA m-2, which was 165.2 times higher than conventional carbon cloth (CC) anodes. Moreover, MFCs equipped with GDC anodes generated a maximum power density of 419.5 mW m-2. Illumina high-throughput sequencing revealed that the GDC anode biofilm was dominated mainly by Geobacter, in contrast with the most abundant Methanobacterium in planktonic cells. It is hypothesized that Methanobacterium reversed the methanogenesis process by transferring electrons to the anodes, and Geobacter generated electricity via the intermediates (e.g., acetate) of anaerobic methane oxidation. Overall, this work provides an effective route in preparing facile and cost-effective anodes for high-performance methane MFCs.

Valuable biochemical production in mixed culture fermentation: fundamentals and process coupling.

The mixed culture fermentation is an important environmental biotechnology that converts biodegradable organic wastes to valuable chemicals such as hydrogen, methane, acetate, ethanol, propionate, and so on. For the multistep process of hydrolysis, acidogenesis, acetogenesis/homoacetogensis, and methanogenesis, the typical metabolic reactions are firstly summarized. And then, since the final metabolites are always a mixture, the separation and purification processes are necessary to couple with anaerobic fermentation. Therefore, several typical coupling technologies including biogas upgrading, two-stage fermentation, gas stripping, membrane technology of pervaporation, membrane distillation, electrodialysis, bipolar membrane electrodialysis, and microbial fuel cells are summarized to separate the metabolites and recover energy. At last, the novel technologies such as the controlled metabolite production, medium chain carboxylic acid production, and high temperature ethanol recovery in thermophilic mixed culture fermentation are also reviewed. However, the novel concepts are still needed to meet the demands of better overall performances and lower total costs.

Simultaneous enrichment of denitrifying anaerobic methane-oxidizing microorganisms and anammox bacteria in a hollow-fiber membrane biofilm reactor.

In this study, the coculture system of denitrifying anaerobic methane oxidation (DAMO) microbes and anaerobic ammonium oxidation (anammox) bacteria was successfully enriched in a hollow-fiber membrane biofilm reactor (HfMBR) using freshwater sediment as the inoculum. The maximal removal rates of nitrate and ammonium were 78 mg N/L/day (131 mg N/m2/day) and 26 mg N/L/day (43 mg N/m2/day), respectively. Due to the high rate of methane mass transfer in HfMBR, the activity of DAMO archaea continued to increase during the enrichment period, indicating that HfMBR could be a powerful tool to enrich DAMO microorganisms. Effects of partial methane pressure, temperature, and pH on the cocultures were obvious. However, the microbial activity in HfMBR could be recovered quickly after the shock change of environmental factors. Furthermore, the result also found that DAMO bacteria likely had a stronger competitive advantage than anammox bacteria under the operating conditions in this study. High-throughput sequencing 16S rRNA genes illustrated that the dominant microbes were NC10, Euryarchaeota, Proteobacteria, Planctomycetes, and Chlorobi with relative abundance of 38.8, 26.2, 13.78, 6.2, and 3.6 %, respectively.

Nitrogen source effects on the denitrifying anaerobic methane oxidation culture and anaerobic ammonium oxidation bacteria enrichment process.

The co-culture system of denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (Anammox) has a potential application in wastewater treatment plant. This study explored the effects of permutation and combination of nitrate, nitrite, and ammonium on the culture enrichment from freshwater sediments. The co-existence of NO3-, NO2-, and NH4+ shortened the enrichment time from 75 to 30 days and achieved a total nitrogen removal rate of 106.5 mg/L/day on day 132. Even though ammonium addition led to Anammox bacteria increase and a higher nitrogen removal rate, DAMO bacteria still dominated in different reactors with the highest proportion of 64.7% and the maximum abundance was 3.07 ± 0.25 × 108 copies/L (increased by five orders of magnitude) in the nitrite reactor. DAMO bacteria showed greater diversity in the nitrate reactor, and one was similar to M. oxyfera; DAMO bacteria in the nitrite reactor were relatively unified and similar to M. sinica. Interestingly, no DAMO archaea were found in the nitrate reactor. This study will improve the understanding of the impact of nitrogen source on DAMO and Anammox co-culture enrichment.

