Correlation of extracellular polymeric substances and microbial community structure in denitrification biofilm exposed to adverse conditions

Microbial community may respond to different adverse conditions and result in the variation of extracellular polymeric substances (EPS) in denitrification biofilm; this study discovered the role of EPS in accordance with the analysis of cyclic diguanylate (c-di-GMP) and electron equilibrium (EE) under low organic loading rate, shock organic loading rate and low temperature conditions. Good nitrate removal performance could be achieved under shock organic loading rate and low temperature conditions; however, owing to the low organic loading rate, the carbon source was preferentially utilized for biomass growth. Tightly bound EPS (TB-EPS) contents progressively increased and facilitated cell adhesion and biofilm formation. The stable TB protein (TB-PN) content in TB-EPS built a cross-linked network to maintain internal biofilm structure and led to the rapid biosynthesis of polysaccharides, which could further enhance microbial adhesion and improve nitrate removal. C-di-GMP played an important role in biomass retention and biofilm formation, based on the correlation analysis of c-di-GMP and EPS. TB polysaccharide (TB-PS) contents presented a significant positive correlation with c-di-GMP content, microbial adhesion and biofilm stabilization was further enhanced through c-di-GMP regulation. In addition, a remarkable negative correlation between electron deletion rate (EDR) and TB-PN and TB-PS was discovered, and TB-PS was required to serve as energy source to enhance denitrification according to EE analysis. Surprisingly, dynamic microbial community was observed due to the drastic community succession under low temperature conditions, and the discrepancy between the dominant species for denitrification was found under shock organic loading rate and low temperature conditions. The notable increase in bacterial strains Simlicispira, Pseudomonas and Chryseobacterium was conducive to biofilm formation and denitrification under shock organic loading rate, while Dechloromonas and Zoogloea dramatically enriched for nitrate removal under low temperature conditions. The high abundance of Dechloromonas improved the secretion of EPS through the downstream signal transduction, and the c-di-GMP conserved in Pseudomonas concurrently facilitated to enhance exopolysaccharide production to shock organic loading rate and low temperature conditions.     

Characterization of functional microbial community in a membrane-aerated biofilm reactor operated for completely autotrophic nitrogen removal

The 16S rDNA-based molecular technique was applied to investigate the functional microbial community of a membrane-aerated biofilm (MAB) that was used for completely autotrophic nitrogen removal over nitrite (CANON). The relationships among two kinds of key bacteria responsible for CANON: aerobic ammonia-oxidizing bacteria (AOB) and Anammox bacteria, and their possible distributions in the MAB were discussed based on the microbial community analysis. FISH analysis showed the existence of two visible active layers in experimental MAB. One is the partial nitrifying layer located in the region of oxygen-rich membrane-biofilm interface, dominated by NSO190-positive AOB. The other is the Anammox active layer located in the region of anoxic liquid-biofilm interface, dominated by PLA46 and AMX820-positive Anammox microorganisms. As a result of this study, the AOB as well as Anammox bacteria were present and active in experimental MABR, and the cooperation between AOB and Anammox bacteria was considered to be responsible for CANON.     

Development of a simultaneous partial nitrification and anaerobic ammonia oxidation process in a single reactor

Up-flow oxygen-controlled biofilm reactors equipped with a non-woven fabric support were used as a single reactor system for autotrophic nitrogen removal based on a combined partial nitrification and anaerobic ammonium oxidation (anammox) reaction. The up-flow biofilm reactors were initiated as either a partial nitrifying reactor or an anammox reactor, respectively, and simultaneous partial nitrification and anammox was established by careful control of the aeration rate. The combined partial nitrification and anammox reaction was successfully developed in both biofilm reactors without additional biomass inoculation. The reactor initiated as the anammox reactor gave a slightly higher and more stable mean nitrogen removal rate of 0.35 (± 0.19) kg-N m⁻³ d⁻¹ than the reactor initiated as the partial nitrifying reactor (0.23 (± 0.16) kg-N m⁻³ d⁻¹). FISH analysis revealed that the biofilm in the reactor started as the anammox reactor were composed of anammox bacteria located in inner anoxic layers that were surrounded by surface aerobic AOB layers, whereas AOB and anammox bacteria were mixed without a distinguishable niche in the biofilm in the reactor started as the partial nitrifying reactor. However, it was difficult to efficiently maintain the stable partial nitrification owing to inefficient aeration in the reactor, which is a key to development of the combined partial nitrification and anammox reaction in a single biofilm reactor.     

