Community analysis of ammonia and nitrite oxidizers during start-up of nitritation reactors

Partial nitrification of ammonium to nitrite under oxic conditions (nitritation) is a critical process for the effective use of alternative nitrogen removal technologies from wastewater. Here we investigated the conditions which promote establishment of a suitable microbial community for performing nitritation when starting from regular sewage sludge. Reactors were operated in duplicate under different conditions (pH, temperature, and dilution rate) and were fed with 50 mM ammonium either as synthetic medium or as sludge digester supernatant. In all cases, stable nitritation could be achieved within 10 to 20 days after inoculation. Quantitative in situ hybridization analysis with group-specific fluorescent rRNA-targeted oligonucleotides (FISH) in the different reactors showed that nitrite-oxidizing bacteria of the genus Nitrospira were only active directly after inoculation with sewage sludge (up to 4 days and detectable up to 10 days). As demonstrated by quantitative FISH and restriction fragment length polymorphism (RFLP) analyses of the amoA gene (encoding the active-site subunit of the ammonium monooxygenase), the community of ammonia-oxidizing bacteria changed within the first 15 to 20 days from a more diverse set of populations consisting of members of the Nitrosomonas communis and Nitrosomonas oligotropha sublineages and the Nitrosomonas europaea-Nitrosomonas eutropha subgroup in the inoculated sludge to a smaller subset in the reactors. Reactors operated at 30 degrees C and pH 7.5 contained reproducibly homogeneous communities dominated by one amoA RFLP type from the N. europaea-N. eutropha group. Duplicate reactors at pH 7.0 developed into diverse communities and showed transient population changes even within the ammonia oxidizer community. Reactors at pH 7.5 and 25 degrees C formed communities that were indistinguishable by the applied FISH probes but differing in amoA RFLP types. Communities in reactors fed with sludge digester supernatant exhibited a higher diversity and were constantly reinoculated with ammonium oxidizers from the supernatant. Therefore, such systems could be maintained at a higher dilution rate (0.75 day(-1) compared to 0.2 day(-1) for the synthetic wastewater reactors). Despite similar reactor performance with respect to chemical parameters, the underlying community structures were different, which may have an influence on stability during perturbations.     

Community Analysis of Ammonia and Nitrite Oxidizers during Start-Up of Nitritation Reactors

Partial nitrification of ammonium to nitrite under oxic conditions (nitritation) is a critical process for the effective use of alternative nitrogen removal technologies from wastewater. Here we investigated the conditions which promote establishment of a suitable microbial community for performing nitritation when starting from regular sewage sludge. Reactors were operated in duplicate under different conditions (pH, temperature, and dilution rate) and were fed with 50 mM ammonium either as synthetic medium or as sludge digester supernatant. In all cases, stable nitritation could be achieved within 10 to 20 days after inoculation. Quantitative in situ hybridization analysis with group-specific fluorescent rRNA-targeted oligonucleotides (FISH) in the different reactors showed that nitrite-oxidizing bacteria of the genus Nitrospira were only active directly after inoculation with sewage sludge (up to 4 days and detectable up to 10 days). As demonstrated by quantitative FISH and restriction fragment length polymorphism (RFLP) analyses of the amoA gene (encoding the active-site subunit of the ammonium monooxygenase), the community of ammonia-oxidizing bacteria changed within the first 15 to 20 days from a more diverse set of populations consisting of members of the Nitrosomonas communis and Nitrosomonas oligotropha sublineages and the Nitrosomonas europaea-Nitrosomonas eutropha subgroup in the inoculated sludge to a smaller subset in the reactors. Reactors operated at 30°C and pH 7.5 contained reproducibly homogeneous communities dominated by one amoA RFLP type from the N. europaea-N. eutropha group. Duplicate reactors at pH 7.0 developed into diverse communities and showed transient population changes even within the ammonia oxidizer community. Reactors at pH 7.5 and 25°C formed communities that were indistinguishable by the applied FISH probes but differing in amoA RFLP types. Communities in reactors fed with sludge digester supernatant exhibited a higher diversity and were constantly reinoculated with ammonium oxidizers from the supernatant. Therefore, such systems could be maintained at a higher dilution rate (0.75 day⁻¹ compared to 0.2 day⁻¹ for the synthetic wastewater reactors). Despite similar reactor performance with respect to chemical parameters, the underlying community structures were different, which may have an influence on stability during perturbations.     

