Microbial Morphology Research Articles

Enhancing electron transfer by ferroferric oxide during the anaerobic treatment of synthetic wastewater with mixed organic carbon

Conductive materials can enhance direct interspecies electron transfer (DIET) during the anaerobic treatment of wastewater and provide ecological advantages for syntrophic methane (CH4) production. The effects of ferroferric oxide (Fe3O4) on the system performance and microbial communities were examined during the anaerobic treatment of synthetic wastewater with mixed organic carbon. The addition of Fe3O4 did not enhance the relative abundance of methanogens, but increased the maximum CH4 production rate by 15.4% and shortened the lag phase by 13.9%. Methanosarcina was the predominant methanogen contributing to DIET. Proteiniclasticum and Kosmotoga were dominant upon the addition of Fe3O4 and were responsible for protein degradation. More extracellular proteins were produced and a filamentous morphology was observed upon the addition of Fe3O4. Short-term Fe3O4 addition also improved the performance of the anaerobic treatment in the control reactor, and the Fe3O4 rather than the microbial morphology was the more important factor enhancing DIET.

CRISPRi engineering E. coli for morphology diversification

Microbial morphology engineering has recently become interesting for biotechnology. Genes ftsZ and mreB encoding proteins of bacterial fission ring and skeletons, respectively, are essential for cell growth, they both are the most important genes keeping the bacterial shapes including the cell length and width, respectively. Clustered regularly interspaced short palindromic repeats interference, abbreviated as CRISPRi, was for the first time used in this study to regulate expression intensities of ftsZ or/and mreB in E. coli. Five sgRNAs associated with CRISPRi were designed and synthesized, respectively, to target five various locations on genes ftsZ or mreB encoded in the E. coli chromosome, resulting in various reduced expression levels of ftsZ or/and mreB, respectively, forming elongated or/and fatter cells. Repressions on gene expressions of ftsZ or/and mreB could be further intensified by combining various sgRNAs together. It was found that the stronger the repression on genes ftsZ or/and mreB, the longer the E. coli fibers, and the larger the E. coli cells. Combined repressions on expressions of ftsZ and mreB generated long and larger E. coli with diverse morphologies including various sizes of gourds, bars, coccus, spindles, multi-angles and ellipsoids. In all cases, accumulations of intracellular biopolyester polyhydroxybutyrate (PHB) were in direct proportional to the intracellular volumes, ranging from 40% to 80% PHB in bacterial cell dry weights, depending on the cell volumes increases by the above CRISPRi applications.

Effects of salinity on simultaneous reduction of perchlorate and nitrate in a methane-based membrane biofilm reactor.

This study builds upon prior work showing that methane (CH4) could be utilized as the sole electron donor and carbon source in a membrane biofilm reactor (MBfR) for complete perchlorate (ClO4-) and nitrate (NO3-) removal. Here, we further investigated the effects of salinity on the simultaneous removal of the two contaminants in the reactor. By testing ClO4- and NO3- at different salinities, we found that the reactor performance was very sensitive to salinity. While 0.2% salinity did not significantly affect the hydrogen-based MBfR for ClO4- and NO3- removals, 1% salinity completely inhibited ClO4- reduction and significantly lowered NO3- reduction in the CH4-based MBfR. In salinity-free conditions, NO3- and ClO4- removal fluxes were 0.171g N/m2-day and 0.091g/m2-day, respectively, but NO3- removal fluxes dropped to 0.0085g N/m2-day and ClO4- reduction was completely inhibited when the medium changed to 1% salinity. Scanning electron microscopy (SEM) showed that the salinity dramatically changed the microbial morphology, which led to the development of wire-like cell structures. Quantitative real-time PCR (qPCR) indicated that the total number of microorganisms and abundances of functional genes significantly declined in the presence of NaCl. The relative abundances of Methylomonas (methanogens) decreased from 31.3 to 5.9% and Denitratisoma (denitrifiers) decreased from 10.6 to 4.4% when 1% salinity was introduced.