厌氧氨氧化颗粒污泥聚集机制研究进展

厌氧氨氧化(anaerobic ammonium oxidation,anammox)工艺被认为是当前污水生物脱氮领域最经济的处理工艺,有利于实现污水处理厂的能源自给。厌氧氨氧化菌是该工艺的核心功能微生物。以厌氧氨氧化菌为主导微生物形成的厌氧氨氧化颗粒污泥具有沉速大、污泥持留能力强及对不利环境抵抗能力强等突出优势,是实现厌氧氨氧化工艺最有前景的污泥形态。本论文围绕厌氧氨氧化颗粒,介绍了厌氧氨氧化菌的特性、种类及代谢途径,综述了厌氧氨氧化颗粒污泥的形成假说及与厌氧氨氧化颗粒污泥聚集密切相关的胞外聚合物(extracellular polymeric substance,EPS)和群体感应研究现状,并对今后厌氧氨氧化颗粒的研究进行了展望,以期为后续厌氧氨氧化颗粒的研究及厌氧氨氧化颗粒工艺的优化提供参考。     

铁矿物强化厌氧氨氧化效能及其微生物机制研究进展

自然界中的氮循环与铁循环相互交联,参与氮循环的厌氧氨氧化（anaerobic ammonium oxidation,anammox）菌的生长代谢及活性发挥也与铁元素紧密关联。自然界广泛存在的铁矿物因具有运行成本低廉、稳定性好、二次污染小等优势,在污水处理领域得到广泛应用。在厌氧氨氧化脱氮系统中引入适量铁矿物,不仅有助于促进anammox菌和铁还原菌的富集,提高功能基因丰度和相关酶活性,还可能通过影响污泥浓度、血红素c含量、胞外聚合物含量和颗粒化程度,改善污泥性能和提高厌氧氨氧化系统的稳定性。同时,铁矿物具有促进体系多种氮素转化途径（如anammox、铁自养反硝化、铁氨氧化、异化硝酸盐还原成铵和反硝化）相耦合的潜能,可以提高anammox污水处理系统的总氮去除率。本文基于铁矿物在促进污水生物脱氮方面的良好性能及其在anammox系统中的变化,从脱氮效能、污泥特性、微生物特征及酶活性等方面,系统综述了铁矿物对厌氧氨氧化系统的强化作用机制,并从anammox菌对铁矿物的利用及铁元素的摄取角度展望了后续的研究方向,以期为铁矿物强化厌氧氨氧化系统的实际应用提供理论和技术指导。     

季节性温度变化对CANON型潮汐流人工湿地脱氮的影响

探究了温度的季节性变化对基于亚硝化的全程自养脱氮(CANON)型潮汐流人工湿地(TFCW)脱氮性能及其微生物特性的影响。CANON型TFCW中的脱氮微生物群落在温度的季节性变化下会发生不同程度的改变,其脱氮途径及性能随之会出现周期性的波动。填料层温度在20.0 ℃以上时对TFCW脱氮性能及其中的优势脱氮菌群无显著影响,CANON作用是系统脱氮的主要途径。当填料层温度低于20.0 ℃时,厌氧氨氧化菌丰度与活性显著降低,在9.3~20.0 ℃时,亚硝酸盐氧化菌(NOB)的增殖及其活性的提高使TFCW中脱氮的主要途径由CANON作用演替为硝化/反硝化作用,系统对总氮(TN)的去除率仅为(34.8±13.0)%;在2.2~9.0 ℃时,TFCW中的厌氧氨氧化菌在受到抑制的同时仍保持着对NOB和反硝化菌群的相对竞争优势,系统脱氮重新依赖于CANON作用,其对TN的去除率为(54.8±4.8)%。该研究可为CANON型TFCW工艺的优化及工程化应用提供参考。     

Function of quorum sensing and cell signaling in the formation of aerobic granular sludge

Aerobic granular sludge (AGS) has recently attracted attention because of its excellent settling ability and treatment efficiency compared with traditional activated sludge. This review provides recent advances on the formation process of AGS and mainly analyzes the function of quorum sensing (QS) and cell signaling during AGS formation. QS and cell signaling play important roles in the formation of AGS. QS can accelerate the synthesis of extracellular polymeric substance (EPS) and increase microbial adhesion to the surface of AGS. Cell signaling can also promote the secretion of EPS and influence biofilm formation. Cyclic diguanylate (c-di-GMP), as a second messenger, acts an important role in granulation. C-di-GMP causes bacteria to adhere to each other and form a biofilm. Adding Ca²⁺ benefits bacterial growth and promotes c-di-GMP secretion. Adding Mn²⁺ reduces c-di-GMP content and triggers AGS disintegration. Finally, the review discusses the possible trends of AGS: QS and cell signaling can lay a theoretical foundation for the formation mechanism of AGS and would be of practical significance for its application in the future.     