Changes in Microbial Communities Associated with the Sea Anemone Anemonia viridis in a Natural pH Gradient

Ocean acidification, resulting from rising atmospheric carbon dioxide concentrations, is a pervasive stressor that can affect many marine organisms and their symbionts. Studies which examine the host physiology and microbial communities have shown a variety of responses to the ocean acidification process. Recently, several studies were conducted based on field experiments, which take place in natural CO₂ vents, exposing the host to natural environmental conditions of varying pH. This study examines the sea anemone Anemonia viridis which is found naturally along the pH gradient in Ischia, Italy, with an aim to characterize whether exposure to pH impacts the holobiont. The physiological parameters of A. viridis (Symbiodinium density, protein, and chlorophyll a+c concentration) and its microbial community were monitored. Although reduction in pH was seen to have had an impact on composition and diversity of associated microbial communities, no significant changes were observed in A. viridis physiology, and no microbial stress indicators (i.e., pathogens, antibacterial activity, etc.) were detected. In light of these results, it appears that elevated CO₂ does not have a negative influence on A. viridis that live naturally in the site. This suggests that natural long-term exposure and dynamic diverse microbial communities may contribute to the acclimation process of the host in a changing pH environment.     

Characterization of virucidal agents in activated sludge.

A comprehensive study was carried out to determine the properties of agents responsible for loss of virus infectivity in mixed-liquor suspended solids (MLSS) of activated sludge. Initial experiments revealed that model enteric viruses (poliovirus-1 and rotavirus SA-11) were irreversibly inactivated in MLSS and released their RNA genomes. Enteric viruses belonging to other genera (echovirus-12, coxsackievirus A13, reovirus-3) were also shown to lose infectivity in MLSS. Although the virucidal activity decreased at reduced temperatures, MLSS still retained significant activity at 4 degrees C. The virucidal agents in MLSS were stable for months at 4 degrees C, but their activity decreased approximately 50% during 4 days of aeration at 26 degrees C. Primary effluent, the nutrient source for activated sludge, also contained virucidal activity. After centrifugation of MLSS, almost all virucidal activity was found in the particulate fraction because of inhibitory substances retained in the supernatant fraction. Decreasing or increasing the solids concentration of the particulate fraction did not increase the virucidal activity of the fraction. The effects of heat and antibiotics on the virucidal activity of MLSS, coupled with the finding that the activity can be produced in autoclaved primary effluent seeded with MLSS, strongly support the conclusion that microorganisms are responsible for this activity. Attempts to characterize the virucidal microbial components of MLSS indicated that treatments that resulted in the inactivation or removal of microorganisms also caused a loss of virucidal activity. Thus, it appears that the virucidal components of microorganisms are either short-lived or active only while bound to the organisms themselves.     

Characterization of virucidal agents in activated sludge

A comprehensive study was carried out to determine the properties of agents responsible for loss of virus infectivity in mixed-liquor suspended solids (MLSS) of activated sludge. Initial experiments revealed that model enteric viruses (poliovirus-1 and rotavirus SA-11) were irreversibly inactivated in MLSS and released their RNA genomes. Enteric viruses belonging to other genera (echovirus-12, coxsackievirus A13, reovirus-3) were also shown to lose infectivity in MLSS. Although the virucidal activity decreased at reduced temperatures, MLSS still retained significant activity at 4 degrees C. The virucidal agents in MLSS were stable for months at 4 degrees C, but their activity decreased approximately 50% during 4 days of aeration at 26 degrees C. Primary effluent, the nutrient source for activated sludge, also contained virucidal activity. After centrifugation of MLSS, almost all virucidal activity was found in the particulate fraction because of inhibitory substances retained in the supernatant fraction. Decreasing or increasing the solids concentration of the particulate fraction did not increase the virucidal activity of the fraction. The effects of heat and antibiotics on the virucidal activity of MLSS, coupled with the finding that the activity can be produced in autoclaved primary effluent seeded with MLSS, strongly support the conclusion that microorganisms are responsible for this activity. Attempts to characterize the virucidal microbial components of MLSS indicated that treatments that resulted in the inactivation or removal of microorganisms also caused a loss of virucidal activity. Thus, it appears that the virucidal components of microorganisms are either short-lived or active only while bound to the organisms themselves.     