Nitrogen Removal Performance of Novel HABR Reactor over CANON Process

The startup of novel hybrid anaerobic baffled reactor (HABR) and the feasibility of completely autotrophic nitrogen removal over nitrite (CANON) process were studied. The reactor was operated by gradually decreasing HRT to improve total nitrogen load. After startup, the change of nitrogen concentrations, conductivity, pH and MLSS was measured along the reactor, and then the microbial morphology and spatial structure of bacteria were defined. The test results indicated several points:under the experimental condition of an average NH4+-N of 40 mg·L-1 in influent, the quick start-up process was successfully achieved by continuous operation within 89 days, and stabilized over 187 days. The average NH4+-N and TN concentrations of effluent were below 2 mg·L-1 and 10 mg·L-1 respectively, the average removal efficiencies of NH4+-N and TN reached above 96% and 83% respectively, and a NRR of 0.15 kg·(m3·d)-1 was obtained. During the stable phase, the concentrations of NH4+-N and TN gradually declined along the reactor, while the concentrations of NO2--N and NO3--N remained constant at relatively lower values. The highest nitrogen removal efficiency of ammonia occurred in compartment 1, and the reason was confirmed by SEM and FISH microbiological analysis that enriched functional bacteria of AOB and AnAOB coexisted in compartment 1.

Controlling the feed rate of propanol to optimize erythromycin fermentation by on-line capacitance and oxygen uptake rate measurement.

The aim of the present study was to optimize the feeding proportion of glucose and propanol for erythromycin biosynthesis by real-time monitoring and exploring its limited ratio by the on-line multi-frequency permittivity measurement. It was found that the capacitance values were sensitive to the variation of biomass concentration and microbial morphology as well as the true state of cell growth. It was most favorable to both cell growth and secondary metabolism to keep the ratio of glucose to propanol at 4.3 (g/g). The specific growth rate calculated by the capacitance measurement correctly and accurately reflected the cell physiological state. An appropriate feed rate of propanol was crucial for cell growth and secondary metabolism, as well as to improve the quality of erythromycin-A. In addition, the erythromycin production titer (10,950 U/mL) was further enhanced by 4 % when the propanol feed was regulated by step-down strategy based on both OUR (oxygen uptake rate) and the on-line monitoring capacitance.

Selection of an actinobacteria mixed culture for chlordane remediation. Pesticide effects on microbial morphology and bioemulsifier production.

Chlordane bioremediation using actinobacteria mixed culture is an attractive clean-up technique. Their ability to produce bioemulsifiers could increase the bioavailability of this pesticide. In order to select a defined actinobacteria mixed culture for chlordane remediation, compatibility assays were performed among six Streptomyces strains. The strains did not show growth inhibition, and they were assayed for chlordane removal, either as pure or as mixed cultures. In pure cultures, all of the strains showed specific dechlorination activity (1.42-24.20 EU mg(-1)) and chlordane removal abilities (91.3-95.5%). The specific dechlorination activity was mainly improved with cultures of three or four microorganisms. The mixed culture consisting of Streptomyces sp. A2-A5-A13 was selected. Their ability to produce bioemulsifiers in the presence of glucose or chlordane was tested, but no significant differences were observed (p > 0.05). However, the stability of the emulsions formed was linked to the carbon source used. Only in chlordane presence the emulsions retained 100% of their initial height. Finally, the selected consortium showed a high degree of sporulation in the pesticide presence. This is the first study on the effects that chlordane exerts on microbe morphology and emulsifier production for a defined mixed culture of Streptomyces with ability to remediate the pesticide.

Automated analysis of filamentous microbial morphology with AnaMorf.

The morphological quantification of filamentous microbes represents an important analytical technique in the optimization of bioprocesses involving such organisms, given the demonstrated links between morphology and metabolite yield. However, in many studies, much of this quantification has required some degree of manual intervention, if it has been conducted at all, burdening biotechnologists with a time-consuming process and potentially introducing bias into analyses. Here, software for the automated quantification of filamentous microbes is presented, implemented as a plug-in for the widely used, freely available image analysis package, ImageJ. The software, together with all related source code, documentation and test data, is freely available to the community via an online repository.

