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Applied Microbiology and Biotechnology (v.92, #2)

UDP-N-acetylglucosamine enolpyruvyl transferase as a potential target for antibacterial chemotherapy: recent developments by Ankur Gautam; Praveen Rishi; Rupinder Tewari (pp. 211-225).

The emergence of antibiotic resistance in bacterial pathogens has foxed the health organizations which are actively scrambling for solutions. The available data indicate an increased morbidity in infections often leading to mortality among patients where drug-resistant pathogens have negated the effect of the medicines. In the context of developing “novel bacterial inhibitors” for killing or arresting the growth of drug-resistant pathogens, UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is an enzyme that provides hope for the future. This enzyme catalyzes the first committed step in the biosynthesis of peptidoglycan, an integral and essential component of the bacterial cell wall. MurA enzyme is neither present nor required by mammals and shows poor homology with human proteins. Therefore, it is an ideal target for antibacterial chemotherapy. Till date, 18 structures of MurA (in native and ligand-bound forms) from different bacterial pathogens have been solved. In the last 2 years, eight structures of bacterial MurA have been submitted to the Protein Data Bank and many inhibitors discovered. The present review discusses the structural and functional features of MurA of bacterial pathogens along with the development of MurA-targeted inhibitors.

Keywords: Antibiotic resistance; Peptidoglycan biosynthesis; UDP-N-acetylglucosamine enolpyruvyl transferase; Antibacterial chemotherapy; Inhibitors

Site-specific recombinases as tools for heterologous gene integration by Nobutaka Hirano; Tetsurou Muroi; Hideo Takahashi; Mitsuru Haruki (pp. 227-239).

Site-specific recombinases are the enzymes that catalyze site-specific recombination between two specific DNA sequences to mediate DNA integration, excision, resolution, or inversion and that play a pivotal role in the life cycles of many microorganisms including bacteria and bacteriophages. These enzymes are classified as tyrosine-type or serine-type recombinases based on whether a tyrosine or serine residue mediates catalysis. All known tyrosine-type recombinases catalyze the formation of a Holliday junction intermediate, whereas the catalytic mechanism of all known serine-type recombinases includes the 180° rotation and rejoining of cleaved substrate DNAs. Both recombinase families are further subdivided into two families; the tyrosine-type recombinases are subdivided by the recombination directionality, and the serine-type recombinases are subdivided by the protein size. Over more than two decades, many different site-specific recombinases have been applied to in vivo genome engineering, and some of them have been used successfully to mediate integration, deletion, or inversion in a wide variety of heterologous genomes, including those from bacteria to higher eukaryotes. Here, we review the recombination mechanisms of the best characterized recombinases in each site-specific recombinase family and recent advances in the application of these recombinases to genomic manipulation, especially manipulations involving site-specific gene integration into heterologous genomes.

Keywords: Genome engineering; Integration; Serine-type recombinase; Site-specific recombination; Tyrosine-type recombinase

Refolding of proteins from inclusion bodies: rational design and recipes by Anindya Basu; Xiang Li; Susanna Su Jan Leong (pp. 241-251).

The need to develop protein biomanufacturing platforms that can deliver proteins quickly and cost-effectively is ever more pressing. The rapid rate at which genomes can now be sequenced demands efficient protein production platforms for gene function identification. There is a continued need for the biotech industry to deliver new and more effective protein-based drugs to address new diseases. Bacterial production platforms have the advantage of high expression yields, but insoluble expression of many proteins necessitates the development of diverse and optimised refolding-based processes. Strategies employed to eliminate insoluble expression are reviewed, where it is concluded that inclusion bodies are difficult to eliminate for various reasons. Rational design of refolding systems and recipes are therefore needed to expedite production of recombinant proteins. This review article discusses efforts towards rational design of refolding systems and recipes, which can be guided by the development of refolding screening platforms that yield both qualitative and quantitative information on the progression of a given refolding process. The new opportunities presented by light scattering technologies for developing rational protein refolding buffer systems which in turn can be used to develop new process designs armed with better monitoring and controlling functionalities are discussed. The coupling of dynamic and static light scattering methodologies for incorporation into future bioprocess designs to ensure delivery of high-quality refolded proteins at faster rates is also discussed.