Microbial behaviors involved in cake fouling in membrane bioreactors under different solids retention times

Biomass characteristics and membrane performances in the MBRs operated at a high flux of 30 L/m² h under different SRTs (10, 30 days, and infinity) were monitored. Results showed that more serious cake-fouling happened in the SRT-infinity MBR, which correlated with the activated sludge characteristics such as smaller floc size and greater EPS amount. DGGE analysis indicated that the microbial community shifted in different ways under various SRTs, which also influenced EPS productions in the MBRs. Different microbial communities were developed on the membrane surfaces at various operating stages and SRTs. Possibly, the activated sludge characteristics (such as MLSS concentration, EPS properties) and hydrodynamic conditions influenced by the SRTs were associated with cake layer development and membrane fouling propensity. Insight into the EPS characteristics and deposition behaviors of bacterial flocs will be crucial to explore appropriate biofouling control strategies in MBRs.     

利用污泥提取液制备微生物絮凝剂

以污水厂剩余污泥作为培养基原料,经过一系列处理,探索微生物絮凝剂产生菌的最适发酵培养基配方,结果表明,污泥预处理条件以pH 12碱解条件最优,碳氮源产出量最大,补加8 g/L葡萄糖后灭菌,微生物絮凝剂产生菌LLin6可正常产絮,絮凝率达91.55%。该结果为降低微生物絮凝剂的制备成本,并实现污泥的减量化和污泥资源化利用提供了基础。     

Study of Biological Activity and Process Stability in Submerged Membrane Bioreactors

Synthetic wastewater was treated in a bench scale submerged membrane bioreactor (SMBR). A long‐term experiment was conducted by varying the sludge residence time (SRT) (10–500 d) and BOD loading (1.3–0.25 kg/m³·d). The biological activity was observed in terms of the oxygen utilization rate (OUR) and adenosine triphosphate (ATP) profile; the process stability was analyzed based on the extent of organic degradation and suction pressure. The microbial population in the SMBR was dependent on the SRT and BOD loading, and its biological activity was increased with an increase in the SRT or BOD loading. At a low feed to microorganism (F/M) ratio (0.06 kg BOD/kg MLSS·d), the sludge production of the reactor was reduced to 0.04 kg MLSS/kg BOD, which is much less than in the conventional activated sludge process (0.4–0.6 kg MLSS/kg BOD). The F/M ratio influenced the biological activity (via ATP and the OUR) significantly at a short SRT (≤90 d). However, the effect of the F/M ratio ceased at a low F/M ratio (≤ 0.07 kg BOD/kg MLSS·d). The accumulation of organics in the SMBR was accompanied with an increase in the supernatant TOC, which caused a high suction pressure and an abrupt change in the operating conditions to process instability. However, the process stability of the SMBR increased with an increase in the SRT and a decrease in the BOD loading along with a concomitant decrease in the biological activity and sludge production.     

Analysis of microbial diversity in tomato paste wastewater through PCR-DGGE

This study was conducted to evaluate the relationship between the flora and the changes in the microbial communities during tomato paste wastewater treatment. The bio-analytical techniques like Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE), adenosine-triphosphate (ATP) analysis, and testing of mixed liquid and suspended solids (MLSS) were simultaneously conducted to analyze the dynamics of the microbial communities during tomato paste wastewater treatment process. The study suggests that the combined approaches of PCR-DGGE, ATP, and MLSS provided a simple and accurate method to evaluate the changes in microbial activity, microbial structure, and population size with the shift in contaminants in different treatment processes. The study also demonstrates that the structure and quantity of a microbial community are influenced by MLSS during the wastewater treatment process, which consequently determines the overall functionality of the system.     

Comparison of p-Nitrophenol Biodegradation in Field and Laboratory Test Systems

Acclimation of microbial communities exposed to p-nitrophenol (PNP) was measured in laboratory test systems and in a freshwater pond. Laboratory tests were conducted in shake flasks with water, shake flasks with water and sediment, eco-cores, and two sizes of microcosm. The sediment and water samples used in the laboratory experiments were obtained from the pond. After a 6-day acclimation period, PNP was biodegraded rapidly in the pond. When the pond was treated with PNP a second time, biodegradation began immediately. The acclimation periods in laboratory test systems that contained sediment were similar to that in the pond. The acclimation period was threefold longer in shake flasks without sediment. PNP was biodegraded more slowly by microbial communities acclimated in the laboratory than it was in the pond, and the rate of biodegradation varied with the type of test. The number of bacteria able to mineralize PNP increased by 3 orders of magnitude in the pond during the acclimation period. Similar increases accompanied acclimation in the laboratory systems.     

