Application of Biofilm-Forming Bacteria on the Enhancement of Organophosphorus Fungicide Degradation

This study aimed to develop technology enhancing the biodegradation efficacy against organophosphorus fungicide with biofilm-forming bacteria in situ. Using the crystal violet staining method, two bacterial strains having biofilm formation capability were isolated and identified as Pseudomonas sp. C7 and Bacillus sp. E5. Compared with the culture of tolclofos-methyl degrader Sphingomonas sp. 224, biofilm formation was improved by co-inoculation with biofilm-forming bacterium Bacillus sp. E5. Evaluated in liquid culture conditions, this two-species mixed consortium was observed to degrade tolclofos-methyl more effectively than Sphingomonas sp. 224 alone, with an approximately 90% degradation efficiency within 48 h of dosing. The improved effectiveness of the consortium biofilm was reflected using soil in situ with an approximately 7% increased degradation ratio over Sphingomonas sp. 224 alone. This is the first report demonstrating improved bioremediation degradation efficacy against tolclofos-methyl exhibited by a consortium biofilm. This work presents a possible effective bioremediation strategy using a specific biofilm composition against pollutants containing organophosphorus compounds in situ.     

Screening of a hypersaline cyanobacterium, <Emphasis Type="Italic">Phormidium</Emphasis><Emphasis Type="Italic">tenue,</Emphasis> for the degradation of aromatic hydrocarbons: naphthalene and anthracene

Hypersaline Phormidium strains were grown in media amended with naphthalene and anthracene. Phormidium tenue was identified as tolerating and effectively degrading polycyclic aromatic hydrocarbons that may be toxic in the environment. GC/MS analysis explained the degradation of these compounds by P. tenue. A dioxygenase enzyme system was evident by the formation of anthracene dione as the first degradation compound. This strain could be used for bioremediation of polycyclic aromatic hydorcarbon pollution on seashores.     

Physiological and metabolic responses for hexadecane degradation in <Emphasis Type="Italic">Acinetobacter oleivorans</Emphasis> DR1

The hexadecane degradation of Acinetobacter oleivorans DR1 was evaluated with changes in temperature and ionic salt contents. Hexadecane degradation of strain DR1 was reduced markedly by the presence of sodium chloride (but not potassium chloride). High temperature (37°C) was also shown to inhibit the motility, biofilm formation, and hexadecane biodégradation. The biofilm formation of strain DR1 on the oil-water interface might prove to be a critical physiological feature for the degradation of hexadecane. The positive relationship between biofilm formation and hexadecane degradation could be observed at 30° C, but not at low temperatures (25°C). Alterations in cell hydrophobicity and EPS production by temperature and salts were not correlated with biofilm formation and hexadecane degradation. Our proteomic analyses have demonstrated that metabolic changes through the glyoxylate pathway are important for efficient degradation of hexadecane. Proteins involved in fatty acid metabolism, gluconeogenesis, and oxidative stress defense proteins appear to be highly expressed during biodégradation of hexadecane. These results suggested that biofilm formation and oxidative stress defense are important physiological responses for hexadecane degradation along with metabolic switch to glyoxylate pathway in strain DR1.     

An extractive membrane biofilm reactor as alternative technology for the treatment of methyl tert‐butyl ether contaminated water

Among the strategies developed for contaminated groundwater bioremediation, those based on the use of bacteria adhering to inert supports and establishing biofilms have gained great importance in this field. Extractive membrane biofilm reactor (EMBFR) technology offers productive solutions for the removal of volatile and semi‐volatile compounds. EMBFR technology is based on the use of extractive semipermeable membranes through which contaminants migrate to the biological compartment in which microorganisms with pollutant biotransformation and/or mineralization capacities can grow, forming an active biofilm on the membrane surface. The objective of this study was to assess the use of three bacterial strains (Paenibacillus sp. SH7 CECT 8558, Agrobacterium sp. MS2 CECT 8557, and Rhodococcus ruber EE6 CECT 8612), as inoculum in a lab‐scale EMBFR running for 28 days under aerobic conditions to eliminate methyl tert‐butyl ether (MTBE) from water samples. Three different hydraulic retention times (1, 6, and 12 h) were employed. MTBE degradation values were determined daily by a gas GC‐MS technique, as well as suspended bacterial growth. The biofilm established by the bacterial strains on the semipermeable membrane was detected by Field‐Emission Scanning Electron Microscopy (FESEM) at the end of each experiment. The acute toxicity of the treated effluents and biomedium was determined by Microtox^© assay (EC₅₀).The results achieved from the MTBE degradation, biofilm formation, and toxicity analysis indicated that bacterial strains MS2 and EE6 were the best options as selective inoculum, although further research is needed, particularly with regard to their possible use as a mixed culture. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1238–1245, 2016     

