Development of high-rate anaerobic ammonium-oxidizing (anammox) biofilm reactors

To promptly establish anaerobic ammonium oxidation (anammox) reactors, appropriate seeding sludge with high abundance and activity of anammox bacteria was selected by quantifying 16S rRNA gene copy numbers of anammox bacteria by real-time quantitative PCR (RTQ-PCR) and batch culture experiments. The selected sludge was then inoculated into up-flow fixed-bed biofilm column reactors with nonwoven fabric sheets as biomass carrier and the reactor performances were monitored over 1 year. The anammox reaction was observed within 50 days and a total nitrogen removal rate of 26.0 kg-Nm(-3)day(-1) was obtained after 247 days. To our knowledge, such a high rate has never been reported before. Hydraulic retention time (HRT) and influent NH(4)(+) to NO(2)(-) molar ratio could be important determinant factors for efficient nitrogen removal in this study. The higher nitrogen removal rate was obtained at the shorter HRT and higher influent NH(4)(+)/NO(2)(-) molar ratio. After anammox reactors were fully developed, the community structure, spatial organization and in situ activity of the anammox biofilms were analyzed by the combined use of a full-cycle of 16S rRNA approach and microelectrodes. In situ hybridization results revealed that the probe Amx820-hybridized anaerobic anammox bacteria were distributed throughout the biofilm (accounting for more than 70% of total bacteria). They were associated with Nitrosomonas-like aerobic ammonia-oxidizing bacteria (AAOB) in the surface biofilm. The anammox bacteria present in this study were distantly related to the Candidatus Brocadia anammoxidans with the sequence similarity of 95%. Microelectrode measurements showed that a high in situ anammox activity (i.e., simultaneous consumption of NH(4)(+) and NO(2)(-)) of 4.45 g-N of (NH(4)(+)+NO(2)(-))m(-2)day(-1) was detected in the upper 800 microm of the biofilm, which was consistent with the spatial distribution of anammox bacteria.     

Impact of inocula and growth mode on the molecular microbial ecology of anaerobic ammonia oxidation (anammox) bioreactor communities

The composition of distinctly inoculated granular anammox and biofilm-based completely autotrophic nitrogen removal over nitrite (CANON) bioreactors was investigated from start-up through continuous long-term operation via denaturing gradient gel electrophoresis (DGGE) and sequencing. The granular anammox reactor was seeded with sludge from an operational anammox reactor in Strass, Austria. The CANON reactor was seeded with activated sludge from a local wastewater treatment plant in New York City. The principal anammox bacteria (AMX) shifted from members related to Kuenenia stuttgartiensis present in the initial inoculum to members related to Candidatus Brocadia fulgida during pre-enrichment (before this study) and to members related to Candidatus Brocadia sp. 40 (during this study) in the granular reactor. AMX related to C. Brocadia sp. 40 were also enriched from activated sludge in the CANON reactor. The estimated doubling times of AMX in the granular and CANON reactors were 5.3 and 8.9 days, respectively, which are lower than the value of 11 days, reported previously. Both the granular anammox and CANON reactors also fostered significant amounts of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). The fractions of AMX and two groups of NOB were generally similar in the granular anammox and CANON reactors. However, the diversity and fractions of AOB in the two reactors was markedly different. Therefore, it is suggested that the composition of the feed and extant substrate concentrations in the reactor likely select for the microbial community composition more than the inocula and reactor configuration. Further, such selection is not equivalent for all resident communities.     

Enrichment of acetogenic bacteria in high rate anaerobic reactors under mesophilic and thermophilic conditions