Nutrient removal in an A2O-MBR reactor with sludge reduction

In the present study, an advanced sewage treatment process has been developed by incorporating excess sludge reduction and phosphorous recovery in an A2O-MBR process. The A2O-MBR reactor was operated at a flux of 17 LMH over a period of 210 days. The designed flux was increased stepwise over a period of two weeks. The reactor was operated at two different MLSS range. Thermo chemical digestion of sludge was carried out at a fixed pH (11) and temperature (75 °C) for 25% COD solubilisation. The released phosphorous was recovered by precipitation process and the organics was sent back to anoxic tank. The sludge digestion did not have any impact on COD and TP removal efficiency of the reactor. During the 210 days of reactor operation, the MBR maintained relatively constant transmembrane pressure. The results based on the study indicated that the proposed process configuration has potential to reduce the excess sludge production as well as it didn’t detoriated the treated water quality.     

Microbial communities acclimate to recurring changes in soil redox potential status

Rapidly fluctuating environmental conditions can significantly stress organisms, particularly when fluctuations cross thresholds of normal physiological tolerance. Redox potential fluctuations are common in humid tropical soils, and microbial community acclimation or avoidance strategies for survival will in turn shape microbial community diversity and biogeochemistry. To assess the extent to which indigenous bacterial and archaeal communities are adapted to changing in redox potential, soils were incubated under static anoxic, static oxic or fluctuating redox potential conditions, and the standing (DNA‐based) and active (RNA‐based) communities and biogeochemistry were determined. Fluctuating redox potential conditions permitted simultaneous CO₂ respiration, methanogenesis, N₂O production and iron reduction. Exposure to static anaerobic conditions significantly changed community composition, while 4‐day redox potential fluctuations did not. Using RNA : DNA ratios as a measure of activity, 285 taxa were more active under fluctuating than static conditions, compared with three taxa that were more active under static compared with fluctuating conditions. These data suggest an indigenous microbial community adapted to fluctuating redox potential.     

Perchlorate reduction by hydrogen autotrophic bacteria and microbial community analysis using high-throughput sequencing

Hydrogen autotrophic reduction of perchlorate have advantages of high removal efficiency and harmless to drinking water. But so far the reported information about the microbial community structure was comparatively limited, changes in the biodiversity and the dominant bacteria during acclimation process required detailed study. In this study, perchlorate-reducing hydrogen autotrophic bacteria were acclimated by hydrogen aeration from activated sludge. For the first time, high-throughput sequencing was applied to analyze changes in biodiversity and the dominant bacteria during acclimation process. The Michaelis–Menten model described the perchlorate reduction kinetics well. Model parameters q _max and K _s were 2.521–3.245 (mg ClO₄ ^?/gVSS h) and 5.44–8.23 (mg/l), respectively. Microbial perchlorate reduction occurred across at pH range 5.0–11.0; removal was highest at pH 9.0. The enriched mixed bacteria could use perchlorate, nitrate and sulfate as electron accepter, and the sequence of preference was: NO₃ ^? > ClO₄ ^? > SO₄ ^2?. Compared to the feed culture, biodiversity decreased greatly during acclimation process, the microbial community structure gradually stabilized after 9 acclimation cycles. The Thauera genus related to Rhodocyclales was the dominated perchlorate reducing bacteria (PRB) in the mixed culture.     

Ascaris lumbricoides: Coproporphyrinogen oxidase activity in eggs and muscle

The possible presence of coproporphyrinogen oxidase (EC 1.3.3.3), an oxygen-requiring enzyme in the porphyrin biosynthetic pathway, was investigated in supernatant fractions of homogenized Ascaris lumbricoides muscle and developing eggs, and in mitochondrial preparations of muscle. Compared with rat liver controls, low levels of enzyme activity were found in A. lumbricoides gut, 6-day eggs, and muscle mitochondria. Enzyme activity in muscle, 8-day, and 25-day eggs was not measurable under the conditions employed.     

The effect of bioaugmentation on the performance of sequencing batch reactor and sludge characteristics in the treatment process of papermaking wastewater

In this paper, the differences between reinforced sequencing batch reactor, which was inoculated with superior mixed flora, and conventional sequencing batch reactor were compared in the process of treating papermaking wastewater under similar conditions. The results showed that the addition of superior mixed flora could not only shorten the sludge acclimation time, but also improve the treatment efficiency of reactor as well as make the reactor have higher ability to withstand high volume loading rate; the phenomenon of aerobic granulation only occurred in reinforced sequencing batch reactor, and superior mixed flora were the key reason that aerobic granular sludge could shape; aerobic granular sludge had many advantages over conventional activated sludge such as it possessed compacter microbial structure, better settling performance, and lower water content.     