Enhancement of acetate productivity in a thermophilic (55 °C) hollow-fiber membrane biofilm reactor with mixed culture syngas (H2/CO2) fermentation.

Conversion of organic wastes to syngas is an attractive way to utilize wastes. The produced syngas can be further used to produce a variety of chemicals. In this study, a hollow-fiber membrane biofilm reactor with mix cultures was operated at 55 °C to convert syngas (H2/CO2) into acetate. A high concentration of acetate (42.4 g/L) was reached in batch experiment while a maximum acetate production rate of 10.5 g/L/day was achieved in the continuous-flow mode at hydraulic retention time (HRT) of 1 day. Acetate was the main product in both batch and continuous-flow experiments. n-Butyrate was the other byproduct in the reactor. Acetate accounted for more than 98.5 and 99.1% of total volatile fatty acids in batch and continuous modes, respectively. Illumina Miseq high-throughput sequencing results showed that microorganisms were highly purified and enriched in the reactor. The main genus was Thermoanaerobacterium (66% of relative abundance), which was usually considered as H2 producer in the literature, however, likely played a role as a H2 consumer in this study. This study provides a new method to generate the high producing rate and purity of acetate from syngas.

Advanced phosphorus recovery using a novel SBR system with granular sludge in simultaneous nitrification, denitrification and phosphorus removal process.

In this study, a novel process for phosphorus (P) recovery without excess sludge production from granular sludge in simultaneous nitrification-denitrification and P removal (SNDPR) system is presented. Aerobic microbial granules were successfully cultivated in an alternating aerobic-anaerobic sequencing batch reactor (SBR) for removing P and nitrogen (N). Dense and stable granular sludge was created, and the SBR system showed good performance in terms of P and N removal. The removal efficiency was approximately 65.22 % for N, and P was completely removed under stable operating conditions. Afterward, new operating conditions were applied in order to enhance P recovering without excess sludge production. The initial SBR system was equipped with a batch reactor and a non-woven cloth filter, and 1.37 g of CH3COONa·3H2O was added to the batch reactor after mixing it with 1 L of sludge derived from the SBR reactor to enhance P release in the liquid fraction, this comprises the new system configuration. Under the new operating conditions, 93.19 % of the P contained in wastewater was released in the liquid fraction as concentrated orthophosphate from part of granular sludge. This amount of P could be efficiently recovered in the form of struvite. Meanwhile, a deterioration of the denitrification efficiency was observed and the granules were disintegrated into smaller particles. The biomass concentration in the system increased firstly and then maintained at 4.0 ± 0.15 gVSS/L afterward. These results indicate that this P recovery operating (PRO) mode is a promising method to recover P in a SNDPR system with granular sludge. In addition, new insights into the granule transformation when confronted with high chemical oxygen demand (COD) load were provided.

In-situ biogas sparging enhances the performance of an anaerobic membrane bioreactor (AnMBR) with mesh filter in low-strength wastewater treatment.

In the recent years, anaerobic membrane bioreactor (AnMBR) technology is being considered as a very attractive alternative for wastewater treatment due to the striking advantages such as upgraded effluent quality. However, fouling control is still a problem for the application of AnMBR. This study investigated the performance of an AnMBR using mesh filter as support material to treat low-strength wastewater via in-situ biogas sparging. It was found that mesh AnMBR exhibited high and stable chemical oxygen demand (COD) removal efficiencies with values of 95 ± 5 % and an average methane yield of 0.24 L CH4/g CODremoved. Variation of transmembrane pressure (TMP) during operation indicated that mesh fouling was mitigated by in-situ biogas sparging and the fouling rate was comparable to that of aerobic membrane bioreactor with mesh filter reported in previous researches. The fouling layer formed on the mesh exhibited non-uniform structure; the porosity became larger from bottom layer to top layer. Biogas sparging could not change the composition but make thinner thickness of cake layer, which might be benefit for reducing membrane fouling rate. It was also found that ultrasonic cleaning of fouled mesh was able to remove most foulants on the surface or pores. This study demonstrated that in-situ biogas sparging enhanced the performance of AnMBRs with mesh filter in low-strength wastewater treatment. Apparently, AnMBRs with mesh filter can be used as a promising and sustainable technology for wastewater treatment.

