Impact of operating history on mixed culture fermentation microbial ecology and product mixture

Mixed culture fermentation is an alternative to pure culture fermentation for production of biofuels and valuable products. A glucose-fed, continuous reactor was operated cyclically to a central pH of 5.5 from a number of precedent pHs, from 4.5 to 7.5. At each pH, stable chemical production was reached after 2 retention times and was held for least 2 further retention times prior to the next change. Bacterial groups were identified by phylogenetic analysis of 16S rRNA gene clones. Bacterial community dynamics were monitored by terminal-restriction fragment length polymorphism. More ethanol was produced at high pH, and more butyrate at lower pH. At pH 5.5, the product spectrum was not measurably influenced by precedent pH but showed seemingly random changes. The impact of precedent pH on community structure was more systematic, with clear indications that when the pH was returned to 5.5, the bacterial group that was dominant at the precedent pH remained at high abundance. This result is important, since it indicates a decoupling between microbial function (as indicated by product spectrum), and community structure. More work is needed to determine the longevity of this hysteresis effect. There was evidence that groups retained their ability to re-emerge even after times of low abundance.     

Anaerobic bio-hydrogen production using pre-heated river sediments as seed sludge.

Anaerobic bio-hydrogen production is the focus in the field of bio-energy resources. In this paper, a series of batch experiments were conducted to investigate the effects of several factors on anaerobic bio-hydrogen producing process carried out by pre-heated river sediments. The results showed that several factors such as substrate and its concentration, temperature and the initial pH value could affect the anaerobic bio-hydrogen production in different levels. At 35 degrees C and the initial pH of 6.5, using glucose of 20,000 mg COD/L as substrate, the highest hydrogen production of 323.8 ml-H2/g TVS in a 100 ml batch reactor was reached, the specific hydrogen production rate was 37.7 ml-H2/g TVS h, and the hydrogen content was 51.2%. Thereafter using the same pre-heated river sediments as seed sludge, continuous anaerobic bio-hydrogen production was achieved successfully in a lab-scale CSTR with gas-separator. At the organic loading rate of 36 kg COD/m3 d, the highest hydrogen production was 6.3-6.7 l-H2/l-reactor d, the specific hydrogen production was 1.3-1.4 mol-H2/mol-glucose, and the hydrogen content in the gas was 52.3%. The effluent of the bio-reactor contained some small molecular organics, mainly including ethanol, acetate, butyrate and their molar proportion is 1:1:0.6.     

Effect of sludge discharge positions on steady-state aerobic granules in sequencing batch reactor (SBR)

We have investigated the effect of sludge discharge location on the steady-state aerobic granules in sequencing batch reactors (SBRs). Two SBRs were operated concurrently with the same sludge retention time using sludge discharge ports at: (a) the reactor bottom in R1; and (b) the reactor middle-lower level in R2. Results indicate that both reactors could maintain sludge granulation and stable operation, but the two different sludge discharge methods resulted in significantly different aerobic granule characteristics. Over 30 days, the chemical oxygen demand (COD) removal of the two reactors was maintained at similar levels (above 96%), and typical bioflocs were not observed. The average aerobic granule size in R2 was twice that in R1, as settling velocity increased in proportion to size increment. Meanwhile, the production yields of polysaccharide and protein content in R2 were always higher than those in R1. However, due to mass transfer limitations and the presence of anaerobes in the aerobic granule cores, larger granules had a tendency to disintegrate in R2. Thus, we conclude that a sludge discharge port situated at the reactor bottom is beneficial for aerobic granule stability, and enhances the potential for long-term aerobic granule SBR operation.     

Polyhydroxyalkanoates production by activated sludge in a SBR using acetate and propionate as carbon sources.