Biodiversity analysis of microbial community in the chem-bioflocculation treatment process

Total DNA was directly extracted from environmental samples and amplified with polymerase chain reaction (PCR) technique. The PCR products were fingerprinted via denaturing gradient gel electrophoresis (DGGE). Significant differences were observed in the microbial community structures between traditional treatment process and chem-bioflocculation process. The microbial community structure shift at different sampling locations in chem-bioflocculation process and on two typical operational conditions was studied. 16S rDNA V3 regions of some dominant species were sequenced and the species were identified. The microbial communities were stable in both the chem-bioflocculation process and the activated sludge process under various experimental conditions presented in this work. The attached growth treatment process was less stable when operational conditions changed.     

The effect of pH on the efficiency of an SBR processing piggery wastewater

To treat piggery wastewater efficiently, the hydrolysis of urea (mainly derived from swine urine) in piggery wastewater with the change of sewage pH must be considered. Using activated sludge, piggery wastewater was treated in a sequencing batch reactor (SBR), and the effects of influent pH on SBR processing efficiency, sludge settle ability, and sludge activity were investigated. The results showed that a high influent pH value contributed to the improvement of the removal rate of ammonia nitrogen and reduction of the chemical oxygen demand (COD). When the influent pH was between 9.0 and 9.5, the removal rate of ammonia nitrogen was higher than 90%, and the reduction of COD from its original value was 80%. The influent pH had a greater influence on sludge concentration and sludge activity. When the influent pH increased from 7.0 to 9.5, the sludge concentration increased from 2,350 to 3,947 mg/L in the reactor, and the activities of ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) first increased and then decreased. When the influent pH was 9.0 and 8.0, the maximum values (0.48 g O₂/(g MLSS/day) and 0.080 g O₂/(g MLSS/day)) were reached, and the sludge settling ratio was nearly steady between 20 and 35% in each reactor.     

Molecular analysis of halophilic bacterial community for high-rate denitrification of saline industrial wastewater

A denitrification system for saline wastewater utilizing halophilic denitrifying bacteria has not been developed so far. In this study, denitrification performance and microbial community under various saline conditions were investigated using denitrifying sludge acclimated under low-salinity condition for a few years as seed sludge. A continuous denitrification experiment showed that denitrification performance and microbial community at 10% salinity was higher than that at 1% salinity. The microbial community in the denitrification sludge that was acclimated under low salinity was monitored by terminal-restriction fragment length polymorphism (T-RFLP) analysis during acclimation to high-salinity condition. T-RFLP profiles and clone analysis based on 16S rRNA-encoding genes in the sludge of the denitrification system with 10% salinity indicated that the γ-Proteobacteria, particularly Halomonas spp., were predominant species, suggesting that these bacterial members were possibly responsible for a high denitrification activity under high-salinity conditions. Furthermore, the investigation of denitrification performance under various saline conditions revealed that 4–10% salinity results in the highest denitrification rate, indicating that this salinity was optimal for predominant bacterial species to exhibit denitrification activity. These results indicate the possibility that an appropriate denitrification system for saline wastewater can be designed using acclimated sludge with a halophilic community.     

Aerobic Reduction of Hexavalent Chromium in Soil by Indigenous Microorganisms

Surface soil containing 25,100 mg/kg total Cr [12,400 mg/kg Cr(VI)] obtained from a Superfund site was used in laboratory microcosm studies to evaluate the potential for aerobic reduction of Cr(VI) by the indigenous soil microbial community. Hexavalent chromium in soil was reduced by as much as 33% (from 1840 to 1240 mg/L) within 21 days under enrichment conditions. Reduction of Cr(VI) in this system was biologically mediated and depended on the availability of usable energy sources. Mass balance studies suggested that the microbial populations removed Cr(VI) from the soil solutions by reduction. Indigenous microbial soil communities even with no history of Cr(VI) contamination were capable of mediating this process. However, Cr(VI) removal was not observed when microbial populations from a sewage sludge sample were added to the soil microcosms. The results suggest that Cr(VI)-reducing microbial populations are widespread in soil, and thus the potential exists for in situ remediation of environmentally significant levels of Cr( VI) contamination.