Characterization and degradation potential of diesel-degrading bacterial strains for application in bioremediation

Bioremediation of polluted soils is a promising technique with low environmental impact, which uses soil organisms to degrade soil contaminants. In this study, 19 bacterial strains isolated from a diesel-contaminated soil were screened for their diesel-degrading potential, biosurfactant (BS) production, and biofilm formation abilities, all desirable characteristics when selecting strains for re-inoculation into hydrocarbon-contaminated soils. Diesel-degradation rates were determined in vitro in minimal medium with diesel as the sole carbon source. The capacity to degrade diesel range organics (DROs) of strains SPG23 (Arthobacter sp.) and PF1 (Acinetobacter oleivorans) reached 17–26% of total DROs after 10 days, and 90% for strain GK2 (Acinetobacter calcoaceticus). The amount and rate of alkane degradation decreased significantly with increasing carbon number for strains SPG23 and PF1. Strain GK2, which produced BSs and biofilms, exhibited a greater extent, and faster rate of alkane degradation compared to SPG23 and PF1. Based on the outcomes of degradation experiments, in addition to BS production, biofilm formation capacities, and previous genome characterizations, strain GK2 is a promising candidate for microbial-assisted phytoremediation of diesel-contaminated soils. These results are of particular interest to select suitable strains for bioremediation, not only presenting high diesel-degradation rates, but also other characteristics which could improve rhizosphere colonization.     

Genetic analysis of benzoate metabolism in Aspergillus niger

Summary The benzoate metabolism of Aspergillus niger was studied as part of a design to clone the benzoate-4-hydroxylase gene of this fungus on the basis of complementation. Filtration enrichment techniques yielded mutants defective for different steps of benzoate degradation: bph (benzoate-4-hydroxylase), phh (4-hydroxybenzoate-3-hydroxylase) and prc (protocatechuate ring cleavage) mutants. In this way the degradation pathway for benzoate, involving the formation of 4-hydroxybenzoate and 3,4-dihydroxybenzoate has been confirmed. In addition a mutant sensitive to benzoate has been found. Complementation tests in somatic diploids showed that the bph mutants belonged to two complementation groups. The major group is probably defective in the structural gene (bphA). All phh mutants tested belonged to one complementation group. The prc mutants could be divided into several groups on the basis of their growth on different aromatic substrates and on the basis of the complementation test. The phh and both bph mutations are shown to be located on different chromosomes.Offprint requests to: C. J. Bos     

奥奈达希瓦氏菌电活性生物被膜的研究进展

面对日益严峻的能源紧缺与环境污染形势,电活性微生物(electroactive microorganisms)的电催化过程为实现绿色生产提供了新的思路。奥奈达希瓦氏菌具有独特的呼吸方式和电子传递能力,在微生物燃料电池、增值化学品的生物电合成、金属废物处理和环境修复系统等领域有着广泛的应用。奥奈达希瓦氏菌(Shewanella oneidensis MR-1)电活性生物被膜是实现电活性微生物电子传递过程的优良载体,其形成过程十分复杂且受到多种因素的影响和调控,在增强细菌环境抗逆性、提高电子传递效率等多方面发挥着十分重要的作用。本文较为系统地综述了奥奈达希瓦氏菌生物被膜的形成过程、影响因素及其在生物能源、生物修复和生物传感中的相关应用,为进一步实现其在更多领域的应用提供了理论基础。     

Dependence of the hydrolytic exoenzyme production level on the way of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> culturing

An 867 mutant has been derived from a B. subtilis 1560 strain capable of biofilm formation during stationary culturing. The mutant was able to form more elastic and resistant to mechanical stress biofilms that could be used in fermentation more than once. Analysis of the hydrolytic activity of the 1560 and 867 strains film and submerged cultures showed that the biofilm culturing permitted to enhance the levels of amylase by 1.5–2.4 times and that of glucanase by three times.     