Rapid startup and high rate nitrogen removal from anaerobic sludge digester liquor using a SNAP process

In this study, a single-stage autotrophic nitrogen removal reactor, packed with a novel acrylic fiber biomass carrier material (Biofix), was applied for nitrogen removal from sludge digester liquor. For rapid start-up, conventional activated sludge was added to the reactor soon after the attachment of anammox biomass on the Biofix carriers, which allowed conventional activated sludge to form a protective layer of biofilm around the anammox biomass. The Nitrogen removal efficiency reached 75% within 1 week at a nitrogen loading rate of 0.46 kg-N/m³/day for synthetic wastewater treatment. By the end of the synthetic wastewater treatment period, the maximum nitrogen removal rate had increased to 0.92 kg-N/m³/day at a nitrogen loading rate of 1.0 kg-N/m³/day. High nitrogen removal rate was also achieved during the actual raw digester liquor treatment with the highest nitrogen removal rate being 0.83 kg-N/m³/day at a nitrogen loading rate of 0.93 kg-N/m³/day. The thick biofilm on Biofix carriers allowed anammox bacteria to survive under high DO concentration of 5–6 mg/l resulting in stable and high nitrogen removal performance. FISH and CLSM analysis demonstrated that anammox bacteria coexisted and surrounded by ammonium oxidizing bacteria.     

c-di-GMP对大肠杆菌生物膜调控的研究进展

大肠杆菌生物膜是由聚集于特定介质上的大肠杆菌菌体细胞相互黏附并分泌胞外基质聚合物(extracellular polymeric substances,EPS)而产生的一种结构复杂的膜状聚集物。感染宿主后的致病性大肠杆菌在形成生物膜后会极大地逃避免疫系统以及环境中各种有害因素对其的影响,对宿主造成持续甚至致命的伤害。环二鸟苷酸(cyclic diguanosine monophosphate,c-di-GMP)是广泛存在于细菌中的第二信使,在调节生物膜形成过程中起到至关重要的作用。基于此,本文对近些年来有关c-di-GMP对大肠杆菌生物膜形成过程中菌体的运动、黏附以及EPS产生机制的研究进行了综述,以期为从c-di-GMP角度抑制大肠杆菌生物膜提供依据和思路。     

Identification of bacteria coexisting with anammox bacteria in an upflow column type reactor

Anammox process has attracted considerable attention in the recent years as an alternative to conventional nitrogen removal technologies. In this study, a column type reactor using a novel net type acrylic fiber (Biofix) support material was used for anammox treatment. The Biofix reactor was operated at a temperature of 25°C (peak summer temperature, 31.5°C). During more than 340 days of operation for synthetic wastewater treatment, the nitrogen loading rates of the reactor were increased to 3.6 kg-N/m³/d with TN removal efficiencies reaching 81.3%. When the reactor was used for raw anaerobic sludge digester liquor treatment, an average TN removal efficiency of 72% was obtained with highest removal efficiency of 81.6% at a nitrogen loading rate of 2.2 kg-N/m³/d. Results of extracellular polymeric substances (EPS) quantification revealed that protein was the most abundant component in the granular sludge and was found to be almost twice than that in the sludge attached to the biomass carriers. The anammox granules in the Biofix reactor illustrated a dense morphology substantiated by scanning electron microscopy and EPS results. The results of DNA analyses indicated that the anammox strain KSU-1 might prefer relatively low nutrient levels, while the anammox strain KU2 strain might be better suited at high nutrient concentration. Other types of bacteria were also identified with the potential of consuming dissolved oxygen in the influent and facilitating survival of anammox bacteria under aerobic conditions.     