In this work, sludge was submitted to aerobic dynamic substrate feeding. Two sequencing batch reactors were operated, with acetate or propionate as carbon substrates. When acetate was used the system only produced a homopolymer of polyhydroxybutyrate (PHB). In order to maximize the PHB production, tests with different concentrations of acetate and ammonia were preformed. The best results (67.2% of PHB by cell dry weight) were obtained for 0.7 Nmmol/l of ammonia and 180 Cmmol/l of acetate. The PHB cell content was further improved by pulse addition of substrate, three times 60 Cmmol/l of acetate, reaching a value of 78.5%. Propionate can be used as a precursor for hydroxyvalerate. In conjunction with other substrates, it allows for the formation of copolymers, which present better processing properties on commercial applications. Tests with different concentrations of propionate and ammonia were performed. Under the operating conditions used, the maximum PHA accumulated inside cells was 34.8%, with 30 Cmmol/l of propionate and no ammonia.     

Microbial community analysis with a high PHA storage capacity.

Activated sludge was submitted to aerobic dynamic substrate feeding for the production of biodegradable plastics. Two sequencing batch reactors were operated with acetate or propionate as sole carbon substrates. With acetate a homopolymer of polyhydroxybutyrate (PHB) was obtained and with propionate a copolymer of hydroxybutyrate and hydroxyvalerate P(HB/HV) was produced. Three main morphotypes were identified in both sludges: two belong to the Alphaproteobacteria class and the third to the Betaproteobacteria class. Bacilli belonging to Betaproteobacteria were shown by FISH analysis, applied in combination with Nile Blue post-staining, to be the main responsible for PHAs storage. The latter were affiliated to Azoarcus genus within Betaproteobacteria.     

Syntrophic acetate oxidation in two-phase (acid-methane) anaerobic digesters

The microbial processes involved in two-phase anaerobic digestion were investigated by operating a laboratory-scale acid-phase (AP) reactor and analyzing two full-scale, two-phase anaerobic digesters operated under mesophilic (35 °C) conditions. The digesters received a blend of primary sludge and waste activated sludge (WAS). Methane levels of 20% in the laboratory-scale reactor indicated the presence of methanogenic activity in the AP. A phylogenetic analysis of an archaeal 16S rRNA gene clone library of one of the full-scale AP digesters showed that 82% and 5% of the clones were affiliated with the orders Methanobacteriales and Methanosarcinales, respectively. These results indicate that substantial levels of aceticlastic methanogens (order Methanosarcinales) were not maintained at the low solids retention times and acidic conditions (pH 5.2-5.5) of the AP, and that methanogenesis was carried out by hydrogen-utilizing methanogens of the order Methanobacteriales. Approximately 43, 31, and 9% of the archaeal clones from the methanogenic phase (MP) digester were affiliated with the orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales, respectively. A phylogenetic analysis of a bacterial 16S rRNA gene clone library suggested the presence of acetate-oxidizing bacteria (close relatives of Thermacetogenium phaeum, 'Syntrophaceticus schinkii,' and Clostridium ultunense). The high abundance of hydrogen consuming methanogens and the presence of known acetate-oxidizing bacteria suggest that acetate utilization by acetate oxidizing bacteria in syntrophic interaction with hydrogen-utilizing methanogens was an important pathway in the second-stage of the two-phase digestion, which was operated at high ammonium-N concentrations (1.0 and 1.4 g/L). A modified version of the IWA Anaerobic Digestion Model No. 1 (ADM1) with extensions for syntrophic acetate oxidation and weak-acid inhibition adequately described the dynamic profiles of volatile acid production/degradation and methane generation observed in the laboratory-scale AP reactor. The model was validated with historical data from the full-scale digesters.     