The staphylococcal nuclease prevents biofilm formation in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and other biofilm-forming bacteria

The staphylococcal nuclease, encoded by the nuc1 gene, is an important virulence factor of Staphylococcus aureus. However, the physiological role of the nuclease has not been fully characterized. The current study observed that biofilm development could be prevented in staphylococcal nuclease-producing strains of S. aureus; however, when the nuc1 gene was knocked out, the ability to form a biofilm significantly increased. Scanning electron and confocal scanning laser microscopy were used to evaluate the role of the nuc1 gene in biofilm formation. Moreover, the nuc1 gene product, staphylococcal nuclease, and recombinant NUC1 protein were found to have a visible effect on other biofilm-forming bacteria, such as Pseudomonas aeruginosa, Actinobacillus pleuropneumoniae, and Haemophilus parasuis. The current study showed a direct relationship between staphylococcal nuclease production and the prevention of biofilm development. The findings from this study underscore the important role of staphylococcal nuclease activity to prevent biofilm formation in S. aureus. They also provided evidence for the biological role of staphylococcal nucleases in other organisms.     

Shear stress effects on production of exopolymeric substances and biofilm characteristics during phenol biodegradation by immobilized Pseudomonas desmolyticum (NCIM2112) cells in a pulsed plate bioreactor

This article reports studies on a continuous pulsed plate bioreactor (PPBR) with the cells of Pseudomonas desmolyticum (NCIM2112) immobilized on granular activated carbon (GAC) used as a biofilm reactor for biodegradation of phenol. Almost complete removal of 200 ppm phenol could be achieved in this bioreactor. Biofilm structure and characteristics are influenced by hydrodynamic and shear conditions in bioreactors. In this article, the effect of shear stress induced by frequency of pulsation on biofilm characteristics during the startup period in the PPBR is reported. The startup time decreased with the increase in frequency of pulsation. The formation of biofilm in PPBR was found to have three phases: accumulation, compaction, and plateau. The effect of frequency on production of exoploymeric substances (EPS) such as, protein, carbohydrate, and humic substance is reported. An increase in shear stress induced by the frequency of pulsation increased the production of exopolymeric substances in the biofilm during startup of the bioreactor. Increase in shear stress caused a decrease in biofilm thickness and an increase in dry density of the biofilm. Increase in shear stress resulted in a smoother and thinner biofilm surface with more compact and dense structure.     

Towards non‐invasive monitoring of pathogen–host interactions during Candida albicans biofilm formation using in vivo bioluminescence

Candida albicans is a major human fungal pathogen causing mucosal and deep tissue infections of which the majority is associated with biofilm formation on medical implants. Biofilms have a huge impact on public health, as fungal biofilms are highly resistant against most antimycotics. Animal models of biofilm formation are indispensable for improving our understanding of biofilm development inside the host, their antifungal resistance and their interaction with the host immune defence system. In currently used models, evaluation of biofilm development or the efficacy of antifungal treatment is limited to ex vivo analyses, requiring host sacrifice, which excludes longitudinal monitoring of dynamic processes during biofilm formation in the live host. In this study, we have demonstrated for the first time that non‐invasive, dynamic imaging and quantification of in vitro and in vivo C. albicans biofilm formation including morphogenesis from the yeast to hyphae state is feasible by using growth‐phase dependent bioluminescent C. albicans strains in a subcutaneous catheter model in rodents. We have shown the defect in biofilm formation of a bioluminescent bcr1 mutant strain. This approach has immediate applications for the screening and validation ofantimycotics under in vivo conditions, for studying host–biofilm interactions in different transgenic mouse models and for testing the virulence of luminescent C. albicans mutants, hereby contributing to a better understanding of the pathogenesis of biofilm‐associated yeast infections.     

Bacterial biofilms and quorum sensing: fidelity in bioremediation technology

Increased contamination of the environment with toxic pollutants has paved the way for efficient strategies which can be implemented for environmental restoration. The major problem with conventional methods used for cleaning of pollutants is inefficiency and high economic costs. Bioremediation is a growing technology having advanced potential of cleaning pollutants. Biofilm formed by various micro-organisms potentially provide a suitable microenvironment for efficient bioremediation processes. High cell density and stress resistance properties of the biofilm environment provide opportunities for efficient metabolism of number of hydrophobic and toxic compounds. Bacterial biofilm formation is often regulated by quorum sensing (QS) which is a population density-based cell–cell communication process via signaling molecules. Numerous signaling molecules such as acyl homoserine lactones, peptides, autoinducer-2, diffusion signaling factors, and α-hydroxyketones have been studied in bacteria. Genetic alteration of QS machinery can be useful to modulate vital characters valuable for environmental applications such as biofilm formation, biosurfactant production, exopolysaccharide synthesis, horizontal gene transfer, catabolic gene expression, motility, and chemotaxis. These qualities are imperative for bacteria during degradation or detoxification of any pollutant. QS signals can be used for the fabrication of engineered biofilms with enhanced degradation kinetics. This review discusses the connection between QS and biofilm formation by bacteria in relation to bioremediation technology.     