Anammox start-up in sequencing batch biofilm reactors using different inoculating sludge

In this study, anammox bacteria were rapidly enriched in sequencing batch biofilm reactors (SBBRs) with different inoculations. The activated sludge taken from a sequencing batch reactor was used and inoculated to SBBR1, while SBBR2 was seeded with stored anaerobic sludge from an upflow anaerobic fixed bed (2-year stored at 5–15 °C). Nitrogen removal performance, anammox activity, biofilm characteristics and variation of the microbial community were evaluated. The maximum total nitrogen loading rate (NLR) of SBBR1 gradually reached to 1.62 kg?N/(m³/day) with a removal efficiency higher than 88 % and the NLR of SBBR2 reached to 1.43 kg?N/(m³/day) with a removal efficiency of 86 %. SBBR2 was more stable compared to SBBR1. These results, combined with molecular techniques such as scanning electron microscope, fluorescence in situ hybridization, and terminal restriction fragment length polymorphism, indicated that different genera of anammox bacteria became dominant. This research also demonstrates that SBBR is a promising bioreactor for starting up and enriching anammox bacteria.     

Accelerating effect of hydroxylamine and hydrazine on nitrogen removal rate in moving bed biofilm reactor

In biological nitrogen removal, application of the autotrophic anammox process is gaining ground worldwide. Although this field has been widely researched in last years, some aspects as the accelerating effect of putative intermediates (mainly N?H? and NH?OH) need more specific investigation. In the current study, experiments in a moving bed biofilm reactor (MBBR) and batch tests were performed to evaluate the optimum concentrations of anammox process intermediates that accelerate the autotrophic nitrogen removal and mitigate a decrease in the anammox bacteria activity using anammox (anaerobic ammonium oxidation) biomass enriched on ring-shaped biofilm carriers. Anammox biomass was previously grown on blank biofilm carriers for 450 days at moderate temperature 26.0 (±0.5) °C by using sludge reject water as seeding material. FISH analysis revealed that anammox microorganisms were located in clusters in the biofilm. With addition of 1.27 and 1.31 mg N L?1 of each NH?OH and N?H?, respectively, into the MBBR total nitrogen (TN) removal efficiency was rapidly restored after inhibitions by NO??. Various combinations of N?H?, NH?OH, NH??, and NO?? were used as batch substrates. The highest total nitrogen (TN) removal rate with the optimum N?H? concentration (4.38 mg N L?1) present in these batches was 5.43 mg N g?1 TSS h?1, whereas equimolar concentrations of N?H? and NH?OH added together showed lower TN removal rates. Intermediates could be applied in practice to contribute to the recovery of inhibition-damaged wastewater treatment facilities using anammox technology.     

Effects of allylthiourea,salinity, and pH on ammonia/ammonium-oxidizing prokaryotes in mangrove sediment incubated in laboratory microcosms

Anaerobic ammonium-oxidizing (anammox) bacteria, aerobic ammonia-oxidizing archaea (AOA) and bacteria (AOB) are three groups of ammonia/ammonium-oxidizing prokaryotes (AOPs) involved in the biochemical nitrogen cycling. In this study, the effects of allylthiourea (ATU), pH, and salinity on these three groups from mangrove sediment were investigated through microcosm incubation in laboratory. ATU treatments (50, 100, and 500 mg L^?1) obviously affected the community structure of anammox bacteria and AOB, but only slightly for AOA. ATU began to inhibit anammox bacteria growth slightly from day 10, but had an obvious inhibition on AOA growth from the starting of the study. At 100 mg L^?1 of ATU or higher, AOB growth was inhibited, but only lasted for 5 days. The pH treatments showed that acidic condition (pH 5) had a slight effect on the community structure of anammox bacteria and AOA, but an obvious effect on AOB. Acidic condition promoted the growth of all groups of AOPs in different extent, but alkaline condition (pH 9) had a weak effect on AOB community structure and a strong effect on both anammox bacteria and AOA. Alkaline condition obviously inhibited anammox bacteria growth, slightly promoted AOA, and slightly promoted AOB in the first 20 days, but inhibited afterward. Salinity treatment showed that higher salinity (20 and 40?‰) resulted in higher anammox bacteria diversity, and both AOA and AOB might have species specificity to salinity. High salinity promoted the growth of both anammox bacteria and AOB, inhibited AOA between 5 and 10 days, but promoted afterward. The results help to understand the role of these microbial groups in biogeochemical nitrogen cycling and their responses to the changing environments.     