Variations of electron flux and microbial community in air-cathode microbial fuel cells fed with different substrates

Microbial fuel cells (MFCs) can convert chemical energy to electricity using microbes as catalysts and a variety of organic wastewaters as substrates. However, electron loss occurs when fermentable substrates are used because fermentation bacteria and methanogens are involved in electron flow from the substrates to electricity. In this study, MFCs using glucose (G-MFC), propionate (P-MFC), butyrate (B-MFC), acetate (A-MFC), and a mix (M-MFC, glucose:propionate:butyrate:acetate = 1:1:1:1) were operated in batch mode. The metabolites and microbial communities were analyzed. The current was the largest electron sink in M-, G-, B-, and A-MFCs; the initial chemical oxygen demands (COD(ini)) involved in current production were 60.1% for M-MFC, 52.7% for G-MFC, 56.1% for B-MFC, and 68.3% for A-MFC. Most of the glucose was converted to propionate (40.6% of COD(ini)) and acetate (21.4% of COD(ini)) through lactate (80.3% of COD(ini)) and butyrate (6.1% of COD(ini)). However, an unknown source (62.0% of COD(ini)) and the current (34.5% of COD(ini)) were the largest and second-largest electron sinks in P-MFC. Methane gas was only detected at levels of more than 10% in G- and M-MFCs, meaning that electrochemically active bacteria (EAB) could out-compete acetoclastic methanogens. The microbial communities were different for fermentable and non-fermentable substrate-fed MFCs. Probably, bacteria related to Lactococcus spp. found in G-MFCs with fermentable substrates would be involved in both fermentation and electricity generation. Acinetobacter-like species, and Rhodobacter-like species detected in all the MFCs would be involved in oxidation of organic compounds and electricity generation.     

Biohydrogen production by mesophilic fermentation of food wastewater.

Batch experiments were conducted to convert molasses wastewater (10-160 g COD/L) into hydrogen at 35 degrees C at various pH (4-8). The maximum hydrogen productivity (HP) and hydrogen production rate (HPR) reached 47.1 mmol-H2/g COD(re) and 97.5 mmol-H2/L-day, respectively, at a substrate concentration of 40 g COD/L and pH 6.0. The methane-free biogas contained up to 50% (v/v) of hydrogen. Fermentation at wastewater concentrations higher than 60 g COD/L required a long acclimation period (more than 20 h). Though the fractions were substrate concentration and pH-dependent, acetate and butyrate were the two main liquid fermentation products. A comparison of the HP and HPR data indicates that defining a hydrogen yield indicator to evaluate hydrogen generation efficiency should be taken into consideration in practical fermentation operations.     

Development and testing of bioelectrochemical reactors converting wastewater organics into hydrogen peroxide

In a bioelectrochemical system, the energy content in dissolved organic matter can be used to power the production of hydrogen peroxide (H(2)O(2)), which is a potentially useful chemical at wastewater treatment plants. H(2)O(2) can be produced by the cathodic reduction of oxygen. We investigated four types of gas-diffusion electrodes (GDEs) for this purpose. A GDE made of carbon nanoparticles bound with 30% polytetrafluoroethylene (PTFE) (wt./wt.C) to a carbon fiber paper performed best and catalyzed H(2)O(2) production from oxygen in air with a coulombic efficiency of 95.1%. We coupled the GDE to biological anodes in two bioelectrochemical reactors. When the anodes were fed with synthetic wastewater containing acetate they generated a current of up to ～0.4 mA/mL total anode compartment volume. H(2)O(2) concentrations of ～0.2 and ～0.5% could be produced in 5 mL catholyte in 9 and 21 h, respectively. When the anodes were fed with real wastewater, the generated current was ～0.1 mA/mL and only 84 mg/L of H(2)O(2) was produced.     

Isolation and characterization of a heterotrophic nitrifier from coke plant wastewater