Enhanced biotransformation of mononitrophenols by Stenotrophomonas maltophilia KB2 in the presence of aromatic compounds of plant origin

Stenotrophomonas maltophilia KB2 used in this study is known to metabolise broad range of aromatic compounds including phenol, some chloro and methylphenols, benzoic acids, catochols and others. To study the applicability of the strain for degradation of mononitrophenols in monosubstrate as well as cometabolic systems its degradation potential in the presence of mononitrophenols or different aromatic compounds of plant origin was tested. Stenotrophomonas maltophilia KB2 strain was not able to degrade any of mononitrophenols used in the single substrate experiments. Effect of additional carbon source on nitrophenols degradation revealed that presence of benzoate, 4-hydroxybenzoate or 3,4-dixydroxybenzoate stimulate transformation of 2-nitrophenol, 3-nitrophenol as well as 4-nitrophenol. Depending on growth substrate and mononitrophenol used, decrease in cometabolite concentration was from 25 to 45%. Obtained results suggest that Stenotrophomonas maltophilia KB2 strain could be potentially used for cometabolic degradation of nitrophenols in the presence of aromatic acids, for the bioremediation of contaminated sites.     

Inhibition of biofilm formation by the antisense peptide nucleic acids targeted at the <Emphasis Type="Italic">motA</Emphasis> gene in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1 strain

Pseudomonas aeruginosa is a ubiquitous bacterium which is able to attach to many abiotic and biotic surfaces and form biofilms resulting in infections. The motA gene was an essential gene in the early phase of biofilm formation of P. aeruginosa PAO1. In this study, antisense peptide nucleic acids (PNAs) and PNAs conjugated with the peptide (KFF)₃K were used to investigate whether they could mediate gene-specific antisense effects in P. aeruginosa PAO1. We found that antisense (KFF)₃K-PNA targeted at motA gene could inhibit biofilm formation in P. aeruginosa PAO1 in a dose-dependent manner. The minimal effective concentration of this antisense agent was 1 μmol l⁻¹, and the inhibited effect could last for at least 8 h. When compared with the control group, the value of OD570 of P. aeruginosa PAO1 reduced apparently when treated with (KFF)₃K-PNA. The expression of motA was sharply reduced when treated with (KFF)₃K-PNA, but reduced slightly when treated with PNA, and had no reduction when treated with (KFF)₃K. Our results demonstrated that the cell-penetrating peptide of (KFF)₃K improved significantly the antisense inhibition effect of PNA. The (KFF)₃K-PNA conjugates might be used as antisense agent for inhibition of the biofilm formation. This provides exciting possibility for developing new tool for microbial genetic treatment.     

Metabolic indicators for detecting in situanaerobic alkylbenzene degradation

Monitoring programs for intrinsic bioremediation of fuel hydrocarbonsrequire indicators that can convincingly demonstrate in situ metabolism. In this evaluation of potential indicators of in situ anaerobic alkylbenzene metabolism, laboratory and field data are reviewed for two classes of aromatic acids: (i) benzylsuccinate, E-phenylitaconate, and their methyl homologs, and (ii) benzoate, and methyl-, dimethyl-, and trimethylbenzoates. The review includes previously unpublished field data from a hydrocarbon-contaminated site in Fallon (Nevada), at which both classes of metabolites were detected in groundwater. The two classes of compounds were evaluated with respect to specificity (i.e., unique biochemical relationship to a specific alkylbenzene), stability, and generation as degradation intermediates versus dead-end products; recent developments in the biochemistry of anaerobic toluene and xylene degradation were incorporated in this evaluation. In general, benzylsuccinates/E-phenylitaconates are superior to benzoates in terms of their very high specificity to their parent hydrocarbons and their lack of commercial and industrial sources. They are also uniquely indicative of anaerobic conditions. All of the benzoates, benzylsuccinates, and E-phenylitaconates are relatively stable chemically and (with the exceptionof benzoate) biologically under anaerobic conditions, based on the limited data available. Although benzoate, benzylsuccinate, and E-phenylitaconate are intermediates of anaerobic toluene mineralization to carbon dioxide, their methyl homologs can be either mineralization intermediates or cometabolic dead-end products of alkylbenzenes, depending on the bacteria involved. Benzoates are far more commonly reported in field studies of hydrocarbon-contaminated aquifers than are benzylsuccinates and E-phenylitaconates, although it is not clear whether this is an accurate representation of the relative occurrenceof these metabolites at contaminated sites, or whether it instead reflects the limited range of target analytes used in most field studies to date.     

