Variable Cell Morphology Approach for Individual-Based Modeling of Microbial Communities

An individual-based, mass-spring modeling framework has been developed to investigate the effect of cell properties on the structure of biofilms and microbial aggregates through Lagrangian modeling. Key features that distinguish this model are variable cell morphology described by a collection of particles connected by springs and a mechanical representation of deformable intracellular, intercellular, and cell-substratum links. A first case study describes the colony formation of a rod-shaped species on a planar substratum. This case shows the importance of mechanical interactions in a community of growing and dividing rod-shaped cells (i.e., bacilli). Cell-substratum links promote formation of mounds as opposed to single-layer biofilms, whereas filial links affect the roundness of the biofilm. A second case study describes the formation of flocs and development of external filaments in a mixed-culture activated sludge community. It is shown by modeling that distinct cell-cell links, microbial morphology, and growth kinetics can lead to excessive filamentous proliferation and interfloc bridging, possible causes for detrimental sludge bulking. This methodology has been extended to more advanced microbial morphologies such as filament branching and proves to be a very powerful tool in determining how fundamental controlling mechanisms determine diverse microbial colony architectures.     

Electrical conductivity in a mixed-species biofilm

Geobacter sulfurreducens can form electrically conductive biofilms, but the potential for conductivity through mixed-species biofilms has not been examined. A current-producing biofilm grown from a wastewater sludge inoculum was highly conductive with low charge transfer resistance even though microorganisms other than Geobacteraceae accounted for nearly half the microbial community.     

Membrane biofouling characterization: effects of sample preparation procedures on biofilm structure and the microbial community

Ensuring the quality and reproducibility of results from biofilm structure and microbial community analysis is essential to membrane biofouling studies. This study evaluated the impacts of three sample preparation factors (ie number of buffer rinses, storage time at 4°C, and DNA extraction method) on the downstream analysis of nitrifying biofilms grown on ultrafiltration membranes. Both rinse and storage affected biofilm structure, as suggested by their strong correlation with total biovolume, biofilm thickness, roughness and the spatial distribution of EPS. Significant variations in DNA yields and microbial community diversity were also observed among samples treated by different rinses, storage and DNA extraction methods. For the tested biofilms, two rinses, no storage and DNA extraction with both mechanical and chemical cell lysis from attached biofilm were the optimal sample preparation procedures for obtaining accurate information about biofilm structure, EPS distribution and the microbial community.     

Development and application of an enzymatic and cell flotation treatment for the recovery of viable microbial cells from environmental matrices such as anaerobic sludge

Efficient dissociation of microorganisms from their aggregate matrix is required to study the microorganisms without interaction with their native environment (e.g., biofilms, flocs, granules, etc.) and to assess their community composition through the application of molecular or microscopy techniques. To this end, we combined enzymatic treatments and a cell extraction by density gradient to efficiently recover anaerobic microorganisms from urban wastewater treatment plant sludge. The enzymes employed (amylase, cellulase, DNase, and pectinase) as a pretreatment softly disintegrated the extrapolymeric substances (EPS) interlocked with the microorganisms. The potential damaging effects of the applied procedure on bacterial and archaeal communities were assessed by studying the variations in density (using quantitative PCR), diversity (using capillary electrophoresis single-strand conformation polymorphism fingerprinting [CE-SSCP]), and activity (using a standard anaerobic activity test) of the extracted microorganisms. The protocol preserved the general capacity of the microbial community to produce methane under anaerobic conditions and its diversity; particularly the archaeal community was not affected in terms of either density or structure. This cell extraction procedure from the matrix materials offers interesting perspectives for metabolic, microscopic, and molecular assays of microbial communities present in complex matrices constituted by bioaggregates or biofilms.     