The objective of the current study was to expand the knowledge of the role of acetogenic Bacteria in high rate anaerobic digesters. To this end, acetogens were enriched by supplying a variety of acetogenic growth supportive substrates to two laboratory scale high rate upflow anaerobic sludge bed (UASB) reactors operated at 37 °C (R1) and 55 °C (R2). The reactors were initially fed a glucose/acetate influent. Having achieved high operational performance and granular sludge development and activity, both reactors were changed to homoacetogenic bacterial substrates on day 373 of the trial. The reactors were initially fed with sodium vanillate as a sole substrate. Although % COD removal indicated that the 55 °C reactor out performed the 37 °C reactor, effluent acetate levels from R2 were generally higher than from R1, reaching values as high as 5023 mg l⁻¹. Homoacetogenic activity in both reactors was confirmed on day 419 by specific acetogenic activity (SAA) measurement, with higher values obtained for R2 than R1.Sodium formate was introduced as sole substrate to both reactors on day 464. It was found that formate supported acetogenic activity at both temperatures. By the end of the trial, no specific methanogenic activity (SMA) was observed against acetate and propionate indicating that the methane produced was solely by hydrogenotrophic Archaea. Higher SMA and SAA values against H₂/CO₂ suggested development of a formate utilising acetogenic population growing in syntrophy with hydrogenotrophic methanogens. Throughout the formate trial, the mesophilic reactor performed better overall than the thermophilic reactor.     

无机碳源浓度对厌氧氨氧化的影响研究

通过试验,分析了ASBR反应器中无机碳源浓度对厌氧氨氧化的影响,指出当无机碳源浓度大于2 g/L时,厌氧氨氧化菌代谢受到严重抑制,但是这种抑制是可逆的;当无机碳源浓度恢复到1 g/L时,厌氧氨氧化菌的代谢恢复正常。     

Identification and quantification of anammox bacteria in eight nitrogen removal reactors

Various studies have revealed anaerobic ammonium oxidation (anammox) as a very attractive alternative process suitable for nitrogen removal from wastewater. Here we investigated anammox bacteria in eight different nitrogen removal reactors. The diversity and abundance of anammox bacteria were determined by the 16S rRNA gene analysis, fluorescence in situ hybridization with specific probes and real-time quantitative PCR (qPCR). In these reactors, at least eight unique near full length anammox 16S rRNA gene sequences were detected, which were distributed over two genera; Candidati Brocadia and Kuenenia. FISH results confirmed that only one anammox bacterium dominated the community in each of the eight reactors investigated in this study. qPCR analysis revealed that anammox bacteria were present in seven of the reactors in the order of 10⁹ cells/ml and 10⁷ cells/ml in reactor A1. The dominant and divergent Brocadia-like anammox phylotype in one reactor represented a novel species for which we propose the name Candidatus Brocadia sinica. Taken together, these results indicated that a single seeding source could be used to seed anammox reactors designed to treat different types of wastewater, which could lead to a faster start-up of bioreactors.     

Influence of manganese and ammonium oxidation on the removal of 17 alpha-ethinylestradiol (EE2)

Flow-through reactors with manganese oxides were examined for their capacity to remove 17α-ethinylestradiol (EE2) at μg L⁻¹ and ng L⁻¹ range from synthetic wastewater treatment plant (WWTP) effluent. The mineral MnO₂ reactors removed 93% at a volumetric loading rate (B_V) of 5 μg EE2 L⁻¹ d⁻¹ and from a B_V of 40 μg EE2 L⁻¹ d⁻¹ on, these reactors showed 75% EE2 removal. With the biologically produced manganese oxides, only 57% EE2 was removed at 40 μg EE2 L⁻¹ d⁻¹. EE2 removal in the ng L⁻¹ range was 84%. The ammonium present in the influent (10 mg N L⁻¹) was nitrified and ammonia-oxidizing bacteria (AOB) were found to be of prime importance for the degradation of EE2. Remarkably, EE2 removal by AOB continued for a period of 4 months after depleting NH₄⁺ in the influent. EE2 removal by manganese-oxidizing bacteria was inhibited by NH₄⁺. These results indicate that the metabolic properties of nitrifiers can be employed to polish water containing EE2 based estrogenic activity.     

