Characterization of an Autotrophic Nitrogen-Removing Biofilm from a Highly Loaded Lab-Scale Rotating Biological Contactor

In this study, a lab-scale rotating biological contactor (RBC) treating a synthetic NH₄⁺ wastewater devoid of organic carbon and showing high N losses was examined for several important physiological and microbial characteristics. The RBC biofilm removed 89% ± 5% of the influent N at the highest surface load of approximately 8.3 g of N m⁻² day⁻¹, with N₂ as the main end product. In batch tests, the RBC biomass showed good aerobic and anoxic ammonium oxidation (147.8 ± 7.6 and 76.5 ± 6.4 mg of NH₄⁺-N g of volatile suspended solids [VSS]⁻¹ day⁻¹, respectively) and almost no nitrite oxidation (< 1 mg of N g of VSS⁻¹ day⁻¹). The diversity of aerobic ammonia-oxidizing bacteria (AAOB) and planctomycetes in the biofilm was characterized by cloning and sequencing of PCR-amplified partial 16S rRNA genes. Phylogenetic analysis of the clones revealed that the AAOB community was fairly homogeneous and was dominated by Nitrosomonas-like species. Close relatives of the known anaerobic ammonia-oxidizing bacterium (AnAOB) Kuenenia stuttgartiensis dominated the planctomycete community and were most probably responsible for anoxic ammonium oxidation in the RBC. Use of a less specific planctomycete primer set, not amplifying the AnAOB, showed a high diversity among other planctomycetes, with representatives of all known groups present in the biofilm. The spatial organization of the biofilm was characterized using fluorescence in situ hybridization (FISH) with confocal scanning laser microscopy (CSLM). The latter showed that AAOB occurred side by side with putative AnAOB (cells hybridizing with probe PLA46 and AMX820/KST1275) throughout the biofilm, while other planctomycetes hybridizing with probe PLA886 (not detecting the known AnAOB) were present as very conspicuous spherical structures. This study reveals that long-term operation of a lab-scale RBC on a synthetic NH₄⁺ wastewater devoid of organic carbon yields a stable biofilm in which two bacterial groups, thought to be jointly responsible for the high autotrophic N removal, occur side by side throughout the biofilm.     

Performance and Microbial Structure of a Combined Biofilm Reactor

A novel combined biofilm reactor was established and applied as a single treatment unit for carbon and nitrogen removal of wastewater. The nitrogen removal performance of the reactor at different levels of organic carbon (COD) loading was investigated when the influent total nitrogen (TN) loading was 0.74 g TN/m² day. Continuous experimental results demonstrated that 80% nitrogen was eliminated when the influent COD loading ranged between 2.06 g and 3.92 g COD/m² day. Microbial composition in the reactor was analyzed using fluorescent in situ hybridization (FISH) and conventional batch tests. The relative abundance of ammonia-oxidizing bacteria in the aerobic zone of the reactor measured by FISH was consistent with the result from conventional batch tests.     

Biological Filtration Limits Carbon Availability and Affects Downstream Biofilm Formation and Community Structure

Carbon removal strategies have gained popularity in the mitigation of biofouling in water reuse processes, but current biofilm-monitoring practices based on organic-carbon concentrations may not provide an accurate representation of the in situ biofilm problem. This study evaluated a submerged microtiter plate assay for direct and rapid monitoring of biofilm formation by subjecting the plates to a continuous flow of either secondary effluent (SE) or biofilter-treated secondary effluent (BF). This method was very robust, based on a high correlation (R² = 0.92) between the biomass (given by the A₆₀₀ in the microtiter plate assay) and the biovolume (determined from independent biofilms developed on glass slides under identical conditions) measurements, and revealed that the biomasses in BF biofilms were consistently lower than those in SE biofilms. The influence of the organic-carbon content on the biofilm community composition and succession was further evaluated using molecular tools. Terminal restriction fragment length polymorphism analysis of 16S rRNA genes revealed a group of pioneer colonizers, possibly represented by Sphingomonadaceae and Caulobacter organisms, to be common in both SE and BF biofilms. However, differences in organic-carbon availabilities in the two water samples eventually led to the selection of distinct biofilm communities. Alphaproteobacterial populations were confirmed by fluorescence in situ hybridization to be enriched in SE biofilms, while Betaproteobacteria were dominant in BF biofilms. Cloning analyses further demonstrated that microorganisms adapted for survival under low-substrate conditions (e.g., Aquabacterium, Caulobacter, and Legionella) were preferentially selected in the BF biofilm, suggesting that carbon limitation strategies may not achieve adequate biofouling control in the long run.     