Assessment of the positive effect of salinity on the nitrogen removal performance and microbial composition during the start-up of CANON process

In this study, a non-woven rotating biological contactor reactor was operated for the start-up of completely autotrophic nitrogen removal over nitrite (CANON) process. In this perfectly attached growth system, nitrite oxidizing was identified, which interfered with the nitrogen removal performance. Batch tests indicated that 10 g NaCl per liter salinity was a preferable definite level to stand out ammonium-oxidizing activity and anammox activity, and selectively suppress nitrite-oxidizing activity under oxygen-limited conditions. Reactor operation showed that the maximum TN removal rate was increased from 425 mg N l(-1) day(-1) to 637 mg N l(-1) day(-1) after the addition of 10 g NaCl per liter salinity on analogous technological parameters. Microbiological community analysis revealed that bacteria strains similar to the genus Nitrospira sp. were specialized nitrite oxidizers existing in CANON reactor, which were then eliminated under salinity exposure for their no salinity-tolerant relative. However, anammox bacteria belonging to Planctomycetes and some aerobic ammonium oxidizers belonging to Nitrosomonas could be highly enriched under this oxygen-limited salinity conditions. Salinity-contained high ammonium wastewater will be so considered as suitable influent for CANON process in further industrial application.     

Characteristics of extracellular polymeric substances from sludge and biofilm in a simultaneous nitrification and denitrification system under high salinity stress

The composition and distribution of extracellular polymeric substance (EPS) both from suspended sludge and attached biofilm were investigated in a simultaneous nitrification and denitrification (SND) system with the increase of the salinity from 1.0 to 3.0 %. Fourier-transform infrared (FTIR) spectroscopy and three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy were used to examine proteins (PN), polysaccharides (PS) and humic substances (HS) present in EPS. High total nitrogen removal (above 83.9 %) via SND was obtained in the salinity range of 1.0–2.5 %. Total EPS in the sludge increased from 150.2 to 200.6 mg/gVSS with the increase of salinity from 1.0 to 3.0 %, whereas the corresponding values in the biofilm achieved the maximum of 288.6 mg/g VSS at 2.0 % salinity. Dominant composition of EPS was detected as HS in both sludge and biofilm, having the percentages of 50.6–68.6 and 41.1–69.9 % in total EPS, respectively. Both PN and PS contents in soluble EPS (S-EPS), loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) of sludge and biofilm increased with the increased salinity. The FTIR spectrum and 3D-EEM fluorescence spectroscopy of S-EPS, LB-EPS and TB-EPS in the sludge and biofilm showed the changes of functional groups and conformations of the compositions in EPS with the increase of salinity. The results demonstrated that the characteristics of EPS varied from sludge to biofilm. The obtained results could provide a better understanding of the salinity effect on the EPS characteristics in a SND system.     

污水短程硝化体系氨氧化菌与亚硝酸盐氧化菌的生态学研究进展

短程硝化(partial nitrification, PN)是一种绿色低碳的生物脱氮创新技术,伴随厌氧氨氧化(anaerobic ammonia oxidation, Anammox)污水脱氮技术的进一步推广,短程硝化作为提供其电子受体的重要环节,已成为了污水脱氮领域的研究热点。氨氧化菌(ammonia-oxidizing bacteria,AOB)和亚硝酸盐氧化菌(nitrite-oxidizing bacteria, NOB)是该技术的核心竞争微生物,掌握这两类微生物的生态学特征,借助生态学理论和手段调控AOB淘汰NOB,提高种群的可预测性,对于实现稳定高效的短程硝化具有重要意义。本文基于生态学角度介绍了AOB和NOB基础分类、生理性能及生态位分离,重点综述了短程硝化系统中AOB和NOB的生长动力学、群落构建、环境因素和相互作用,最后对这两类微生物的未来研究重点和研究方法进行了展望,为短程硝化工艺的快速启动和稳定运行提供理论指导。     

In situ activity and spatial organization of anaerobic ammonium-oxidizing (anammox) bacteria in biofilms

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that "Brocadia"-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 microm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH(4)(+) and NO(2)(-) consumption rates decreased from 0.68 and 0.64 micromol cm(-2) h(-1) at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 micromol cm(-2) h(-1) at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH(4)(+) and NO(2)(-) and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O(2) or organic compounds, which consequently established suitable microenvironments for anammox bacteria.     