Phenols in anaerobic digestion processes and inhibition of ammonia oxidising bacteria (AOB) in soil

This study focuses on the presence of phenols in digestate from seven Swedish large-scale anaerobic digestion processes and their impact on the activity of ammonia oxidising bacteria (AOB) in soil. In addition, the importance of feedstock composition and phenol degradation capacity for the occurrence of phenols in the digestate was investigated in the same processes. The results revealed that the content of phenols in the digestate was related to the inhibition of the activity of AOB in soil (EC(50)=26 microg phenols g(-1) d.w. soil). In addition, five pure phenols (phenol, o-, p-, m-cresol and 4-ethylphenol) inhibited the AOB to a similar extent (EC(50)=43-110 microg g(-1) d.w. soil). The phenol content in the digestate was mainly dependent on the composition of the feedstock, but also to some extent by the degradation capacity in the anaerobic digestion process. Swine manure in the feedstock resulted in digestate containing higher amounts of phenols than digestate from reactors with less or no swine manure in the feedstock. The degradation capacity of phenol and p-cresol was studied in diluted small-scale batch cultures and revealed that anaerobic digestion at mesophilic temperatures generally exhibited a higher degradation capacity compared to digestion at thermophilic temperature. Although phenol, p-cresol and 4-ethylphenol were quickly degraded in soil, the phenols added with the digestate constitute an environmental risk according to the guideline values for contaminated soils set by the Swedish Environmental Protection Agency. In conclusion, the management of anaerobic digestion processes is of decisive importance for the production of digestate with low amounts of phenols, and thereby little risks for negative effects of the phenols on the soil ecosystem.     

Modeling the decay of ammonium oxidizing bacteria

A bench-scale sequencing batch reactor was used to study factors affecting the endogenous decay of the ammonium oxidizing biomass (AOB) in different operating conditions. AOB decay was very sensitive to oxygen concentration, and increased up to 0.4 d⁻¹ for oxygen concentration of 7 mg O₂ L⁻¹. The decay in anaerobic conditions was shown to be very low (0.03 d⁻¹) when compared to literature data.The effect of nitrite and nitrate on AOB decay was also studied. The correlation was quite weak suggesting that both nitrate and nitrite absence had little impact on decay which is contrary to what is typically assumed in some of the existing process models. A simple expression for the decay of AOB was proposed, calibrated and validated using the results of batch kinetic tests and of the continuous sequencing batch reactor monitoring.     

Partial ammonium oxidation to nitrite of high ammonium content urban landfill leachates

In an effort to treat N-rich streams in a more sustainable way, recent years have seen the development of new technologies, most of which are based on autotrophic denitrification via nitrite (anammox). In order to attain a suitable influent for that process, the wastewater must be treated by partially oxidising the ammonium to nitrite. In that aspect, this article presents the start-up and operation of a Partial Nitritation Sequencing Batch Reactor (PN-SBR) treating urban landfill leachates. Stable partial nitritation has been reached treating high ammonium loads (1-1.5 kg Nm(-3)d(-1)), demonstrating the feasibility of this technology as a previous step of anammox process. This study has also given away the importance of pH influence over ammonium oxidising bacteria (AOB) activity, thus it has been possible to determine the values of the half inhibition constants for free ammonia (k(I,FA)=605.48+/-87.18 mg N-NH L(-1)) and free nitrous acid (k(I,FNA)=0.49+/-0.09 mg N-HNO2 L(-1)), as well as the half-saturation constant for bicarbonate (k(HCO3-) = 0.01 +/- 0.16 mg CL(-1)).     

Phosphine generation by mixed- and monoseptic-cultures of anaerobic bacteria

A microbial basis for bioreductive generation of phosphine is proposed, which could account at least in part for the presence of this toxic gas in natural anaerobic environments and in sewage and landfill gases. Phosphine generation under anaerobic growth conditions was dependent upon both the culture inoculum source (animal faeces) and enrichment culture conditions. Phosphine was detected in headspace gases from mixed cultures under conditions promoting fermentative growth of mixed acid and butyric acid bacteria, either in the presence or absence of methane generation. Monoseptic cultures of certain mixed acid fermentors (Escherichia coli, Salmonella gallinarum, and Salmonella arizonae) and solvent fermentors (Clostridium sporogenes, Clostridium acetobutyricum and Clostridium cochliarium) also generated phosphine. Such fermentative bacteria participate in the multi-stage process of methanogenesis in nature. Generation of phosphine by these bacteria, rather than by methanoarchaea themselves, could explain the apparent correlation between methanogenesis and the formation of phosphine in nature.     