Sulfur-cycling fossil bacteria from the 1.8-Ga Duck Creek Formation provide promising evidence of evolution's null hypothesis

The recent discovery of a deep-water sulfur-cycling microbial biota in the ∼ 2.3-Ga Western Australian Turee Creek Group opened a new window to life's early history. We now report a second such subseafloor-inhabiting community from the Western Australian ∼ 1.8-Ga Duck Creek Formation. Permineralized in cherts formed during and soon after the 2.4- to 2.2-Ga "Great Oxidation Event," these two biotas may evidence an opportunistic response to the mid-Precambrian increase of environmental oxygen that resulted in increased production of metabolically useable sulfate and nitrate. The marked similarity of microbial morphology, habitat, and organization of these fossil communities to their modern counterparts documents exceptionally slow (hypobradytelic) change that, if paralleled by their molecular biology, would evidence extreme evolutionary stasis.

Effect of increasing total solids contents on anaerobic digestion of food waste under mesophilic conditions: performance and microbial characteristics analysis.

The total solids content of feedstocks affects the performances of anaerobic digestion and the change of total solids content will lead the change of microbial morphology in systems. In order to increase the efficiency of anaerobic digestion, it is necessary to understand the role of the total solids content on the behavior of the microbial communities involved in anaerobic digestion of organic matter from wet to dry technology. The performances of mesophilic anaerobic digestion of food waste with different total solids contents from 5% to 20% were compared and the microbial communities in reactors were investigated using 454 pyrosequencing technology. Three stable anaerobic digestion processes were achieved for food waste biodegradation and methane generation. Better performances mainly including volatile solids reduction and methane yield were obtained in the reactors with higher total solids content. Pyrosequencing results revealed significant shifts in bacterial community with increasing total solids contents. The proportion of phylum Chloroflexi decreased obviously with increasing total solids contents while other functional bacteria showed increasing trend. Methanosarcina absolutely dominated in archaeal communities in three reactors and the relative abundance of this group showed increasing trend with increasing total solids contents. These results revealed the effects of the total solids content on the performance parameters and the behavior of the microbial communities involved in the anaerobic digestion of food waste from wet to dry technologies.

Changes in the microbial community structure of filaments and floc formers in response to various carbon sources and feeding patterns.

Filamentous bulking is a complicated problem in wastewater treatment plants treating various wastewaters, leading to the deterioration of the settling properties and the effluent quality. This study systematically investigated long-term effects of various carbon sources and feeding patterns on the growth of filamentous bacteria, in order to reveal the mechanism of filamentous bulking. Sludge volume index (SVI), microscopic observations, staining (Gram and Neisser staining), scan electron microscopic, and fluorescent in situ hybridization (FISH) were used to monitor the bulking and track the changes of microbial morphology and community structure of activated sludge in six lab-scale sequencing batch reactors (SBRs) fed with different carbon sources. Filamentous bulking was not observed in all SBRs under anoxic feeding pattern with a short fill time, in which SVI remained below 150 mL/g. In contrast, serious bulking (SVI > 500 mL/g) occurred under aerobic feeding pattern when fed with ethanol, propionate, acetate, and glucose, in which Thiothrix and Sphaerotilus natans proliferated as dominant filaments. Compared to glucose-fed reactor, relatively light bulking was caused in starch-fed reactor with the growth of Nostocoida limicola II. In addition, flocs in starch-fed reactor were more open and fluffy than flocs formed on readily biodegradable substrates. Finally, a framework integrating kinetic selection, diffusion selection, storage selection, and protozoa capture mechanism was proposed to explain filamentous bulking.

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.

Qualitative examination of diatom diversity in black fly (Diptera: Simuliidae) gut contents across a mortality gradient after exposure to Bacillus thuringiensis subsp. israelensis de Barjac, 1978

While Bacillus thuringiensis subsp. israelensis de Barjac, 1978 (Bti) is generally effective in reducing Simulium Latreille, 1802 pest levels, impaired toxicity has been identified. Diatoms are typically abundant in Simulium larval gut contents and thought to affect feeding behaviour and/or Bti ingestion. We sought to determine diatom composition in larval Simulium gut contents and periphyton in relation to mortality subsequent in situ Bti application across three stream reaches. Generally, lower larval mortality after Bti exposure was associated with comparatively greater diatom diversity inside gut contents, corresponding with an inverse relationship between periphyton diatom diversity and larval mortality across our study reaches. High profile diatoms inside gut contents were positively correlated with mortality, suggesting microbial morphology may affect Bti efficacy. Diatoms inside live larval guts were most similar to periphyton which indicates that these larvae may have been feeding less on lower quality detrital material and/or exhibiting comparatively greater benthic feeding.