Keywords: Refolding; Inclusion bodies; Chromatography; Static light scattering; Dynamic light scattering

Extracellular metalloproteases from bacteria by Ji-Wei Wu; Xiu-Lan Chen (pp. 253-262).

Bacterial extracellular metalloproteases (BEMPs) are a large group of metal-containing proteases secreted by heterotrophic bacteria. In this review, the diversity, structural characteristics, mechanisms of maturation, physiological roles, and applications of BEMPs are described. BEMPs are distributed among nine families of metalloproteases because of differences in primary sequences and structural characteristics. Until now, all of the BEMPs identified have been endoproteases harboring one catalytic Zn²⁺ in the active centers. BEMPs are usually synthesized as inactive zymogens with a propeptide that is covalently linked to and inhibits the catalytic domain. The removal of the propeptides of BEMPs is dependent on other proteases or an autocleavage process. The main physiological function of BEMPs is to degrade environmental proteins and peptides for bacterial heterotrophic nutrition. As extracellular proteases, BEMPs vary greatly in enzymology properties to adapt to their respective environments. BEMPs have been widely used in the food and pharmaceutical industries. In order to broaden the application of BEMPs, it is essential to explore novel BEMPs and apply gene/protein engineering to improve the production and properties of promising BEMPs.

Keywords: Bacterial extracellular metalloproteases; Diversity; Structural characteristics; Maturation mechanisms; Properties; Applications

Biological souring and mitigation in oil reservoirs by Lisa M. Gieg; Tom R. Jack; Julia M. Foght (pp. 263-282).

Souring in oilfield systems is most commonly due to the action of sulfate-reducing prokaryotes, a diverse group of anaerobic microorganisms that respire sulfate and produce sulfide (the key souring agent) while oxidizing diverse electron donors. Such biological sulfide production is a detrimental, widespread phenomenon in the petroleum industry, occurring within oil reservoirs or in topside processing facilities, under low- and high-temperature conditions, and in onshore or offshore operations. Sulfate reducers can exist either indigenously in deep subsurface reservoirs or can be “inoculated” into a reservoir system during oilfield development (e.g., via drilling operations) or during the oil production phase. In the latter, souring most commonly occurs during water flooding, a secondary recovery strategy wherein water is injected to re-pressurize the reservoir and sweep the oil towards production wells to extend the production life of an oilfield. The water source and type of production operation can provide multiple components such as sulfate, labile carbon sources, and sulfate-reducing communities that influence whether oilfield souring occurs. Souring can be controlled by biocides, which can non-specifically suppress microbial populations, and by the addition of nitrate (and/or nitrite) that directly impacts the sulfate-reducing population by numerous competitive or inhibitory mechanisms. In this review, we report on the diversity of sulfate reducers associated with oil reservoirs, approaches for determining their presence and effects, the factors that control souring, and the approaches (along with the current understanding of their underlying mechanisms) that may be used to successfully mitigate souring in low-temperature and high-temperature oilfield operations.

Keywords: Souring; Sulfide; Sulfate-reducing prokaryotes; Oil reservoirs; Water flooding; Nitrate; Biocides

Species and material considerations in the formation and development of microalgal biofilms by Tyler E. Irving; D. Grant Allen (pp. 283-294).

The development of microalgal biofilms has received very limited study despite its relevance in the design of photobioreactors where film growth may be advantageous for biomass separation or disadvantageous in fouling surfaces. Here, the effects of species selection, species control, and substrate properties on biofilms of Scenedesmus obliquus and Chlorella vulgaris were investigated. Experiments were conducted in batch culture and in continuous culture modes in a flow cell. Cell growth was monitored using confocal laser scanning microscopy and gravimetrically. Species selection and species control had significant effects on biofilm development. On non-sterile wastewater, C. vulgaris shifted from primarily planktonic (23.7% attachment) to primarily sessile (79.8% attachment) growth. The biofilms that developed in non-sterile conditions were thicker (52 ± 19 μm) than those grown in sterile conditions (7 ± 6 μm). By contrast, S. obliquus attained similar thicknesses (54 ± 31 and 53 ± 38 μm) in both sterile and non-sterile conditions. Neither species was able to dominate a non-sterile biofilm. The effect of substrate surface properties was minimal. Both species grew films of similar thickness (∼30 μm for S. obliquus, <10 μm for C. vulgaris) on materials ranging from hydrophilic (glass) to hydrophobic (polytetrafluoroethylene). Surface roughness created by micropatterning the surface with 10 μm grooves did not translate into long-term increases in biofilm thickness. The results indicate that species selection and control are more important than surface properties in the development of microalgal biofilms.