This study investigated some factors affecting ammonium removal and nitrite accumulation by Alcaligenes faecalis C16, which was isolated from the activated sludge of a coking wastewater treatment plant. Nitrite was produced from ammonium only in the presence of citrate, acetate, meat extract, peptone or ethanol. The highest amount of nitrite was found with citrate as carbon source. A. faecalis C16 could not use glucose, fructose, sucrose and methanol. Under the optimum conditions of initial pH 6.0, C/N 14, 30 °C and 120 rpm, a maximum nitrite accumulation of 28.29 mg/L NO(2)(-)-N was achieved when the organism grew with citrate in four days. Nitrite accumulation increased with the increase of NH(4)(+)-N. Furthermore, A. faecalis C16 was shown to have phenol-degrading capacity during ammonium removal. Metabolism of phenol resulted in acidification of the media, which is not favorable for nitrification, whereas many other carbon sources made the medium more alkaline. However, no inhibitory effect by phenol was observed when phenol and acetate were used as mixed carbon source at different phenol/sodium acetate (P/S) ratios and their pH values were all controlled above 9.2 or P/S ratios below 5:5. These results suggested that A. faecalis C16 has some potential application in industrial wastewater treatment systems.     

Effect of pH control strategies and substrate concentration on the hydrogen yield from fermentative hydrogen production in large laboratory-scale

A series of batch experiments investigating two different pH control strategies, initial pH adjustment and continuous pH control, have been carried out in large laboratory-scale reactors with working volumes of 30 L. In both cases, pH was varied between 5 and 7.5. Sucrose concentrations were also varied starting from 0 up to 30 g/L. Higher hydrogen production yields can be achieved by batch experiments through continuous pH control than by simple initial pH adjustment. In the case of continuous pH control, maximization of hydrogen yield was acquired for slightly acidic pH of 6.5. Continuous pH control in the neutral pH range of 7.0 and in pH lower than 6.5, induced a reduction in the hydrogen production yield. Sucrose can be completely degraded only for a pH higher than 6. Lower pH values seem to inhibit the hydrogen-producing bacteria. Under the conditions of continuous pH adjustment at pH 6.5 and a sucrose concentration of 25 g/L the maximum hydrogen yield of 1.79 mol H(2)/mol hexose was obtained. These conditions could be applied for the batch start-up of large fermentors.     

Continuous hydrogen production from organic waste.

The antibiotic effects of lactic acid bacteria, Lactobacillus paracasei, on hydrogen production were investigated using glucose as the substrate for the batch experiments. The effects of lactic acid bacteria on hydrogen fermentation depended on pH and the inhibition of hydrogen-producing bacteria was prevented by keeping the pH over 5.0. Then, a continuous hydrogen production experiment was conducted by using bean curd manufacturing waste as an actual organic waste at pH 5.5 at 35 degrees C. The increase of the substrate concentration and the addition of nitrogen gave precedence to acetic and butyric acids production in the metabolic pathway and suppressed propionic acid production. As the result, continuous hydrogen production from municipal organic waste was enabled.     

Effect of denitrification type on pH profiles in the sequencing batch reactor process.

Laboratory batch experiments were conducted to investigate pH profiles during partial and complete denitrification with sufficient organic carbon source. Five stirred tank-type glass vessels, with a 7 L working volume for each, were used as SBR reactors that were all operated in denitrification mode. Five levels of initial proportion of nitrogen substances, i.e. nitrate and nitrite, were used in five reactors, respectively. Results showed that, at given temperature and mixed liquor suspended solids (MLSS), partial denitrification could attain a higher pH value than complete denitrification at the end of denitrification with the same initial NOx- concentration. The larger proportion the nitrite took in initial NOx- concentration, the higher pH peak would be obtained on pH profiles during denitrification despite the same total alkalinity produced. It was found that different types of alkalinity were produced during biological denitrification with different nitrogen substances. Partial denitrification could more carbonate alkalinity produce than complete denitrification. Furthermore, some characteristic points were identified on pH profiles which could indicate the disappearance of not only nitrate, but also nitrite in system. When computers are used to detect these features, they can provide rapid, real-time information, regarding the biological state of the system.     

Thermal pretreatment of the solid fraction of manure: impact on the biogas reactor performance and microbial community.