Nitrous oxide production in autotrophic nitrogen removal granular sludge: A modeling study

The sustainability of autotrophic granular system performing partial nitritation and anaerobic ammonium oxidation (anammox) for complete nitrogen removal is impaired by the production of nitrous oxide (N₂O). A systematic analysis of the pathways and affecting parameters is, therefore, required for developing N ₂O mitigation strategies. To this end, a mathematical model capable of describing different N ₂O production pathways was defined in this study by synthesizing relevant mechanisms of ammonium-oxidizing bacteria (AOB), nitrite-oxidizing bacteria, heterotrophic bacteria (HB), and anammox bacteria. With the model validity reliably tested and verified using two independent sets of experimental data from two different autotrophic nitrogen removal biofilm/granular systems, the defined model was applied to reveal the underlying mechanisms of N ₂O production in the granular structure as well as the impacts of operating conditions on N ₂O production. The results show that: (a) in the aerobic zone close to the granule surface where AOB contribute to N ₂O production through both the AOB denitrification pathway and the NH ₂OH pathway, the co-occurring HB consume N ₂O produced by AOB but indirectly enhance the N ₂O production by providing NO from NO ₂ ⁻ reduction for the NH ₂OH pathway, (b) the inner anoxic zone of granules with the dominance of anammox bacteria acts as a sink for NO ₂ ⁻ diffusing from the outer aerobic zone and, therefore, reduces N ₂O production from the AOB denitrification pathway, (c) operating parameters including bulk DO, influent NH ₄ ⁺, and granule size affect the N ₂O production in the granules mainly by regulating the NH ₂OH pathway of AOB, accounting for 34–58% of N ₂O turnover, and (d) the competition between the NH ₂OH pathway and heterotrophic denitrification for nitric oxide leads to the positive role of HB in reducing N ₂O production in the autotrophic nitrogen removal granules, which could be further enhanced in the presence of a proper level of influent organics.     

Rapid start-up of nitrifying MBBRs at low temperatures: nitrification,biofilm response and microbiome analysis

The moving bed biofilm reactor (MBBR), operated as a post carbon removal system, requires long start-up times in comparison to carbon removal systems due to slow growing autotrophic organisms. This study investigates the use of carriers seeded in a carbon rich treatment system prior to inoculation in a nitrifying MBBR system to promote the rapid development of nitrifying biofilm in an MBBR system at temperatures between 6 and 8 °C. Results show that nitrification was initiated by the carbon removal carriers after 22 h of operation. High throughput 16S-rDNA sequencing indicates that the sloughing period was a result of heterotrophic organism detachment and the recovery and stabilization period included a growth of Nitrosomonas and Nitrospira as the dominant ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) in the biofilm. Peripheral microorganisms such as Myxococcales, a rapid EPS producer, appear to have contributed to the recovery and stabilization of the biofilm.

     

Cyclic-di-GMP regulates extracellular polysaccharide production, biofilm formation, and rugose colony development by Vibrio vulnificus

Vibrio vulnificus is a human and animal pathogen that carries the highest death rate of any food-borne disease agent. It colonizes shellfish and forms biofilms on the surfaces of plankton, algae, fish, and eels. Greater understanding of biofilm formation by the organism could provide insight into approaches to decrease its load in filter feeders and on biotic surfaces and control the occurrence of invasive disease. The capsular polysaccharide (CPS), although essential for virulence, is not required for biofilm formation under the conditions used here. In other bacteria, increased biofilm formation often correlates with increased exopolysaccharide (EPS) production. We exploited the translucent phenotype of acapsular mutants to screen a V. vulnificus genomic library and identify genes that imparted an opaque phenotype to both CPS biosynthesis and transport mutants. One of these encoded a diguanylate cyclase (DGC), an enzyme that synthesizes bis-(3'-5')-cyclic-di-GMP (c-di-GMP). This prompted us to use this DGC, DcpA, to examine the effect of elevated c-di-GMP levels on several developmental pathways in V. vulnificus. Increased c-di-GMP levels induced the production of an EPS that was distinct from the CPS and dramatically enhanced biofilm formation and rugosity in a CPS-independent manner. However, the EPS could not compensate for the loss of CPS production that is required for virulence. In contrast to V. cholerae, motility and virulence appeared unaffected by elevated levels of c-di-GMP.