Critical analysis of hydrogen production from mixed culture fermentation under thermophilic condition (60 °C).

Bio-hydrogen production from mixed culture fermentation (MCF) of glucose was studied by conducting a comprehensive product measurement and detailed mass balance analysis of their contributions to the final H2 yield. The culture used in this study was enriched on glucose at 60 °C through a sequential batch operation consisting of daily glucose feeds, headspace purging and medium replacement every third day in serum bottles for over 2 years. 2-Bromoethanesulfonate (BES) was only required during the first three 3-day cycles to permanently eliminate methanogenic activity. Daily glucose feeds were fully consumed within 24 h, with a persistent H2 yield of 2.7 ± 0.1 mol H2/mol glucose, even when H2 was allowed to accumulate over the 3-day cycle. The measured H2 production exceeded by 14 % the theoretical production of H2 associated with the fermentation products, dominated by acetate and butyrate. Follow-up experiments using acetate with a (13)C-labelled methyl group showed that the excess H2 production was not due to acetate oxidation. Chemical formula analysis of the biomass showed a more reduced form of C5H11.8O2.1N1.1 suggesting that the biomass formation may even consume produced H2 from fermentation.

Characterization of microbial compositions in a thermophilic chemostat of mixed culture fermentation.

The microbial community compositions of a chemostat enriched in a thermophilic (55 °C) mixed culture fermentation (MCF) for hydrogen production under different operational conditions were revealed in this work by integrating denaturing gradient gel electrophoresis (DGGE), Illumina Miseq high-throughput sequencing, and 16S rRNA clone library sequencing. The results showed that the community structure of the enriched cultures was relatively simple. Clones close to the genera of Thermoanaerobacter and/or Bacillus mainly dominated the bacteria. And homoacetogens and archaea were washed out and not detected even by Illumina Miseq high-throughput sequencing which supported the benefit for hydrogen production. On the other hand, the results revealed that the metabolic shift was clearly associated with the change of dominated bacterial groups. The effects of hydrogen partial pressure (PH2) and pH from 4.0 to 5.5 on the microbial compositions were not notable and Thermoanaerobacter was dominant, thus, the metabolites were also not changed. While Bacillus, Thermoanaerobacter and Propionispora hippei dominated the bacteria communities at neutral pH, or Bacillus and Thermoanaerobacter dominated at high influent glucose concentrations, consequently the main metabolites shifted to acetate, ethanol, propionate, or lactate. Thereby, the effect of microbial composition on the metabolite distribution and shift shall be considered when modeling thermophilic MCF in the future.

Environmental evaluation of coexistence of denitrifying anaerobic methane-oxidizing archaea and bacteria in a paddy field.

The nitrate-dependent denitrifying anaerobic methane oxidation (DAMO) process, which is metabolized together by anaerobic methanotrophic archaea and NC10 phylum bacteria, is expected to be important for the global carbon and nitrogen cycles. However, there are little studies about the existence of this process and the functional microbes in environments. Therefore, the coexistence of DAMO archaea and bacteria in a paddy field was evaluated in this study. Next-generation sequencing showed that the two orders, Methanosarcinales and Nitrospirales, to which DAMO archaea and DAMO bacteria belong, were detected in the four soil samples. Then the in vitro experiments demonstrated both of nitrite- and nitrate-dependent DAMO activities, which confirmed the coexistence of DAMO archaea and DAMO bacteria. It was the first report about the coexistence of DAMO archaea and bacteria in a paddy field. Furthermore, anammox bacteria were detected in two of the four samples. The in vitro experiments did not show anammox activity in the initial period but showed low anammox activity after 20 days' enrichment. These results implicated that anammox bacteria may coexist with DAMO microorganisms in this field, but at a very low percentage.

Experimental evaluation of the metabolic reversibility of ANME-2d between anaerobic methane oxidation and methanogenesis.