Application of thermal treatment at 100-140 degrees C as a pretreatment method prior to anaerobic digestion of a mixture of cattle and swine manure was investigated. In a batch test, biogasification of manure with thermally pretreated solid fraction proceeded faster and resulted in the increase of methane yield. The performances of two thermophilic continuously stirred tank reactors (CSTR) treating manure with solid fraction pretreated for 40 minutes at 140 degrees C and non-treated manure were compared. The digester fed with the thermally pretreated manure had a higher methane productivity and an improved removal of the volatile solids (VS). The properties of microbial communities of both reactors were analysed. The specific methanogenic activity (SMA) test showed that both biomasses had significant activity towards hydrogen and formate, while the activity with the VFA - acetate, propionate and butyrate - was low. The kinetic parameters of the VFA conversion revealed a reduced affinity of the microbial community from the CSTR fed with thermally pre-treated manure for acetate, propionate and butyrate. The bacterial and archaeal populations identified by t-RLFP analysis of 16S rRNA genes were found to be identical in both systems. However, a change in the abundance of the species present was detected.     

Comparison of different conditions, substrates and operation modes by dynamic simulation of a full-scale anaerobic SBR plant

Simulation studies for a full-scale anaerobic unit of a wastewater treatment plant (WWTP) were performed using the anaerobic digestion model no. 1 (ADM1). The anaerobic full-scale plant consists of one mesophilic and one thermophilic digester, operated in an anaerobic sequential batch reactor (ASBR) mode, and sludge enrichment reactors (SER) for each digester. The digesters are fed with a mixture of vegetable waste and process wastewater from the food factory. Characteristics such as COD(total), N(total) and NH(4)-N concentrations in the influent and effluent of the digester and SERs were measured and used for input fractionation. Parameters such as level, pH, biogas amount and composition in the digester were measured online and used for calibration. For simulation studies, different temperatures and operation modes with varying chemical oxygen demand (COD) input loads corresponding to feedstocks such as fruits, vegetables and grain were analysed and compared. Higher gas production and digestion efficiency in the thermophilic reactor and in shorter cycles were found and confirmed at full scale. Serial operation mode increased the gas production, but pH inhibition occurred earlier. Feeding only biosolids into digester I and the effluent of digester I together with process water into digester II further improved gas production in serial operation mode.     

Effects of heat treatment on microbial communities of granular sludge for biological hydrogen production

Dark fermentation shares many features with anaerobic digestion with the exception that to maximize hydrogen production, methanogens and hydrogen-consuming bacteria should be inhibited. Heat treatment is widely applied as an inoculum pre-treatment due to its effectiveness in inhibiting methanogenic microflora but it may not exclusively select for hydrogen-producing bacteria. This work evaluated the effects of heat treatment on microbial viability and structure of anaerobic granular sludge. Heat treatment was carried out on granular sludge at 100 °C with four residence times (0.5, 1, 2 and 4 h). Hydrogen production of treated sludges was studied from glucose by means of batch test at different pH values. Results indicated that each heat treatment strongly influenced the granular sludge resulting in microbial communities having different hydrogen productions. The highest hydrogen yields (2.14 moles of hydrogen per mole of glucose) were obtained at pH 5.5 using the sludge treated for 4 h characterized by the lowest CFU concentration (2.3 × 10(3)CFU/g sludge). This study demonstrated that heat treatment should be carefully defined according to the structure of the sludge microbial community, allowing the selection of highly efficient hydrogen-producing microbes.     

Application of enzymes in anaerobic digestion.