Effects of feeding pattern and dissolved oxygen concentration on microbial morphology and community structure: The competition between floc-forming bacteria and filamentous bacteria

Understanding of the competition between floc-formers and filaments is critical to prevent filamentous bulking in practice. This study aimed to investigate the effects of feeding pattern and dissolved oxygen (DO) concentration on their competition in four sequencing batch reactors (SBRs). Short feeding under anoxic condition (fill time<10min) resulted in a well-settling sludge (sludge volume index (SVI)<100mL/g), in spite of DO concentrations. Sludge settleability deteriorated (SVI>200mL/g) and filamentous bulking was observed when the substrate was added in a limiting rate by prolonging the anoxic fill time up to 90min. In contrast, sludge settleability in fully aerobic systems was quite poor (SVI>500mL/g) in spite of the feeding length. Compared to the systems with an anoxic fill phase, more types and abundant filamentous bacteria were identified in fully aerobic systems. Microscopic observation, staining reactions and fluorescence in situ hybridization analysis indicated that the extensive filaments, including Thiothrix nivea, Type 021N, Type 1851 and Microthrix parvicella, proliferated in fully aerobic systems. The results of this study indicated that substrate gradients played an important role on the competition between filaments and floc-formers. It is recommend the adoption of plug-flow selector configurations, with anoxic conditions in order to maintain good and robust sludge settleability.

Optimization of polyhydroxyalkanoates fermentations with on-line capacitance measurement

The aim of this work was to provide an effective methodology for optimization of the polyhydroxyalkanoates (PHAs) fermentation with Ralstonia eutropha by the on-line capacitance measurement. The present study found the capacitance values could reflect variations of microbial morphology and viability. Furthermore, oxygen uptake rate, specific oxygen uptake rate and specific growth rate were measured in real-time and compared with the capacitance value. In addition, a fed-batch control strategy based on the on-line capacitance measurement was proposed to improve the PHAs production by 22%.

A single betaproteobacterium dominates the microbial community of the crambescidine-containing sponge Crambe crambe

Crambe crambe is a marine sponge that produces high concentrations of the pharmacologically significant pentacyclic guanidine alkaloids (PGAs), Crambescines and Crambescidines. Although bio-mimetic chemical synthesis of PGAs suggests involvement of microorganisms in their biosynthesis, there are conflicting reports on whether bacteria are associated with this sponge or not. Using 16S rRNA gene pyrosequencing we show that the associated bacterial community of C. crambe is dominated by a single bacterial species affiliated to the Betaproteobacteria. Microscopy analysis of sponge tissue sections using a specific probe and in situ hybridization confirmed its dominance in the sponge mesohyl and a single microbial morphology was observed by transmission electron microscopy. If confirmed the presence of a simple bacteria community in C. crambe makes this association a very pertinent model to study sponge-bacteria interactions and should allow further research into the possible implication of bacteria in PGA biosynthesis.

Isolation of Endophytic Fungi from &lt;i&gt;Xylocarpus granatum&lt;/i&gt; and Study of Antimicrobial Activities

Endophytic fungi isolation from the Meliaceae plant Xylocarpus granatum was investigated and 20 potential pure strains were isolated. According to microbial taxonomy and morphology method, 10 species Rhizoctonia DC of Deuteromycotina, Penicillium 6 species, 1 species of Aspergillus, 1 species Trichoderma and 2 species Mucor of Zygomycotina were identified with microscopic examination. Antibacterial experiments were carried out with Oxford cup assay and 10 strains metabolites with strong antibacterial activity were found.