The "reverse methanogenesis" hypothesis as the metabolic pathway of AOM has recently been supported in the novel ANME lineage ANME-2d in denitrifying anaerobic methane oxidation (DAMO). However, no previous studies have experimentally evaluated the reversal of methane oxidation and methane production in this archaea. In the present study, the metabolic reversibility of ANME-2d from AOM to methanogenesis was evaluated using H2/CO2 and acetate as substrates. The results showed that the system produced methane from H2/CO2 but not from acetate. However, the clone library and real-time PCR analysis of the culture showed that both the percentage and quantity of ANME-2d decreased significantly under this condition, while methanogen abundance increased. Further high-throughput sequencing results showed that the archaea community did not change at the fourth day after H2/CO2 was supplied, but changed profoundly after methanogenesis took place for 3 days. The percentage of DAMO archaea in the total archaea decreased obviously, while more methanogens grew up during this period. Comparatively, the bacteria community changed profoundly at the fourth day. These results indicated that ANME-2d might not reverse its metabolism to produce methane from H2/CO2 or acetate. After archaea were returned to DAMO conditions, DAMO activity decreased and the amount of ANME-2d continued to fall, implying that the lineage had suffered from severe injury and required a long recovery time.

Role of sufficient phosphorus in biodiesel production from diatom Phaeodactylum tricornutum.

In order to study the role of sufficient phosphorus (P) in biodiesel production by microalgae, Phaeodactylum tricornutum were cultivated in six different media treatments with combination of nitrogen (N) sufficiency/deprivation and phosphorus sufficiency/limitation/deprivation. Profiles of N and P, biomass, and fatty acids (FAs) content and compositions were measured during a 7-day cultivation period. The results showed that the FA content in microalgae biomass was promoted by P deprivation. However, statistical analysis showed that FA productivity had no significant difference (p = 0.63, >0.05) under the treatments of N deprivation with P sufficiency (N-P) and N deprivation with P deprivation (N-P-), indicating P sufficiency in N deprivation medium has little effect on increasing biodiesel productivity from P. triornutum. It was also found that the P absorption in N-P medium was 1.41 times higher than that in N sufficiency and P sufficiency (NP) medium. N deprivation with P limitation (N-P-l) was the optimal treatment for producing biodiesel from P. triornutum because of both the highest FA productivity and good biodiesel quality.

Changes in glucose fermentation pathways by an enriched bacterial culture in response to regulated dissolved H2 concentrations.

It is well established that metabolic pathways in the fermentation of organic waste are primarily controlled by dissolved H2 concentrations, but there is no reported study that compares observed and predicted shifts in fermentation pathways induced by manipulating the dissolved H2 concentration. A perfusion system is presented that was developed to control dissolved H2 concentrations in the continuous fermentation of glucose by a culture highly enriched towards Thermoanaerobacterium thermosaccharolyticum (86 ± 9% relative abundance) from an originally diverse consortia in the leachate of a laboratory digester fed with municipal solid waste. Media from a 2.5 L CSTR was drawn through sintered steel membrane filters to retain biomass, allowing vigorous sparging in a separate chamber without cellular disruption. Through a combination of sparging and variations in glucose feeding rate from 0.8 to 0.2 g/L/d, a range of steady state fermentations were performed with dissolved H2 concentrations as low as an equivalent equilibrated H2 partial pressure of 3 kPa. Trends in product formation rates were simulated using a H2 regulation partitioning model. The model correctly predicted the direction of products redistribution in response to H2 concentration changes and the acetate and butyrate formation rates when H2 concentrations were less than 6 kPa. However, the model over-estimated acetate, ethanol and butanol productions at the expense of butyrate production at higher H2 concentrations. The H2 yield at the lowest dissolved H2 concentration was 2.67 ± 0.08 mol H2 /mol glucose, over 300% higher than the yield achieved in a CSTR operated without sparging.

The role of paraffin oil on the interaction between denitrifying anaerobic methane oxidation and Anammox processes.