Keywords: Microalgae; Photobioreactor; Biofilm; Flow cell; Confocal laser scanning microscopy (CLSM)

High-level production of poly (β-l-malic acid) with a new isolated Aureobasidium pullulans strain by Huili Zhang; Jin Cai; Jiaqi Dong; Danping Zhang; Lei Huang; Zhinan Xu; Peilin Cen (pp. 295-303).

Poly (β-l-malic acid) (PMLA) is a water-soluble polyester with many attractive properties in chemical industry and medicine development. However, the low titer of PMLA in the available producer strains limits further industrialization efforts and restricts its many potential applications. In order to solve this problem, a new strain with the distinguished high productivity of PMLA was isolated from fresh plants samples. It was characterized as the candidate of Aureobasidium pullulans based on the morphology and phylogenetic analyses of the internal transcribed spacer sequences. After the optimization of culture conditions, the highest PMLA concentration (62.27 g l⁻¹) could be achieved in the shake flask scale. In addition, the contribution of the carbon flux to exopolysaccharide (EPS) and PMLA could be regulated by the addition of CaCO₃ in the medium. This high-level fermentation process was further scaled up in the 10 l benchtop fermentor with a high PMLA concentration (57.2 g l⁻¹) and productivity (0.35 g l⁻¹ h⁻¹), which are the highest level in all the literature. Finally, the suitable acid hydrolysis conditions of PMLA were also investigated with regard to the production of l-malic acid, and the kinetics of PMLA acid hydrolysis was modeled to simulate the whole degradation process. The present work paved the road to produce this multifunctional biomaterial (PMLA) at industrial scale and promised one alternative method to produce l-malic acid in the future.

Keywords: Poly (β-l-malic acid); Aureobasidium pullulans ; l-malic acid; Exopolysaccharide

A novel low-temperature-active β-glucosidase from symbiotic Serratia sp. TN49 reveals four essential positions for substrate accommodation by Junpei Zhou; Rui Zhang; Pengjun Shi; Huoqing Huang; Kun Meng; Tiezheng Yuan; Peilong Yang; Bin Yao (pp. 305-315).

A 2,373-bp full-length gene (bglA49) encoding a 790-residue polypeptide (BglA49) with a calculated mass of 87.8 kDa was cloned from Serratia sp. TN49, a symbiotic bacterium isolated from the gut of longhorned beetle (Batocera horsfieldi) larvae. The deduced amino acid sequence of BglA49 showed the highest identities of 80.1% with a conceptually translated protein from Pantoea sp. At-9b (EEW02556), 38.3% with the identified glycoside hydrolase (GH) family 3 β-glucosidase from Clostridium stercorarium NCBI 11754 (CAB08072), and <15.0% with the low-temperature-active GH 3 β-glucosidases from Shewanella sp. G5 (ABL09836) and Paenibacillus sp. C7 (AAX35883). The recombinant enzyme (r-BglA49) was expressed in Escherichia coli and displayed the typical characteristics of low-temperature-active enzymes, such as low temperature optimum (showing apparent optimal activity at 35°C), activity at low temperatures (retaining ∼60% of its maximum activity at 20°C and ∼25% at 10°C). Compared with the thermophilic GH 3 β-glucosidase, r-BglA49 had fewer hydrogen bonds and salt bridges and less proline residues. These features might relate to the increased structure flexibility and higher catalytic activity at low temperatures of r-BglA49. The molecular docking study of four GH 3 β-glucosidases revealed five conserved positions contributing to substrate accommodation, among which four positions of r-BglA49 (R192, Y228, D260, and E449) were identified to be essential based on site-directed mutagenesis analysis.