Light and Electron Microscopic Studies of Microorganisms Growing in Rotating Biological Contactor Biofilms

The biofilms growing in the first compartments of two rotating biological contactors used to treat municipal wastewater were examined by light and electron microscopy. The biofilms were found to contain a complex and varied microbial community that included filamentous and unicellular bacteria, protozoa, metazoa, and (possibly) bacteriophage. The predominant microorganism among these appeared to be a filamentous bacterium that was identical to Sphaerotilus in both morphological and ultrastructural characteristics. It was possible to isolate a Sphaerotilus-like bacterium from each contactor. Both the Sphaerotilus filaments and the wide variety of unicellular bacteria present tended to contain poly-β-hydroxybutyrate inclusions, a probable indication that these organisms were removing carbon from the wastewater and storing it. The microbial population of the biofilms appeared to be metabolically active, as evidenced by the presence of microcolonies and dividing cells.     

Soil Water Content and Organic Carbon Availability Are Major Determinants of Soil Microbial Community Composition

Exploration of environmental factors governing soil microbial community composition is long overdue and now possible with improved methods for characterizing microbial communities. Previously, we observed that rice soil microbial communities were distinctly different from tomato soil microbial communities, despite management and seasonal variations within soil type. Potential contributing factors included types and amounts of organic inputs, organic carbon content, and timing and amounts of water inputs. Of these, both soil water content and organic carbon availability were highly correlated with observed differences in composition. We examined how organic carbon amendment (compost, vetch, or no amendment) and water additions (from air dry to flooded) affect microbial community composition. Using canonical correspondence analysis of phospholipid fatty acid data, we determined flooded, carbon-amended (+C) microcosm samples were distinctly different from other +C samples and unamended (–C) samples. Although flooding without organic carbon addition influenced composition some, organic carbon addition was necessary to substantially alter community composition. Organic carbon availability had the same general effects on microbial communities regardless of whether it was compost or vetch in origin. In addition, flooded samples, regardless of organic carbon inputs, had significantly lower ratios of fungal to bacterial biomarkers, whereas under drier conditions and increased organic carbon availability the microbial communities had higher proportions of fungal biomass. When comparing field and microcosm soil, flooded +C microcosm samples were most similar to field-collected rice soil, whereas all other treatments were more similar to field-collected tomato soil. Overall, manipulating water and carbon content selected for microbial communities similar to those observed when the same factors were manipulated at the field scale.     

Functional Gene Composition,Diversity and Redundancy in Microbial Stream Biofilm Communities

We surveyed the functional gene composition and diversity of microbial biofilm communities in 18 New Zealand streams affected by different types of catchment land use, using a comprehensive functional gene array, GeoChip 3.0. A total of 5,371 nutrient cycling and energy metabolism genes within 65 gene families were detected among all samples (342 to 2,666 genes per stream). Carbon cycling genes were most common, followed by nitrogen cycling genes, with smaller proportions of sulphur, phosphorus cycling and energy metabolism genes. Samples from urban and native forest streams had the most similar functional gene composition, while samples from exotic forest and rural streams exhibited the most variation. There were significant differences between nitrogen and sulphur cycling genes detected in native forest and urban samples compared to exotic forest and rural samples, attributed to contrasting proportions of nitrogen fixation, denitrification, and sulphur reduction genes. Most genes were detected only in one or a few samples, with only a small minority occurring in all samples. Nonetheless, 42 of 65 gene families occurred in every sample and overall proportions of gene families were similar among samples from contrasting streams. This suggests the existence of functional gene redundancy among different stream biofilm communities despite contrasting taxonomic composition.     

Impacts of Labile Organic Carbon Concentration on Organic and Inorganic Nitrogen Utilization by a Stream Biofilm Bacterial Community

In aquatic ecosystems, carbon (C) availability strongly influences nitrogen (N) dynamics. One manifestation of this linkage is the importance in the dissolved organic matter (DOM) pool of dissolved organic nitrogen (DON), which can serve as both a C and an N source, yet our knowledge of how specific properties of DOM influence N dynamics are limited. To empirically examine the impact of labile DOM on the responses of bacteria to DON and dissolved inorganic nitrogen (DIN), bacterial abundance and community composition were examined in controlled laboratory microcosms subjected to various combinations of dissolved organic carbon (DOC), DON, and DIN treatments. Bacterial communities that had colonized glass beads incubated in a stream were treated with various glucose concentrations and combinations of inorganic and organic N (derived from algal exudate, bacterial protein, and humic matter). The results revealed a strong influence of C availability on bacterial utilization of DON and DIN, with preferential uptake of DON under low C concentrations. Bacterial DON uptake was affected by the concentration and by its chemical nature (labile versus recalcitrant). Labile organic N sources (algal exudate and bacterial protein) were utilized equally well as DIN as an N source, but this was not the case for the recalcitrant humic matter DON treatment. Clear differences in bacterial community composition among treatments were observed based on terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes. C, DIN, and DON treatments likely drove changes in bacterial community composition that in turn affected the rates of DON and DIN utilization under various C concentrations.     