Sponge swabs increase sensitivity of sterility testing of processed bone and tendon allografts

Sterility testing is the final, and critical, step in quality control of tissue banking. It informs the decision whether to release the tissue allografts for clinical use, or not. The most common method for sterility testing of structural bone and tendon allografts is to swab using cotton tip streaks. This method provides low recovery efficiency; and therefore may pass allografts with low bioburden, providing false negatives. Our pilot data revealed organism recovery efficiencies of 60, 30 and 100% from cotton swab, membrane filtration and sponge swaps, respectively. Our aim was to develop a high sensitivity sterility test for structural bone and tendon allografts using a sponge sampling method. Eighty-one bone and tendon allograft samples were inoculated with organism suspensions (10(2) or less organisms per 0.1 mL) of Clostridium sporogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis, Aspergillus niger, Staphylococcus epidermidis and Micrococcus spp. Nasco sponges (4 × 8 cm) were used to aseptically sample the whole surface of allograft samples. The sponges were cut in half and cultured in either tryptone soya or fluid thioglycollate broths for 14 days. Positive culture samples were further examined for microbial morphology. The results showed that the sensitivity of the method, and negative predictive value, is 100% for all inoculated organisms incubated with thioglycollate. We conclude that this sponge sampling method should be applied as the standard for sterility testing of structural bone and tendon allografts.

The preservation and degradation of filamentous bacteria and biomolecules within iron oxide deposits at Rio Tinto, Spain

One of the keys to understanding and identifying life on other planets is to study the preservation of organic compounds and their precursor micro-organisms on Earth. Rio Tinto in southwestern Spain is a well documented site of microbial preservation within iron sulphates and iron oxides over a period of 2.1 Ma. This study has investigated the preservation of filamentous iron oxidising bacteria and organics through optical microscopy, scanning electron microscopy (SEM) and Fourier transform infra-red (FTIR) spectroscopy, from laboratory cultures of natural samples to contemporary natural materials to million-year old river terraces. Up to 40% elemental carbon and >7% nitrogen has been identified within microbial filaments and cell clusters in all samples through SEM EDS analyses. FTIR spectroscopy identified C-H(x) absorption bands between 2960 and 2800 cm(-1), Amide I and II absorption bands at 1656 and 1535 cm(-1), respectively and functional group vibrations from within nucleic acids at 917, 1016 and 1124 cm(-1). Absorption bands tracing the diagenetic transformation of jarosite to goethite to hematite through the samples are also identified. This combination of mineralogy, microbial morphology and biomolecular evidence allows us to further understand how organic fossils are created and preserved in iron-rich environments, and ultimately will aid in the search for the earliest life on Earth and potential organics on Mars.

Operation of household wastewater treatment plant equipped with membrane module

The goal of the contribution is to present a practical experience with operation of a household MBR plant under the real conditions. The tested household MBR plant is installed in the garden of a four-person house. All the relevant indicators and parameters were analyzed, i.e. quality of influent and effluent, pH, temperature, membrane flux; microbial morphology, physiology and activity of sludge etc.

Microbial morphology and community structure in a suspended carrier biofilm reactor as a function of substrate loading rates

An aerobic suspended carrier biofilm reactor was efficient in simultaneous organic carbon and nitrogen removal, with COD removal efficiencies of 87.1–99.0% and simultaneous nitrification and denitrification (SND) efficiencies about 96.7-98.8%. The effects of substrate loading on microbial morphology and community structure were investigated by environmental scanning electron microscopy (ESEM), denaturing gel gradient electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). Biofilms formed at different substrate loadings had different morphology and community structures. A higher substrate concentration resulted in denser and thinner biofilms, while a lower substrate concentration resulted in looser and thicker biofilms with significant presence of filamentous bacteria. Both sequence analysis of DGGE bands and FISH analysis indicated the dominance of β-Proteobacteria in the biofilm communities, especially Zoogloea. FISH analysis revealed that the relative abundance of β-proteobacteria ammonia oxidizing bacteria (AOB) was positively correlated with ammonium concentrations, whereas Nitrospira-like nitrite-oxidizing bacteria (NOB) were negatively affected by ammonia and nitrite concentrations. The presence of denitrifying bacteria, Hydrogenophaga spp.,Hyphomicrobium spp. and Rhizobium spp. suggested that not only the oxygen microgradients within the biofilm but also aerobic denitrifiers may be responsible for SND in the aerobic biofilm. Key words: Microbial community structure, microbial morphology, substrate loading, suspended carrier biofilm.