Keywords: Longhorned beetle; Symbiotic Serratia sp.; Low-temperature-active β-glucosidase; Gut; Ligand docking

Molecular and biochemical characterization of a new alkaline β-propeller phytase from the insect symbiotic bacterium Janthinobacterium sp. TN115 by Rui Zhang; Peilong Yang; Huoqing Huang; Tiezheng Yuan; Pengjun Shi; Kun Meng; Bin Yao (pp. 317-325).

A phytase-encoding gene (phyA115) was cloned from Janthinobacterium sp. TN115, a symbiotic bacterial strain isolated from the gut contents of Batocera horsfieldi larvae (Coleoptera: Cerambycidae), and expressed in Escherichia coli. The 1,884-bp full-length gene encodes a 28-residue putative signal peptide and a 599-residue mature protein with a calculated mass of 64 kDa. The deduced PhyA115 shares low identity with known sequences (47% at most) and contains an N-terminal incomplete domain (residues 29–297; domain N) and a typical β-propeller phytase domain at the C terminus (residues 298–627; domain C). Distinct from other β-propeller phytases that have neutral pH optima (pH 6.0–7.5), purified recombinant PhyA115 exhibits maximal activity at pH 8.5 and 45°C in the presence of 1 mM Ca²⁺ and is highly active over a wider pH range (pH 6.0–9.0). These results indicate that PhyA115 is a β-propeller phytase that has application potential in aquaculture feed. To our knowledge, this is the first report of cloning of a phytase gene from the symbiotic microbes of an insect digestive tract and from the genus Janthinobacterium. The N-terminal incomplete domain is found to have no phytase activity but can influence the pH property of PhyA115.

Keywords: β-Propeller phytase; Janthinobacterium sp. TN115; Alkaline phytase; Batocera horsfieldi (Coleoptera: Cerambycidae)

High-level production of a kringle domain variant by high-cell-density cultivation of Escherichia coli by Seung Hoon Jang; Chang Han Lee; Yong Sung Kim; Ki Jun Jeong (pp. 327-336).

Human kringle domains (KDs) are ubiquitously expressed binding modulators that fold into seven flexible loops and it has been previously demonstrated that KDs can be engineered toward target-specific binding proteins as a non-antibody protein scaffold. Here, we report a method for efficient expression of a KD derivative (KD548)—a promising anti-cancer agent—by high-cell-density culture of Escherichia coli at a preparative scale production. The correct folding of KD548 requires three disulfide bonds. Nevertheless, cytoplasmic expression of KD548 in E. coli led to good yields of highly soluble proteins with high activity. For efficient expression, four sets of expression systems consisting of different promoters (lac or T7) and fusion tags (His or FLAG) were examined. Of these, the expression system using a combination of the T7 promoter with the FLAG tag resulted in the highest production in shake flask cultivation as well as in high-cell-density cultivation performed in a 6.6-L jar bioreactor. When protein expression was induced at high-cell density (optical density [OD] = 100) and when complex feeding solutions were supplemented, cell density (maximum OD = 184) and production yield (∼5.4 g/L) were significantly enhanced to values that were much higher than those found previously with Pichia cultivation (<8 mg/L).

Keywords: Kringle domain; Escherichia coli ; High-cell-density culture; Fusion tag

Overexpression of the ABC transporter AvtAB increases avermectin production in Streptomyces avermitilis by Jingfan Qiu; Ying Zhuo; Dongqing Zhu; Xiufen Zhou; Lixin Zhang; Linquan Bai; Zixin Deng (pp. 337-345).