Analysis of Microbial Community during Biofilm Development in an Anaerobic Wastewater Treatment Reactor

The formation, structure, and biodiversity of a multispecies anaerobic biofilm inside an Upflow Anaerobic Sludge Bed (UASB) reactor fed with brewery wastewater was examined using complementary microbial ecology methods such us fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), and cloning. The biofilm development can be roughly divided into three stages: an initial attachment phase (0-36 h) characterized by random adhesion of the cells to the surface; a consolidation phase (from 36 h to 2 weeks) defined by the appearance of microcolonies; and maturation phase (from 2 weeks to 2 months). During the consolidation period, proteobacteria with broad metabolic capabilities, mainly represented by members of alpha-Proteobacteria class (Oleomonas, Azospirillum), predominated. Beta-, gamma-, delta- (both syntrophobacteria and sulfate-reducing bacteria) and epsilon- (Arcobacter sp.) Proteobacteria were also noticeable. Archaea first appeared during the consolidation period. A Methanospirillum-like methanogen was detected after 36 h, and this was followed by the detection of Methanosarcina, after 4 days of biofilm development. The mature biofilm displayed a hill and valley topography with cells embedded in a matrix of exopolymers where the spatial distribution of the microorganisms became well-established. Compared to the earlier phases, the biodiversity had greatly increased. Although alpha-Proteobacteria remained as predominant, members of the phyla Firmicutes, Bacteroidete, and Thermotogae were also detected. Within the domain Archaea, the acetoclastic methanogen Methanosaeta concilii become dominant. This study provides insights on the trophic web and the shifts in population during biofilm development in an UASB reactor.     

Microbial Community Analysis of Anaerobic Reactors Treating Soft Drink Wastewater

The anaerobic packed-bed (AP) and hybrid packed-bed (HP) reactors containing methanogenic microbial consortia were applied to treat synthetic soft drink wastewater, which contains polyethylene glycol (PEG) and fructose as the primary constituents. The AP and HP reactors achieved high COD removal efficiency (>95%) after 80 and 33 days of the operation, respectively, and operated stably over 2 years. 16S rRNA gene pyrotag analyses on a total of 25 biofilm samples generated 98,057 reads, which were clustered into 2,882 operational taxonomic units (OTUs). Both AP and HP communities were predominated by Bacteroidetes, Chloroflexi, Firmicutes, and candidate phylum KSB3 that may degrade organic compound in wastewater treatment processes. Other OTUs related to uncharacterized Geobacter and Spirochaetes clades and candidate phylum GN04 were also detected at high abundance; however, their relationship to wastewater treatment has remained unclear. In particular, KSB3, GN04, Bacteroidetes, and Chloroflexi are consistently associated with the organic loading rate (OLR) increase to 1.5 g COD/L-d. Interestingly, KSB3 and GN04 dramatically decrease in both reactors after further OLR increase to 2.0 g COD/L-d. These results indicate that OLR strongly influences microbial community composition. This suggests that specific uncultivated taxa may take central roles in COD removal from soft drink wastewater depending on OLR.     

Characterization of Microbial Communities and Composition in Constructed Dairy Wetland Wastewater Effluent

Constructed wetlands have been recognized as a removal treatment option for high concentrations of contaminants in agricultural waste before land application. The goal of this study was to characterize microbial composition in two constructed wetlands designed to remove contaminants from dairy washwater. Water samples were collected weekly for 11 months from two wetlands to determine the efficiency of the treatment system in removal of chemical contaminants and total and fecal coliforms. The reduction by the treatment was greatest for biological oxygen demand, suspended solids, chemical oxygen demand, nitrate, and coliforms. There was only moderate removal of total nitrogen and phosphorus. Changes in the total bacterial community and ammonia-oxidizing bacterial composition were examined by using denaturing gradient gel electrophoresis (DGGE) and sequencing of PCR-amplified fragments of the gene carrying the α subunit of the ammonia monooxygenase gene (amoA) recovered from soil samples and DGGE bands. DGGE analysis of wetlands and manure samples revealed that the total bacterial community composition was dominated by bacteria from phylogenetic clusters related to Bacillus, Clostridium, Mycoplasma, Eubacterium, and Proteobacteria originally retrieved from the gastrointestinal tracts of mammals. The population of ammonia-oxidizing bacteria showed a higher percentage of Nitrosospira-like sequences from the wetland samples, while a higher percentage of Nitrosomonas-like sequences from manure, feces, raw washwater, and facultative pond was found. These results show that the wetland system is a natural process dependent upon the development of healthy microbial communities for optimal wastewater treatment.     