Methane is sparingly soluble in water, resulting in a slow reaction rate in the denitrifying anaerobic methane oxidation (DAMO) process. The slow rate limits the feasibility of research to examine the interaction between the DAMO and the anaerobic ammonium oxidation (Anammox) process. In this study, optimized 5 % (v/v) paraffin oil was added as a second liquid phase to improve methane solubility in a reactor containing DAMO and Anammox microbes. After just addition, methane solubility was found to increase by 25 % and DAMO activity was enhanced. After a 100-day cultivation, the paraffin reactor showed almost two times higher consumption rates of NO3 (-) (0.2268 mmol/day) and NH4 (+) (0.1403 mmol/day), compared to the control reactor without paraffin oil. The microbes tended to distribute in the oil-water interface. The quantitative (q) PCR result showed the abundance of gene copies of DAMO archaea, DAMO bacteria, and Anammox bacteria in the paraffin reactor were higher than those in the control reactor after 1 month. Fluorescence in situ hybridization revealed that the percentages of the three microbes were 55.5 and 77.6 % in the control and paraffin reactors after 100 days, respectively. A simple model of mass balance was developed to describe the interactions between DAMO and Anammox microbes and validate the activity results. A mechanism was proposed to describe the possible way that paraffin oil enhanced DAMO activity. It is quite clear that paraffin oil enhances not only DAMO activity but also Anammox activity via the interaction between them; both NO3 (-) and NH4 (+) consumption rates were about two times those of the control.

New primers for detecting and quantifying denitrifying anaerobic methane oxidation archaea in different ecological niches.

The significance of ANME-2d in methane sink in the environment has been overlooked, and there was no any study evaluating the distribution of ANME-2d in the environment. New primers were thus needed to be designed for following research. In this paper, a pair of primers (DP397F and DP569R) was designed to quantify ANME-2d. The specificity and amplification efficiency of this primer pair were acceptable. PCR amplification of another pair of primers (DP142F and DP779R) generated a single, bright targeted band from the enrichment sample, but yielded faint, multiple bands from the environmental samples. Nested PCR was conducted using the primers DP142F/DP779R in the first round and DP142F/DP569R in the second round, which generated a bright targeted band. Further phylogenetic analysis showed that these targeted bands were ANME-2d-related sequences. Real-time PCR showed that the copies of the 16s ribosomal RNA gene of ANME-2d in these samples ranged from 3.72 × 10(4) to 2.30 × 10(5) copies µg(-1) DNA, indicating that the percentage of ANME-2d was greatest in a polluted river sample and least in a rice paddy sample. These results demonstrate that the newly developed real-time PCR primers could sufficiently quantify ANME-2d and that nested PCR with an appropriate combination of the new primers could successfully detect ANME-2d in environmental samples; the latter finding suggests that ANME-2d may spread in environments.

Polyphosphate during the Regreening of Chlorella vulgaris under Nitrogen Deficiency.

Polyphosphate (Poly-P) accumulation has been reported in Chlorella vulgaris under nitrogen deficiency conditions with sufficient P supply, and the process has been demonstrated to have great impact on lipid productivity. In this article, the utilization of polyphosphates and the regreening process under N resupplying conditions, especially for lipid production reviving, were investigated. This regreening process was completed within approximately 3-5 days. Polyphosphates were first degraded within 3 days in the regreening process, with and without an external P supply, and the degradation preceded the assimilation of phosphate in the media with an external P offering. Nitrate assimilation was markedly influenced by the starvation of P after polyphosphates were exhausted in the medium without external phosphates, and then the reviving process of biomass and lipid production was strictly impeded. It is, thus, reasonable to assume that simultaneous provision of external N and P is essential for overall biodiesel production revival during the regreening process.

The chemostat study of metabolic distribution in extreme-thermophilic (70°C) mixed culture fermentation.