Density of founder cells affects spatial pattern formation and cooperation in Bacillus subtilis biofilms

In nature, most bacteria live in surface-attached sedentary communities known as biofilms. Biofilms are often studied with respect to bacterial interactions. Many cells inhabiting biofilms are assumed to express ‘cooperative traits'', like the secretion of extracellular polysaccharides (EPS). These traits can enhance biofilm-related properties, such as stress resilience or colony expansion, while being costly to the cells that express them. In well-mixed populations cooperation is difficult to achieve, because non-cooperative individuals can reap the benefits of cooperation without having to pay the costs. The physical process of biofilm growth can, however, result in the spatial segregation of cooperative from non-cooperative individuals. This segregation can prevent non-cooperative cells from exploiting cooperative neighbors. Here we examine the interaction between spatial pattern formation and cooperation in Bacillus subtilis biofilms. We show, experimentally and by mathematical modeling, that the density of cells at the onset of biofilm growth affects pattern formation during biofilm growth. At low initial cell densities, co-cultured strains strongly segregate in space, whereas spatial segregation does not occur at high initial cell densities. As a consequence, EPS-producing cells have a competitive advantage over non-cooperative mutants when biofilms are initiated at a low density of founder cells, whereas EPS-deficient cells have an advantage at high cell densities. These results underline the importance of spatial pattern formation for competition among bacterial strains and the evolution of microbial cooperation.     

Anaerobic ammonium oxidation (Anammox) in immobilized activated sludge biofilms during the treatment of weak wastewater

This work studied the formation of molecular nitrogen by the microbial population of immobilized activated sludge of the domestic wastewater treatment plants (WWTP) that employ the technology developed by ZAO ECOS Company. The technology includes physicochemical water pretreatment and treated water recycling. A hard flexible fibrous brush carrier is used for the immobilization of microorganisms. The presence of both aerobic and anaerobic microorganisms and functioning of the methanogenic microbial community was shown in the biofilms developing on the carrier fibers and in suspended sludge. The high efficiency of nitrogen removal at a low C/N ratio was established to be due to the conjugated nitrification, denitrification, and anammox processes, whose functioning was demonstrated by laboratory cultivation methods and by studying the processes in batch and continuous reactors. Fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes (FISH) revealed bacteria belonging to the order Planctomycetales, particularly their anammox group. This work is the first evidence of the important role of the anammox process in the combined system of physicochemical and biological treatment of weak wastewater (BCDEAMOX).     

Time-course correlation of biofilm properties and electrochemical performance in single-chamber microbial fuel cells

The relationship between anode microbial characteristics and electrochemical parameters in microbial fuel cells (MFCs) was analyzed by time-course sampling of parallel single-bottle MFCs operated under identical conditions. While voltage stabilized within 4 days, anode biofilms continued growing during the six-week operation. Viable cell density increased asymptotically, but membrane-compromised cells accumulated steadily from only 9% of total cells on day 3 to 52% at 6 weeks. Electrochemical performance followed the viable cell trend, with a positive correlation for power density and an inverse correlation for anode charge transfer resistance. The biofilm architecture shifted from rod-shaped, dispersed cells to more filamentous structures, with the continuous detection of Geobacter sulfurreducens-like 16S rRNA fragments throughout operation and the emergence of a community member related to a known phenazine-producing Pseudomonas species. A drop in cathode open circuit potential between weeks two and three suggested that uncontrolled biofilm growth on the cathode deleteriously affects system performance.     

Microbial Community Analysis of Fresh and Old Microbial Biofilms on Bayon Temple Sandstone of Angkor Thom, Cambodia