Chromium (VI) reduction in activated sludge bacteria exposed to high chromium loading: Brits culture (South Africa)

A mixed-culture of bacteria collected from a wastewater treatment plant in Brits, North-West Province (South Africa) biocatalytically reduced Cr(VI) at much higher concentrations than previously observed in cultures isolated in North America. Cr(VI) reduction rate up to 8 times higher than the rate in previous cultures was achieved by the Brits culture under aerobic conditions. Near complete Cr(VI) reduction was observed in batches under initial concentrations up to 200 mg Cr(VI)/L after incubation for 65 h in aerobic cultures. Under anaerobic conditions up to 150 mg Cr(VI)/L was completely removed after incubating for 130-155 h. In the previous cultures, complete removal was only achieved in cultures at an initial Cr(VI) concentration lower than 30 mg/L after incubation for 96-110 h. Consortium cultures were characterised using 16S rRNA partial sequence analysis. Results showed that the Gram-positive Bacillus genera predominated under aerobic conditions with a small composition of the Gram-negative Microbacterium sp. More biodiversity was observed in anaerobic cultures with the marked appearance of Enterococcus, Arthrobacter, Paenibacillus and Oceanobacillus species. Experiments run on purified individual species did not achieve the same level of Cr(VI) reduction as observed in the original consortium from sludge indicating possible existence of interspecies interactions necessary for optimum Cr(VI) reduction. All Cr(VI) reduced was accounted for as Cr(III) with a small error range (2-6%).     

Photocatalytic disinfection of marine bacteria using fluorescent light

Photocatalytic oxidation (PCO) using fluorescent light was used to disinfect two marine bacteria: Alteromonas alvinellae and Photobacterium phosphoreum. Results showed that P. phosphoreum exhibited a lower susceptibility towards PCO than A. alvinellae, which was related to their fatty acid profiles and levels of superoxide dismutase (SOD) and catalase (CAT). Results also revealed that CAT and SOD levels would be triggered by PCO reaction. In addition, PCO disinfection efficiency could be enhanced by acidic pH levels and increased temperatures, while different growth phases demonstrated diverse effects to the studied bacteria, probably due to their morphological difference or change in physiological state. Lastly intracellular leakage of potassium ion (K(+)), in line with the loss of cell viability, was observed during PCO. Mineralization of bacteria by PCO was monitored by total organic carbon analysis. From the regrowth study, the tested strains failed to reactivate within 96h after PCO treatment, indicating PCO caused irreversible bacterial inactivation.     

注浆法在治理自燃矸石山工程中的应用

通过分析矸石自燃原理,提出采用注浆法对自燃矸石山进行治理,并结合某煤矿工程中矸石山治理实例,依据相关检测标准,证明了注浆法是治理自燃矸石山的一种有效方法。     

Enrichment of anaerobic polychlorinated biphenyl dechlorinators from sediment with iron as a hydrogen source

Little is known about anaerobic polychlorinated biphenyl (PCB) dechlorination, although it is believed that some microorganisms are capable of respiring PCBs, gaining energy for growth from PCB dechlorination. If this is the case, the amendment of appropriate electron donors to contaminated sediment should stimulate dechlorination. The effect of elemental iron (Fe0) addition, an easily amended electron donor, on the microbial dechlorination of the PCB congeners 3,4,5-trichlorobiphenyl (3,4,5-CB) and 2,2',3,4,4',5,5'-heptachlorobiphenyl (2,2',3,4,4',5,5'-CB) was investigated in microcosms containing estuarine sediment from Baltimore Harbor. Results showed that the addition of 0.1 g Fe0/g sediment reduced the lag time for removal of doubly flanked para chlorines by approximately 100 days. Because Fe0 is a source of cathodic hydrogen (H2), the effect of direct H2 addition to sediment microcosms was also tested. The addition of 0.001 atm H2 in the headspace generated the same dechlorination activity and reduction in lag time as the addition of 0.1g Fe0/g. Higher concentrations of Fe0 or H2 increased the lag prior to dechlorination. Additional results showed that an alkaline pH (> or = 7.5), high [Fe2+] (3.3 g/L), or HS- (0.1 mg/L total sulfide) inhibited dechlorination. Elevated concentrations of Fe2+, OH-, and HS- are products of Fe0 oxidation or increased microbial activity (methanogenesis, homoacetogenesis, and sulfate reduction), both of which would result from the amendment of large quantities of Fe0 or H2 to sediment. This research shows that not only can PCB dechlorination be stimulated through the addition of electron donor, but implies that the dechlorinators are enriched by the continuous addition of low concentrations of H2, similar to other known dechlorinators, such as the dehalorespirer Dehalococcoides ethenogenes. These results suggest that the direct addition of controlled amounts of Fe0 to sediments may be an effective remediation tool to reduce the lag period prior to dechlorination at PCB-impacted sites. They also suggest that PCB dechlorinators may be enriched using techniques similar to those used with known dehalorespirers.     