Avermectins are 16-membered macrocyclic polyketides with potent antiparasitic activities, produced by Streptomyces avermitilis. Upstream of the avermectin biosynthetic gene cluster, there is the avtAB operon encoding the ABC transporter AvtAB, which is highly homologous to the mammalian multidrug efflux pump P-glycoprotein (Pgp). Inactivation of avtAB had no effect, but increasing the concentration of avtAB mRNA 30–500-fold, using a multi-copy plasmid in S. avermitilis, enhanced avermectin production about two-fold both in the wild-type and in a high-yield producer strain on agar plates. In liquid industrial fermentation medium, the overall productivity of avermectin B1a in the engineered high-yield producer was improved for about 50%, from 3.3 to 4.8 g/l. In liquid YMG medium, moreover, the ratio of intracellular to extracellular accumulation of avermectin B1a was dropped from 6:1 to 4.5:1 in response to multiple copies of avtAB. Additionally, the overexpression of avtAB did not cause any increased expression of the avermectin biosynthetic genes through RT-PCR analysis. We propose that the AvtAB transporter exports avermectin, and thus reduces the feedback inhibition on avermectin production inside the cell. This strategy may be useful for enhancing the production of other antibiotics.

Keywords: Antibiotic overproduction; Avermectin; Drug efflux pump; ABC transporter

Reconstruction and verification of a genome-scale metabolic model for Synechocystis sp. PCC6803 by Katsunori Yoshikawa; Yuta Kojima; Tsubasa Nakajima; Chikara Furusawa; Takashi Hirasawa; Hiroshi Shimizu (pp. 347-358).

In terms of generating sustainable energy resources, the prospect of producing energy and other useful materials using cyanobacteria has been attracting increasing attention since these processes require only carbon dioxide and solar energy. To establish production processes with a high productivity, in silico models to predict the metabolic activity of cyanobacteria are highly desired. In this study, we reconstructed a genome-scale metabolic model of the cyanobacterium Synechocystis sp. PCC6803, which included 465 metabolites and 493 metabolic reactions. Using this model, we performed constraint-based metabolic simulations to obtain metabolic flux profiles under various environmental conditions. We evaluated the simulated results by comparing these with experimental results from ¹³C-tracer metabolic flux analyses, which were obtained under heterotrophic and mixotrophic conditions. There was a good agreement of simulation and experimental results under both conditions. Furthermore, using our model, we evaluated the production of ethanol by Synechocystis sp. PCC6803, which enabled us to estimate quantitatively how its productivity depends on the environmental conditions. The genome-scale metabolic model provides useful information for the evaluation of the metabolic capabilities, and prediction of the metabolic characteristics, of Synechocystis sp. PCC6803.

Keywords: Synechocystis sp. PCC6803; Genome-scale metabolic model; Flux balance analysis

Willow volatiles influence growth, development, and secondary metabolism in Aspergillus parasiticus by Ludmila V. Roze; Anna V. Koptina; Maris Laivenieks; Randolph M. Beaudry; Daniel A. Jones; Albert V. Kanarsky; John E. Linz (pp. 359-370).

Aflatoxin is a mycotoxin and the most potent naturally occurring carcinogen in many animals. Aflatoxin contamination of food and feed crops causes a significant global burden on human and animal health. However, available methods to eliminate aflatoxin from food and feed are not fully effective. Our goal is to discover novel, efficient, and practical methods to control aflatoxin contamination in crops during storage. In the present study, we tested the effect of volatiles produced by willow (Salix acutifolia and Salix babylonica) and maple (Acer saccharinum) bark on fungal growth, development, and aflatoxin production by the fungus Aspergillus parasiticus, one economically important aflatoxin producer. S. acutifolia bark volatiles nearly eliminated aflatoxin accumulation (>90% reduction) by A. parasiticus grown on a minimal agar medium. The decrease in aflatoxin accumulation correlated with a twofold reduction in ver-1 (encodes a middle aflatoxin pathway enzyme) transcript level. Expression data also indicate that one histone H4 acetyltransferase, MYST3, may play a role in epigenetic control of aflatoxin gene transcription in response to volatile exposure. Volatiles derived from wood bark samples also increased fungal growth up to 20% and/or enhanced conidiospore development. Solid-phase microextraction–gas chromatographic–mass spectrometric analysis of bark samples identified sets of shared and unique volatile compounds that may mediate the observed regulatory effects on growth, development, and aflatoxin synthesis. This work provides an experimental basis for the use of willow industry by-products to control aflatoxin contamination in food and feed crops.