The temples of Angkor monuments including Angkor Thom and Bayon in Cambodia and surrounding countries were exclusively constructed using sandstone. They are severely threatened by biodeterioration caused by active growth of different microorganisms on the sandstone surfaces, but knowledge on the microbial community and composition of the biofilms on the sandstone is not available from this region. This study investigated the microbial community diversity by examining the fresh and old biofilms of the biodeteriorated bas-relief wall surfaces of the Bayon Temple by analysis of 16S and 18S rRNA gene sequences. The results showed that the retrieved sequences were clustered in 11 bacterial, 11 eukaryotic and two archaeal divisions with disparate communities (Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Proteobacteria; Alveolata, Fungi, Metazoa, Viridiplantae; Crenarchaeote, and Euyarchaeota). A comparison of the microbial communities between the fresh and old biofilms revealed that the bacterial community of old biofilm was very similar to the newly formed fresh biofilm in terms of bacterial composition, but the eukaryotic communities were distinctly different between these two. This information has important implications for understanding the formation process and development of the microbial diversity on the sandstone surfaces, and furthermore to the relationship between the extent of biodeterioration and succession of microbial communities on sandstone in tropic region.     

Biofilm reactors for industrial bioconversion processes: employing potential of enhanced reaction rates

This article describes the use of biofilm reactors for the production of various chemicals by fermentation and wastewater treatment. Biofilm formation is a natural process where microbial cells attach to the support (adsorbent) or form flocs/aggregates (also called granules) without use of chemicals and form thick layers of cells known as "biofilms." As a result of biofilm formation, cell densities in the reactor increase and cell concentrations as high as 74 gL^-1 can be achieved. The reactor configurations can be as simple as a batch reactor, continuous stirred tank reactor (CSTR), packed bed reactor (PBR), fluidized bed reactor (FBR), airlift reactor (ALR), upflow anaerobic sludge blanket (UASB) reactor, or any other suitable configuration. In UASB granular biofilm particles are used. This article demonstrates that reactor productivities in these reactors have been superior to any other reactor types. This article describes production of ethanol, butanol, lactic acid, acetic acid/vinegar, succinic acid, and fumaric acid in addition to wastewater treatment in the biofilm reactors. As the title suggests, biofilm reactors have high potential to be employed in biotechnology/bioconversion industry for viable economic reasons. In this article, various reactor types have been compared for the above bioconversion processes.     

Long-term effect of dissolved oxygen on partial nitrification performance and microbial community structure

In this study, the performance of partial nitrification via nitrite and microbial community structure were investigated and compared in two sequencing batch reactors (SBR) with different dissolved oxygen (DO) levels. Both reactors achieved stable partial nitrification with nitrite accumulation ratio of above 95% by using real-time aeration duration control. Compared with high DO (above 3 mg/l on average) SBR, simultaneous nitrification and denitrification (SND) via nitrite was carried out in low DO (0.4–0.8 mg/l) SBR. The average efficiencies of SND in high DO and low DO reactor were 7.7% and 44.9%, and the specific SND rates were 0.20 and 0.83 mg N/(mg MLSS h), respectively. Low DO did not produce sludge with poorer settling properties but attained lower turbidities of the effluent than high DO. Fluorescence in situ hybridization (FISH) analysis in both the reactors showed that ammonia-oxidizing bacteria (AOB) were the dominant nitrifying bacteria and nitrite-oxidizing bacteria (NOB) did not be recovered in spite of exposing nitrifying sludge to high DO. The morphology of the sludge from both two reactors according to scanning electron microscope indicated that small rod-shaped and spherical clusters were dominant, although filamentous bacteria and few long rod-shaped coexisted in the low DO reactor. By selecting properly DO level and adopting process control method is not only of benefit to the achievement of novel biological nitrogen removal technology, but also favorable to sludge population optimization.     

Overall functional gene diversity of microbial communities in three full-scale activated sludge bioreactors