高负荷厌氧氨氧化的稳定性试验研究

为探讨高负荷条件下复合式UASB反应器中厌氧氨氧化反应的脱氮性能和稳定性,在已经成功启动并稳定运行的厌氧氨氧化反应器中,通过提高进水总氮浓度和缩短水力停留时间两种方式提高总氮负荷,考察脱氮效果。结果表明,通过逐步提高进水总氮浓度和缩短HRT可以提高厌氧氨氧化反应的总氮负荷,并获得理想的脱氮效果和运行稳定性,但过高的亚硝态氮浓度会对反应产生一定的抑制作用。     

Quantification of anaerobic ammonium-oxidizing bacteria in enrichment cultures by real-time PCR

The anaerobic ammonium-oxidizing (ANAMMOX) bacteria were enriched from a rotating disk reactor (RDR) biofilm in semi-batch cultures. Based on fluorescence in situ hybridization (FISH) analysis, this enrichment led to a relative population size of 36% ANAMMOX bacteria. Phylogenetic analysis revealed that all the detected clones were related to the previously reported ANAMMOX bacteria, Candidatus Brocadia anammoxidans (AF375994), with 92% sequence similarity. Furthermore, we successfully developed a real-time polymerase chain reaction (PCR) assay to quantify populations of ANAMMOX bacteria in the enrichment cultures. For this real-time PCR assay, PCR primer sets targeting 16S ribosomal RNA genes of ANAMMOX bacteria were designed and used. The quantification range of this assay was 6 orders of magnitude, from 8.9x10(1) to 8.9x10(6) copies per PCR, corresponding to the detection limit of 3.6x10(3) target copies mL(-1). A significant correlation was found between the increase in copy numbers of 16S rRNA gene of ANAMMOX bacteria and the increase in nitrogen removal rates in the enrichment cultures. Quantifying ANAMMOX bacterial populations in the enrichment culture made it possible to estimate the doubling time of the enriched ANAMMOX bacteria to be 3.6 to 5.4 days. The real-time PCR assay gave comparable population sizes in the enrichment cultures with the FISH results. These results suggest that the real-time PCR assay developed in this study is useful and reliable for quantifying the populations of ANAMMOX bacteria in environmental and engineering samples.     

Evaluation on the microbial interactions of anaerobic ammonium oxidizers and heterotrophs in Anammox biofilm

Anaerobic ammonium oxidation (Anammox) is a cost-effective new process to treat high-strength nitrogenous wastewater. In this work, the microbial interactions of anaerobic ammonium oxidizers and heterotrophs through the exchange of soluble microbial products (SMP) in Anammox biofilm and the affecting factors were evaluated with both experimental and modeling approaches. Fluorescent in situ hybridization (FISH) analysis illustrated that Anammox bacteria and heterotrophs accounted for 77% and 23% of the total bacteria, respectively, even without addition of an external carbon source. Experimental results showed the heterotrophs could grow both on SMP and decay released substrate from the metabolism of the Anammox bacteria. However, heterotrophic growth in Anammox biofilm (23%) was significantly lower than that of nitrifying biofilm (30-50%). The model predictions matched well with the experimental observations of the bacterial distribution, as well as the nitrogenous transformations in batch and continuous experiments. The modeling results showed that low nitrogen surface loading resulted in a lower availability of SMP leading to low heterotrophic growth in Anammox biofilm, but high nitrogen surface loading would lead to relative stable biomass fractions although the absolute heterotrophic growth increased. Meanwhile, increasing biofilm thickness increased heterotrophic growth but has little influence on the relative biomass fractions.