蜘蛛丝的组成结构与生物学功能

蜘蛛是纺丝种类最多的一种节肢动物,目前共发现有8种丝腺,各纺出具有不同生物学功能的丝纤维,可分别用于织网、捕食、逃避、扩散、织制卵袋等行为活动。蜘蛛丝是一种天然的动物蛋白纤维,是随蜘蛛4亿年进化的结果,也是为蜘蛛的生存与繁殖所设计的,蜘蛛丝的适应与进化使蜘蛛丝具有多样化的生物学功能。但蜘蛛不是唯一能纺丝的节肢动物,除蛛形纲以外,还有其它很多节肢动物,如昆虫纲和多足纲的动物都有具有丝腺,能纺出一种或多种丝蛋白纤维。本文将以昆虫作为比较来概述蜘蛛丝腺的起源与种类,蜘蛛丝的化学组成、结构、种类与其生物学功能。     

Microbial Community Comparison of Different Biological Processes for Treating the Same Sewage

The microbial communities, including ammonia-oxidizing bacterial (AOB), eubacterial, actinomycetic and yeast communities, were investigated in two different systems by PCR-DGGE (denaturing gradient gel electrophoresis) using amplified 16S rRNA gene fragments of bacteria and 26S rRNA gene fragments of yeast. The two systems, which used an anoxic-anaerobic-aerobic process (A₂O) and an anoxic-aerobic process (AO), respectively, received identical sewage, operated under the same conditions and demonstrated similar treatment performance. The AOB communities of the two systems showed almost identical structures corresponding to similar ammonium removal, while bacterial, actinomycetic and yeast communities demonstrated obvious differences. The A₂O system showed richer eubacterial, actinomycetic and yeast communities than the AO system. FISH results showed that the AOB cells in the A₂O system made up 3.6 ± 0.2% of the total bacterial population, while those in the AO system accounted for 1.9 ± 0.2%. Thus the existence of an anaerobic environment in the A₂O system resulted in a marked increase in biodiversity.     

Structure and Composition of Biological Slimes on Paper and Board Machines

Biological slimes (biofilms) collected from the wet end of paper and board machines were examined by electron microscopy and analyzed for fatty acid composition, neutral sugar composition, and ATP. Electron microscopy revealed minuscule prokaryotic organisms (diameter, 0.2 to 0.4 μm). Larger cells morphologically resembling Sphaerotilus and Leptothrix spp. were found in slimes from machines using recycled fiber or unbleached pulp. The bacteria were embedded in a slimy matrix and often contained reserve materials microscopically resembling poly-β-hydroxybutyrate and glycogen. Fatty acid analysis of the slimes revealed bacterial signature fatty acids in concentrations equivalent to the presence of 2 × 10¹⁰ to 2.6 × 10¹² (average, 7 × 10¹¹) bacterial cells (live and dead) per g (dry weight) of slime. The slimes contained several known components of bacterial polysaccharides in addition to glucose, indicating that the slime body consisted of bacterial polysaccharides. The slimes contained uronic acids equivalent to a binding capacity of 12.5 to 50 μmol of divalent cations per g (dry weight) of slime. The uronic acid-containing polysaccharides may be responsible for the accumulation of heavy metals in the slime. Calculation of the ATP contents of the slimes resulted in an estimate of 5 × 10¹² cells per g (dry weight) of slime when calibrated with pure bacterial cultures isolated from the slimes. From electron micrographs, an estimate ranging from 1 × 10¹⁰ to 1.5 × 10¹² (average, 4 × 10¹¹) cells per g (dry weight) of slime was obtained.     