Owing to the very low economic value of brewer's spent grains, its utilisation for biogas production is very promising. The hydrolysis of ligno-cellulose is the rate limiting step in anaerobic digestion. Enzymatic pre-treatment promotes the hydrolysis of ligno-cellulose, breaking it down to lower molecular weight substances which are ready to be utilised by the bacteria. A cheap raw multi-enzyme produced by a solid state fermentation (SSF) process is a good substitute for expensive conventional enzyme. The SSF enzyme application to spent grain has been investigated by carrying out enzymatic solubility tests, hydrolytic experiments and two-step anaerobic fermentation of spent grain. Gas chromatograph analysis was conducted to quantify fatty acids concentrations, while CH(4), CO(2), O(2), H(2) and H(2)S were measured to determine biogas quality by means of a gas analyser. DS, oDS, pH were also measured to analyse the anaerobic digestion. The result shows that enzyme application promotes the hydrolysis of ligno-cellulose, indicated by higher enzymatic solubility and fatty acid concentration in a hydrolytic bioreactor. Moreover, biogas production is also increased. The quality of the gases produced is also enhanced. Since the anaerobic digestion can be operated in a stable performance, it can also be concluded that SSF enzyme is compatible with anaerobic digestion.     

Sludge-sludge interaction in the enhanced biological phosphorus removal process.

Metabolisms related to enhanced biological phosphorus removal (EBPR) were found to be affected when two activated sludges with different EBPR activities were mixed together. In the present study, two laboratory scale EBPR processes were operated in parallel, one of them with higher and another with lower EBPR activities. The activated sludges from the two reactors were mixed together at different mixing ratios. The supernatant was made the same for all mixing ratios, anaerobic-aerobic batch experiments were performed, and acetate uptake rate and phosphate release rate under anaerobic conditions and phosphate uptake rate under aerobic condition were determined. The metabolic rates measured were expected to be linear to the mixing ratios, as the supernatant was the same for all mixing ratios, whereas the metabolic rates were either promoted or inhibited by mixing of sludges. As an indicator for the sludge mixing effect on the metabolic rates, mixing effect intensity (MEI) was introduced. Chemical substances that are produced by microorganisms in activated sludge are proposed to be one of the possible causes of the sludge mixing effect.     

EGSB反应器的启动运行研究

采用两个小试规模的EGSB(ExpandedGranularSludgeBed)反应器 ,分别接种厌氧絮状污泥和颗粒污泥来研究其启动规律。试验结果表明 ,接种厌氧絮状污泥的R1反应器 ,由于出水循环的采用导致严重的污泥流失 ,首先应按UASB反应器的运行方式培养出颗粒污泥后 ,再按EGSB反应器方式运行 ,共需 78d才能完成启动 ;接种厌氧颗粒污泥的R2反应器 ,采用适宜的回流比有利于细菌的生长和反应器运行效果的改善 ,在经过短暂的无回流驯化后 ,即可按EGSB运行方式启动运行 ,仅需 32d即完成启动。     

Investigation of the diversity of homoacetogenic bacteria in mesophilic and thermophilic anaerobic sludges using the formyltetrahydrofolate synthetase gene.

Homoacetogenic bacteria are strict anaerobes capable of autotrophic growth on H(2)/CO(2) or CO, and of heterotrophic growth on a wide range of sugars, alcohols, methoxylated aromatic compounds and one carbon compounds, yielding acetate as their sole metabolic end-product. Batch activity tests on anaerobic granular sludge, using H(2)/CO(2) as a substrate and 2-bromoethanesulfonate (BES) as a specific methanogenic inhibitor revealed that H(2)/CO(2) conversion and concomitant acetate production commenced only after a lag period of 60-100 h. This finding suggests that the homoacetogenic population of digester sludge could be maintained by heterotrophic growth on sugars or other organic compounds, rather than by autotrophic growth on H(2)/CO(2). In the present study, two upflow anaerobic sludge bed (UASB) reactors were operated at 37 degrees C and 55 degrees C for two distinct trial periods, each characterised by the application of influents designed to enrich for homoacetogenic bacteria. Specific primers designed for the amplification of the functional gene encoding formyltetrahydrofolate synthetase (FTHFS), a key enzyme in the acetyl-CoA pathway of acetogenesis, were used as a specific probe for acetogenic bacteria. The diversity of acetogens in the granular sludge cultivated in each reactor was revealed by application of FTHFS targeted PCR. Results show that biomass acetogenic composition was dependent upon the operational temperature of the reactor and the substrate supplied as influent.