The effects of pH, hydrogen partial pressure (PH2), and influent glucose concentration on the metabolic distribution in chemostat were investigated in this work in extreme-thermophilic mixed culture fermentation (MCF) process. The results showed that acetate, ethanol, and hydrogen were the main metabolites. A shift of ethanol to acetate and hydrogen was observed as pH increasing from 4.0 to 7.0 or PH2 decreasing from 0.64 to 0.05 atm. The maximum hydrogen yield was 3.16 ± 0.16 mol/mol glucose at PH2 0.05 atm. Lactate was only accumulated at low pH or high influent glucose concentration, while others such as butyrate and formate were rather low. Thermodynamic analysis illustrated that a mixture of acetate, ethanol, and/or lactate was essential for hydrogen production in extreme-thermophilic MCF. The hydrogen-producing rate was also calculated, and the maximum value was 2.2 ± 0.1 L/(L-reactor/day) at PH2 0.05 atm. Except hydrogen, other metabolites, such as liquid fatty acids and biofuels, could also be the producing targets in extreme-thermophilic MCF.

Simultaneous enrichment of denitrifying methanotrophs and anammox bacteria.

Interaction between denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (anammox) processes may play an important role in global carbon and nitrogen cycles. In this study, a coculture of denitrifying methanotrophs (DAMO archaea and DAMO bacteria) and anammox bacteria, initially sourced from the environment, was enriched with a supply of methane, nitrate, and ammonium. After a 4.5-month enrichment, simultaneous oxidation of methane and ammonium and reduction of nitrate were observed. The highest rate of nitrate reduction in the suspended DAMO culture was 4.84 mmol/L/day, and simultaneously, the highest ammonium removal rate was 4.07 mmol/L/day. Fluorescence in situ hybridization and analysis of 16S rRNA gene clone libraries revealed the coexistence of DAMO archaea, DAMO bacteria, and anammox bacteria. The development of anammox bacteria might reduce the enrichment time of DAMO microorganisms and promote the activity of DAMO archaea. The activity of the reactor fluctuated during the long-term operation, which might be caused by the formation of microbial clusters whereby DAMO archaea grew in aggregates that were surrounded by anammox and DAMO bacteria. This study is the first to demonstrate that it is feasible to establish a coculture of DAMO archaea, DAMO bacteria, and anammox bacteria from environmental inocula.

Precise and economical dredging model of sediments and its field application: case study of a river heavily polluted by organic matter, nitrogen, and phosphorus.

Environmental dredging is an efficient means to counteract the eutrophication of water bodies caused by endogenous release of nitrogen and/or phosphorus from polluted sediments. The huge operational cost and subsequent disposal cost of the dredged polluted sediments, as well as the adverse effect on the benthic environment caused by excessive dredging, make the currently adopted dredging methods unfavorable. Precise dredging, i.e., determining the dredging depth based on the pollution level, not only significantly decreases the costs but also leaves a uniform favorable environment for benthos. However, there is still no feasible process to make this promising method executable. Taking a river heavily polluted by organic compounds as an example, we proposed an executable precise dredging process, including sediment survey, model establishment, data interpolation, and calculation of dredging amount. Compared with the traditional dredging method, the precise one would save 16 to 45% of cost according to different pollutant removal demands. This precise dredging method was adopted by the National Water Project of China to treat the endogenous pollution of Nanfei River in 2010. This research provides a universal scientific and engineering basis for sediment dredging projects.

Evaluation of the after-effects of cyanobacterial cell removal and lysis by photocatalysis using Ag/AgBr/TiO2.

This study aimed to evaluate the after-effects of cyanobacterial cell removal and lysis by photocatalysis in water. A low concentration of 50 mg/L Ag/AgBr/TiO2 was applied to inactivate Microcystis aeruginosa under visible light irradiation. Most of the M. aeruginosa was killed within 5 h while microcystins-LR (MC-LR) was released into water and accumulated to a high concentration of 100 µg/L. Organic constituents released from cell damage led to 70 mg/L of total organic carbon (TOC) in water. The release of MC-LR and TOC would affect the biostability in the receiving water. Further, mineralization of cell lysis after photocatalysis over a long time resulted in the release of nutrients in water which would be a risk to cause cyanobacterial blooming again. Therefore, these after-effects should not be ignored when photochemical catalysis is applied to mitigate cyanobacterial blooming. Perhaps the best treatment is to remove intact cyanobacterial cells from water and then treat them off-site, for example by anaerobic digestion.