Degradation of chlorotoluenes and chlorobenzenes by the dual-species biofilm of Comamonas testosteroni strain KT5 and Bacillus subtilis strain DKT

The cooperation of Bacillus subtilis strain DKT and Comamonas testosteroni KT5 was investigated for biofilm development and toluenes and chlorobenzenes degradation. Bacillus subtilis strain DKT and C. testosteroni KT5 were co-cultured in liquid media with toluenes and chlorobenzenes to determine the degradation of these substrates and formation of dual-species biofilm used for the degradation process. Bacillus subtilis strain DKT utilized benzene, mono- and dichlorinated benzenes as carbon and energy sources. The catabolism of chlorobenzenes was via hydroxylation, in which chlorine atoms were replaced by hydroxyl groups to form catechol, followed by ring fission via the ortho-cleavage pathway. The investigation of the dual-species biofilm composed of B. subtilis DKT and C. testosteroni KT5 (a toluene and chlorotoluene-degrading isolate with low biofilm formation) showed that B. subtilis DKT synergistically promoted C. testosteroni KT5 to develop biofilm. The bacterial growth in dual-species biofilm overcame the inhibitory effects caused by monochlorobenzene and 2-chlorotoluene. Moreover, the dual-species biofilm showed effective degradability toward the mixture of these substrates. This study provides knowledge about the commensal relationships in a dual-culture biofilm for designing multispecies biofilms applied for the biodegradation of toxic organic substrates that cannot be metabolized by single-organism biofilms.

     

Formation of <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> biofilms on multiple surfaces on <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Summary A liquid-based assay was used to evaluate the ability of Yersinia pseudotuberculosis to form a bacterial biofilm on the nematode Caenorhabditis elegans. After 3 days of incubation in the liquid assay a biofilm was clearly visible by light microscopy on both the head and vulva region of the worms. At times, the biofilm formation on the vulva appeared to prevent the laying of eggs by the adult hermaphrodite; the eggs would later hatch inside of the worm. One possible explanation for the biofilm formation observed on the vulva may be the increased motion of the cuticle surrounding the vulva when the worm is immersed in a liquid culture. This is the first report of biofilm formation on the vulva of C. elegans.     

Probiotic Lactobacilli Interfere with <Emphasis Type="Italic">Streptococcus mutans</Emphasis> Biofilm Formation In Vitro

In clinical studies, probiotic bacteria have decreased the counts of salivary mutans streptococci (MS). We compared the effects of probiotic Lactobacillus strains on the biofilm formation of Streptococcus mutans. The bacterial strains used included four S. mutans strains (reference strains NCTC 10449 and Ingbritt and clinical isolates 2366 and 195) and probiotic strains Lactobacillus rhamnosus GG, L. plantarum 299v, and L. reuteri strains PTA 5289 and SD2112. The ability of MS to adhere and grow on a glass surface, reflecting biofilm formation, was studied in the presence of the lactobacilli (LB). The effect of LB culture supernatants on the viability of the MS was studied as well. All of the LB inhibited the biofilm formation of the clinical isolates of MS (P < 0.001). The biofilm formation of the reference strains of MS was also inhibited by the LB, but L. plantarum and L. reuteri PTA 5289 showed a weaker inhibition when compared to L. reuteri SD2112 and L. rhamnosus GG. Viable S. mutans cells could be detected in the biofilms and culture media only when the experiments were performed with the L. reuteri strains. The L. reuteri strains were less efficient in killing the MS also in the tests performed with the culture supernatants. The pHs of the supernatants of L. reuteri were higher compared to those of L. rhamnosus GG and L. plantarum; P < 0.001. In conclusion, our results demonstrated that four commonly used probiotics interfered with S. mutans biofilm formation in vitro, and that the antimicrobial activity against S. mutans was pH-dependent.