Understanding microbial community composition is thought to be crucial for improving process functioning and stabilities of full-scale activated sludge reactors in wastewater treatment plants (WWTPs). However, functional gene compositions of microbial communities within them have not been clearly elucidated. To gain a complete picture of microbial community, in this study, GeoChip 4.2 was used to profile the overall functional genes of three full-scale activated sludge bioreactors, the 16S rRNA gene diversities of which had been unveiled by 454-pyrosequencing in our previous investigation. Triplicate activated sludge samples from each system were analyzed, with the detection of 38,507 to 40,654 functional genes. A high similarity of 77.3–81.2 % shared functional genes was noted among the nine samples, verified by the high 16S rRNA gene similarity with shared operational taxonomic units (OTUs) constituting 66.4–70.0 % of the detected sequences in each system. Correlation analyses showed that the abundances of a wide array of functional genes were associated with system performances. For example, the abundances of carbon degradation genes were strongly correlated to chemical oxygen demand (COD) removal efficiencies (r?=?0.8697, P?<?0.01). Lastly, we found that sludge retention time (SRT), influent total nitrogen concentrations (TN inf), and dissolved oxygen (DO) concentrations were key environmental factors shaping the overall functional genes. Together, the results revealed vast functional gene diversity and some links between the functional gene compositions and microbe-mediated processes.     

Starter culture of <Emphasis Type="Italic">Pseudomonas veronii</Emphasis> strain B for aerobic granulation

Aggregation of bacterial cells is used in formation of microbial granules. Aerobically grown microbial granules can be used as the bio-agents in the treatment of wastewater. However, there are problems with start up of microbial granulation and biosafety of this process. Aim of this research was selection and testing of safe microbial strain with high cell aggregation ability to shorten period of microbial granules formation. Five bacterial strains with cell aggregation index higher than 50% have been isolated from the granules. Strain of Pseudomonas veronii species was considered as most probably safe starter culture for granulation because other strains belonged to the species known as human pathogens. The microbial granules were formed after 3 days of cultivation in case when P. veronii strain B was applied to start-up aerobic granulation process using model wastewater. The granules were produced from activated sludge after 9 days of cultivation. Microbial aggregates produced from starter culture of P. veronii strain B were more compact (sludge volume index was 70 ml/g) than those produced from activated sludge (sludge volume index was 106 ml/g). It is a first proof that application of selected safe starter pure culture with high cell aggregation ability can accelerate and enhance formation of microbial granules.     

Specific aerobic granules can be developed in a completely mixed tank reactor by bioaugmentation using micro-mycelial pellets of Phanerochaete chrysosporium

Aerobic granules were firstly developed in a completely mixed tank reactor (CMTR) by seeding micro-mycelial pellets (MMPs) of Phanerochaete chrysosporium. During phenol wastewater treatment, sludge granulation rate reached 67 % after 15-day operation. The granules in CMTR are different from aerobic granules described in literature in morphology, and a majority of them are rod-shaped or rodlike sludge besides spherical granules. The polymorphic granules, having no essential difference with aerobic granules previously reported, achieve advantages over conventional activated sludge in settling ability, biomass concentration, density, integrity coefficient and removal ability to phenol wastewater. The optimized parameters for sludge granulation in CMTR including temperature, inoculum quantity, rotary speed and superficial air upflow velocity are 30 °C, 5–7 g/l, 150 rpm, and 0.5 cm/s, respectively. Analysis on sludge granulation mechanism indicates that MMPs not only result in the formation of aerobic granules containing MMPs as nuclei, but also induce the formation of biogranules which do not have MMP at their cores. The work challenges the general belief that the homogenous circular flow pattern of microbial aggregates is necessary for aerobic sludge granulation.     

Application of Image Analysis Techniques in Activated Sludge Wastewater Treatment Processes

Image analytical techniques have been extensively developed to evaluate complex microbial aggregates such as sludge flocs and biofilms. This review covers the latest contributions concerning the application of image analysis to the activated sludge systems with respect to the most frequently used morphological parameters and relations between them and traditional wastewater treatment parameters. Recent developments have indicated that image analysis can be successfully used for the quantification of flocs and filamentous bacteria in the operating wastewater treatment plants, which enables prediction of bulking events and pinpoint flocs formation.     