Keywords: Aspergillus parasiticus ; Aflatoxin; Willow volatiles; Salix acutifolia ; Aflatoxin genes; Gas chromatography/mass spectrometry

Optimization of fermentation medium for triterpenoid production from Antrodia camphorata ATCC 200183 using artificial intelligence-based techniques by Zhen-Ming Lu; Jian-Yong Lei; Hong-Yu Xu; Jing-Song Shi; Zheng-Hong Xu (pp. 371-379).

In this study, alteration in morphology of submergedly cultured Antrodia camphorata ATCC 200183 including arthroconidia, mycelia, external and internal structures of pellets was investigated. Two optimization models namely response surface methodology (RSM) and artificial neural network (ANN) were built to optimize the inoculum size and medium components for intracellular triterpenoid production from A. camphorata. Root mean squares error, R ², and standard error of prediction given by ANN model were 0.31%, 0.99%, and 0.63%, respectively, while RSM model gave 1.02%, 0.98%, and 2.08%, which indicated that fitness and prediction accuracy of ANN model was higher when compared to RSM model. Furthermore, using genetic algorithm (GA), the input space of ANN model was optimized, and maximum triterpenoid production of 62.84 mg l⁻¹ was obtained at the GA-optimized concentrations of arthroconidia (1.78 × 10⁵ ml⁻¹) and medium components (glucose, 25.25 g l⁻¹; peptone, 4.48 g l⁻¹; and soybean flour, 2.74 g l⁻¹). The triterpenoid production experimentally obtained using the ANN–GA designed medium was 64.79 ± 2.32 mg l⁻¹ which was in agreement with the predicted value. The same optimization process may be used to optimize many environmental and genetic factors such as temperature and agitation that can also affect the triterpenoid production from A. camphorata and to improve the production of bioactive metabolites from potent medicinal fungi by changing the fermentation parameters.

Keywords: Antrodia camphorata ; Artificial neural network; Genetic algorithm; Media optimization; Response surface methodology; Triterpenoid

Optimization of fixation methods for observation of bacterial cell morphology and surface ultrastructures by atomic force microscopy by Yuanqing Chao; Tong Zhang (pp. 381-392).

Fixation ability of five common fixation solutions, including 2.5% glutaraldehyde, 10% formalin, 4% paraformaldehyde, methanol/acetone (1:1), and ethanol/acetic acid (3:1) were evaluated by using atomic force microscopy in the present study. Three model bacteria, i.e., Escherichia coli, Pseudomonas putida, and Bacillus subtilis were applied to observe the above fixation methods for the morphology preservation of bacterial cells and surface ultrastructures. All the fixation methods could effectively preserve cell morphology. However, for preserving bacterial surface ultrastructures, the methods applying aldehyde fixations performed much better than those using alcohols, since the alcohols could detach the surface filaments (i.e., flagella and pili) significantly. Based on the quantitative and qualitative assessments, the 2.5% glutaraldehyde was proposed as a promising fixation solution both for observing morphology of both bacterial cell and surface ultrastructures, while the methonal/acetone mixture was the worst fixation solution which may obtain unreliable results.

Keywords: Fixation methods; Cell morphology; Ultrastructure; Atomic force microscopy

Molecular determinants of azo reduction activity in the strain Pseudomonas putida MET94 by Sónia Mendes; Luciana Pereira; Carlos Batista; Lígia O. Martins (pp. 393-405).

Azo dyes are the major group of synthetic colourants used in industry and are serious environmental pollutants. In this study, Pseudomonas putida MET94 was selected from 48 bacterial strains on the basis of its superior ability to degrade a wide range of structurally diverse azo dyes. P. putida is a versatile microorganism with a well-recognised potential for biodegradation or bioremediation applications. P. putida MET94 removes, in 24 h and under anaerobic growing conditions, more than 80% of the majority of the structurally diverse azo dyes tested. Whole cell assays performed under anaerobic conditions revealed up to 90% decolourisation in dye wastewater bath models. The involvement of a FMN dependent NADPH: dye oxidoreductase in the decolourisation process was suggested by enzymatic measurements in cell crude extracts. The gene encoding a putative azoreductase was cloned from P. putida MET94 and expressed in Escherichia coli. The purified P. putida azoreductase is a 40 kDa homodimer with broad substrate specificity for azo dye reduction. The presence of dioxygen leads to the inhibition of the decolourisation activity in agreement with the results of cell cultures. The kinetic mechanism follows a ping-pong bi–bi reaction scheme and aromatic amine products were detected in stoichiometric amounts by high-performance liquid chromatography. Overall, the results indicate that P. putida MET94 is a promising candidate for bioengineering studies aimed at generating more effective dye-reducing strains.