Analysis of the Microbial Community in an Acidic Hollow-Fiber Membrane Biofilm Reactor (Hf-MBfR) Used for the Biological Conversion of Carbon Dioxide to Methane

Hydrogenotrophic methanogens can use gaseous substrates, such as H₂ and CO₂, in CH₄ production. H₂ gas is used to reduce CO₂. We have successfully operated a hollow-fiber membrane biofilm reactor (Hf-MBfR) for stable and continuous CH₄ production from CO₂ and H₂. CO₂ and H₂ were diffused into the culture medium through the membrane without bubble formation in the Hf-MBfR, which was operated at pH 4.5–5.5 over 70 days. Focusing on the presence of hydrogenotrophic methanogens, we analyzed the structure of the microbial community in the reactor. Denaturing gradient gel electrophoresis (DGGE) was conducted with bacterial and archaeal 16S rDNA primers. Real-time qPCR was used to track changes in the community composition of methanogens over the course of operation. Finally, the microbial community and its diversity at the time of maximum CH₄ production were analyzed by pyrosequencing methods. Genus Methanobacterium, related to hydrogenotrophic methanogens, dominated the microbial community, but acetate consumption by bacteria, such as unclassified Clostridium sp., restricted the development of acetoclastic methanogens in the acidic CH₄ production process. The results show that acidic operation of a CH₄ production reactor without any pH adjustment inhibited acetogenic growth and enriched the hydrogenotrophic methanogens, decreasing the growth of acetoclastic methanogens.     

Impact of Nutrient Composition on a Degradative Biofilm Community

A microbial community was cultivated in flow cells with 2,4,6-trichlorobenzoic acid (2,4,6-TCB) as sole carbon and energy source and was examined with scanning confocal laser microscopy and fluorescent molecular probes. The biofilm community which developed under these conditions exhibited a characteristic architecture, including a basal cell layer and conspicuous mounds of bacterial cells and polymer (approximately 20 to 30 (mu)m high and 25 to 40 (mu)m in diameter) occurring at 20- to 200-(mu)m intervals. When biofilms grown on 2,4,6-TCB were shifted to a labile, nonchlorinated carbon source (Trypticase soy broth), the biofilms underwent an architectural change which included the loss of mound structures and the formation of a more homogeneous biofilm. Neutrally charged fluorescent dextrans, which upon hydration become cationic, were observed to bind to mounds, as well as to the basal cell layer, in 14-day biofilms. In contrast, polyanionic dextrans bound only to the basal cell layer, indicating that this material incorporated sites with both positive and negative charge. The results from this study indicate that nutrient composition has a significant impact on both the architecture and the physicochemistry of degradative biofilm communities.     

造纸废水活性污泥的微生物群落结构及多样性分析

为探究造纸废水活性污泥中微生物群落结构多样性以及对造纸废水处理效果的影响，利用Illumina MiSeq 高通量测序方法，分析在处理造纸废水过程中，同一运行阶段两个并联氧化沟内活性污泥的微生物群落与多样性组成。结果表明，系统中处理造纸废水的活性污泥在同一废水条件下微生物群落结构总体稳定，优势细菌为绿弯菌门(Chloroflexi)、拟杆菌门（Bacteroidota）、变形菌门（Proteobacteria）、Myxococcota、放线菌门(Actinobacteria）、厚壁菌门(Firmicutes）等。最重要的优势细菌类群为Chloroflexi，相对丰度占比为47.67%~48.22%，远远高于其他废水中Chloroflexi的占比，其中厌氧绳菌纲（Anaerolineae）是其主要成员，占比84.39%~88.34%，可针对性地去除造纸废水中的污染物。造纸废水活性污泥样品中存在大量特殊功能菌群，其在废水中污染物尤其是木质素的去除中发挥着重要作用。     

不同富集和分离培养条件下的含酚废水处理生物膜微生物群落结构与活性比较分析

采用Biolog和变性梯度凝胶电泳(DGGE)技术研究了不同苯酚浓度培养对焦化废水处理厂反硝化池生物膜样品中微生物群落结构和代谢类型的影响。DGGE结果表明, 不同浓度苯酚和不同培养方式富集培养后, 细菌16S rDNA的部分条带分布谱形发生改变, 还有部分条带只受到了苯酚浓度变化的影响; 富集培养过程中由于碳源组成相对焦化废水简单, DGGE条带所代表的优势微生物多样性有所降低。Biolog试验结果表明, 生物膜样本的细菌群落代谢能力最强; 低浓度苯酚富集后的样品能利用的底物碳源类型最丰富。对Biolog试验结果的主成分分析显示, 相同浓度苯酚富集培养后的细菌群落代谢功能多样性相似, 但从DGGE结果看出其结构组成产生了变化。富集培养使样品微生物群落的代谢功能发生改变, 低浓度的苯酚富集增加了群落中微生物的代谢类型。而不同条件获得的分离物其苯酚降解能力的初步分析也表明, 富集与分离条件对苯酚降解菌的分离能力和得到的菌株特性具有差别。