Detection of anaerobic processes and microorganisms in immobilized activated sludge of a wastewater treatment plant with intense aeration

Attached activated sludge from the Krasnaya Polyana (Sochi) wastewater treatment plant was studied after the reconstruction by increased aeration and water recycle, as well as by the installation of a bristle carrier for activated sludge immobilization. The activated sludge biofilms developing under conditions of intense aeration were shown to contain both aerobic and anaerobic microorganisms. Activity of a strictly anaerobic methanogenic community was revealed, which degraded organic compounds to methane, further oxidized by aerobic methanotrophs. Volatile fatty acids, the intermediates of anaerobic degradation of complex organic compounds, were used by both aerobic and anaerobic microorganisms. Anaerobic oxidation of ammonium with nitrite (anammox) and the presence of obligate anammox bacteria were revealed in attached activated sludge biofilms. Simultaneous aerobic and anaerobic degradation of organic contaminants by attached activated sludge provides for high rates of water treatment, stability of the activated sludge under variable environmental conditions, and decreased excess sludge formation.     

Systematic analysis of biochemical performance and the microbial community of an activated sludge process using ozone-treated sludge for sludge reduction

Two lab-scale bioreactors (reactors 1 and 2) were employed to examine the changes in biological performance and the microbial community of an activated sludge process fed with ozonated sludge for sludge reduction. During the 122 d operation, the microbial activities and community in the two reactors were evaluated. The results indicated that, when compared with the conventional reactor (reactor 1), the reactor that was fed with the ozonated sludge (reactor 2) showed good removal of COD, TN and cell debris, without formation of any excess sludge. In addition, the protease activity and intracellular ATP concentration of reactor 2 were increased when compared to reactor 1, indicating that reactor 2 had a better ability to digest proteins and cell debris. DGGE analysis revealed that the bacterial communities in the two reactors were different, and that the dissimilarity of the bacterial population was nearly 40%. Reactor 2 also contained more protozoa and metazoa, which could graze on the ozone-treated sludge debris directly.     

rRNA-targeted寡核苷酸探针识别活性污泥微生物群落结构与功能研究进展

活性污泥中微生物群落内部关系非常复杂 ,及时对活性污泥中优势菌群和群落内部关系进行监测是污水处理中采取正确措施的关键。历史研究表明传统培养方法经常导致活性污泥优势菌群检测的失败 ,而r RNA- targeted寡核苷酸探针作为一种快速原位监测活性污泥微生物群落结构和功能的新工具被引入 ,使我们对参与污水净化的微生物群落结构和优势菌群能有较全面的了解。就该方法在识别除磷污泥、脱氮污泥、污泥泡沫和膨胀污泥中微生物群落结构和功能的典型应用进行综述 ,分析了该方法存在的优点和缺点 ,并对目前已建立且应用于活性污泥微生物检测的 r RNA- targeted寡核苷酸探针进行了详细总结     

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.     

Microbial community structure and biomass in developing drinking water biofilms

Traditional techniques to study microbes, such as culturable counts, microbial biomass, or microbial activity, do not give information on the microbial ecology of drinking water systems. The aim of this study was to analyze whether the microbial community structure and biomass differed in biofilms collected from two Finnish drinking water distribution systems (A and B) receiving conventionally treated (coagulation, filtration, disinfection) surface water. Phospholipid fatty acid methyl esters (PLFAs) and lipopolysaccharide 3-hydroxy fatty acid methyl esters (LPS 3-OH-FAs) were analyzed from biofilms as a function of water residence time and development time. The microbial communities were rather stabile through the distribution systems, as water residence time had minor effects on PLFA profiles. In distribution system A, the microbial community structure in biofilms, which had developed in 6 weeks, was more complex than those grown for 23 or 40 weeks. The microbial communities between the studied distribution systems differed, possibly reflecting the differences in raw water, water purification processes, and distribution systems. The viable microbial biomass, estimated on the basis of PLFAs, increased with increasing water residence time in both distribution systems. The quantitative amount of LPS 3-OH-FAs increased with increasing development time of biofilms of distribution system B. In distribution system A, LPS 3-OH-FAs were below the detection limit.