Keywords: Pseudomonas putida ; Azoreductase; Azo dyes; Decolourisation; Whole cell catalysis

Effects of entrapment on nucleic acid content, cell morphology, cell surface property, and stress of pure cultures commonly found in biological wastewater treatment by Sudipta Pramanik; Rohit Khanna; Kalpana Katti; John McEvoy; Eakalak Khan (pp. 407-418).

The effects of cell entrapment on nucleic acid content, cell morphology, cell surface property, and stress of major groups of bacteria (betaproteobacteria and gammaproteobacteria) in biological municipal wastewater treatment were investigated. Three different entrapment media (alginate, carrageenan, and polyvinyl alcohol) were examined. Results indicated that the entrapment and type of entrapment media affected nucleic acid content, cell morphology, cell surface property, and stress of the three representative species (Alcaligenes faecalis, Comamonas testosteroni, and Pseudomonas putida) studied. The highest deoxyribonucleic acid and ribonucleic acid increases were observed with the alginate and polyvinyl alcohol (PVA) entrapment, respectively. A cell morphological change from bacilli to coccoidal was observed in the case of alginate entrapment while the PVA-entrapped cells had a slim morphology when compared to non-entrapped cells and formed putative nanowires. The entrapment increased or decreased the surface roughness of cells depending on the type of entrapment media. Expression of a nitrosative stress gene, which is linked to oxygen deprivation, was observed more in the alginate-entrapped cells. These research findings advance the fundamental understanding of the entrapped cell physiology which can lead to more efficient entrapped cell-based wastewater treatment.

Keywords: Entrapped cells; Nanowire; Physiology; Stress; Wastewater treatment

Operational temperature regulates anodic biofilm growth and the development of electrogenic activity by Iain S. Michie; Jung Rae Kim; Richard M. Dinsdale; Alan J. Guwy; Giuliano C. Premier (pp. 419-430).

The operational temperature of microbial fuel cell reactors influences biofilm development, and this has an impact on anodic biocatalytic activity. In this study, we compared three microbial fuel cell (MFC) reactors acclimated at 10°C, 20°C and 35°C to investigate the effect on biomass development, methanogenesis and electrogenic activity over time. The start-up time was inversely influenced by temperature, but the amount of biomass accumulation increased with increased temperatures, the 10°C, 20°C and 35°C acclimated biofilms resulted in 0.57, 0.82 and 5.43 g biomass (volatile suspended solids) per litre respectively at 56 weeks of operation. Biofilm build-up on the 35°C anode was further demonstrated by scanning electron microscopy, which showed large aggregations of biomass accumulating on the anode when compared to 10°C and 20°C biofilms. Biomass accumulation had a direct impact on biocatalytic performance, with the maximum power at 35°C after 60 weeks of operation being 2.14 W m⁻³ and power densities for the 10°C and 20°C reactors being and 4.29 W m⁻³. Methanogenic activity was also shown to be higher at 35°C, with a rate of 10.1 mmol CH₄ biofilm per gram of volatile suspended solid (VSS) per day, compared to 0.28 mmol CH₄ per gram of VSS per day produced at 20°C. These results demonstrate that higher MFC operating temperatures could be detrimental to the biocatalytic performance of electrochemically active bacteria in anodic biofilms due to biomass accumulation with enhanced development of non-electrogenic communities (e.g. methanogens and fermenters), meaning that, over time, psychro- or mesophilic operation can have beneficial effects for the development of electrogenically active populations in the reactor.

Keywords: Microbial fuel cell (MFC); Tubular reactor; Biofilm; Temperature; Electricity generation

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Applied Microbiology and Biotechnology (v.92, #2)

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Applied Microbiology and Biotechnology (v.92, #2)