Enhancing polypropylene bioconversion and lipogenesis by Yarrowia lipolytica using a chemical/biological hybrid process.

Plastic waste can serve as a feedstock for microbial bioconversion using a chemical/biological hybrid strategy. We developed a polypropylene (PP) upcycling process that coupled pyrolysis with bioconversion by the oleaginous yeast Yarrowia lipolytica. Using virgin PP, we optimized pH, inoculum density, C/N ratio, and osmolarity and increased the fatty acid titer nearly four-fold to 1.9 g L-1, with 41 percent cellular fatty acid content, the highest content reported to date for plastic-to-lipid microbial bioconversion. The highest fatty acid titer was achieved with an inoculum density of 3 (OD 600 nm), pH = 6.0 and C/N ratio of 80:1. Increasing the medium osmolarity by adding sodium chloride adversely affected cell growth and did not improve the fatty acid titer. The maximum fatty acid titer occurred under conditions that balanced cell growth versus lipogenesis. Using postconsumer PP, the fatty acid titer was significantly lower (0.13 g L-1). Overall, the work demonstrates the potential and the challenges associated with microbial bioconversion of plastics.

4-Ethoxybenzoic acid inhibits Staphylococcus aureus biofilm formation and potentiates biofilm sensitivity to vancomycin.

The adverse health effects of Staphylococcus aureus biofilm infections coupled with an increased global prevalence of antibiotic resistance highlight the need for novel anti-pathogenic, anti-biofilm compounds. The authors recently determined that ethyl-4-ethoxybenzoic acid (EEB) had anti-pathogenic, anti-biofilm activity. Based on this finding, a structure-activity analysis was undertaken to identify more effective compounds. Microtitre crystal violet assays followed by plate counts were conducted to measure the dose-dependent anti-biofilm and antimicrobial activities of 13 phenolic compounds related to EEB. By displaying these characteristics on a two-component plot, 4-ethoxybenzoic acid (4EB) and methyl gallate were identified as two anti-pathogenic, anti-biofilm compounds of interest. To characterize their mechanisms of activity, their effects on cell hydrophobicity, hemolysis activity, membrane integrity, extracellular polymeric substance production and vancomycin sensitivity were examined. Both 4EB and methyl gallate inhibited up to 87% of biofilm formation with minimal impact on the viability of stationary-phase cells or bacterial growth. Combination treatments of 4EB and vancomycin decreased the viability of biofilm-dwelling cells by up to 85% compared with vancomycin alone, indicating a synergistic effect. Methyl gallate did not potentiate vancomycin. 4EB decreased the percentage of hydrophobic cells in culture from 78% to 49%, indicating that 4EB may prevent biofilm formation by altering cell membrane hydrophobicity. These findings suggest that 4EB has potential as an anti-pathogenic, anti-biofilm agent for the prevention of S. aureus biofilms, or as a treatment for established biofilms when combined with antibiotics.

Microbial bioconversion of thermally depolymerized polypropylene by Yarrowia lipolytica for fatty acid production.

Plastic production and waste generation will continue to rise as nations worldwide grow economically. In this work, we detail a pyrolysis-based bioconversion process for polypropylene (PP) to produce value-added fatty acids (FAs). PP pellets were depolymerized by pyrolysis, generating oil that consisted of mainly branched chain fatty alcohols and alkenes. The oil was mixed with biodegradable surfactants and trace nutrients and mechanically homogenized. The resulting medium, OP4, was used for fermentation by Yarrowia lipolytica strain 78-003. Y. lipolytica assimilated > 80% of the substrate over 312 h, including 86% of the fatty alcohols. Y. lipolytica produced up to 492 mg L-1 lipids, compared with 216 mg L-1 during growth in surfactant-based control medium. C 18 compounds, including oleic acid, linoleic acid, and stearic acid, were the predominant products, followed by C 16 compounds palmitic and palmitoleic acids. Two percent of the products was C 20 compounds. The majority of the products were unsaturated FAs. Growth on hydrophobic substrates (OP4 medium, hexadecane) was compared with growth on hydrophilic substrates (glucose, starch). The resulting FA profiles revealed an absence of short-chain fatty acids during growth on hydrophobic media, findings consistent with ex novo FA biosynthesis. Overall, FA profiles by Y. lipolytica during growth in OP4 medium were similar to FA profiles while growing on natural substrates. The process described here offers an alternative approach to managing postconsumer plastic waste.

Rhamnus prinoides (gesho): A source of diverse anti-biofilm activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhamnus prinoides (gesho) is an evergreen shrub from East Africa traditionally used for the treatment of illnesses including atopic dermatitis, ear, nose and throat infections, pneumonia, arthritis, brucellosis, flu, indigestion and fatigue. AIM OF THE STUDY: Several of the conditions for which gesho is traditionally used are associated with communities of surface-attached microorganisms, or biofilms. We hypothesized that gesho has anti-biofilm activity. The principal aim of this study was to evaluate gesho-associated anti-biofilm activity and identify active compounds. MATERIALS AND METHODS: Lyophilized ethanol and aqueous extracts were prepared from dried Rhamnus prinoides stems and leaves. Biofilm inhibition was measured by crystal violet staining and subsequent viability assays were conducted on growth agar. Chemical fractionation, chemical testing, Fourier transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC-MS) were used to isolate and identify active compounds. RESULTS: Leaf and stem ethanol extracts significantly inhibited Staphylococcus aureus, Bacillus subtilis and Streptococcus mutans biofilm formation up to 99.9% and reduced planktonic cell growth up to 10 log units relative to untreated controls. The anti-biofilm activity of the ethanol stem extracts was due to a biocidal or bacteriostatic mechanism while bacteriostatic or anti-pathogenic mechanisms were attributed to the leaf ethanol extract. Gesho extracts showed activity against all three species tested but the treatment efficacy and mechanism were species dependent. Chemical fractionation and activity screens of the leaf ethanol extract identified ethyl 4-ethoxybenzoate and 4-hydroxy 4-methyl pentanone to be compounds with anti-biofilm activity. Ethyl 4-ethoxybenzoate activity was potentiated by DMSO. Notably, concentrations of both compounds were identified where biofilm formation was prevented without inhibition of cell growth; i.e. anti-pathogenic characteristics were evident. CONCLUSION: Gesho leaf ethanol extract contains chemicals with anti-biofilm and bactericidal activities. This work lends support to the traditional use of gesho for treating topical infections and warrants further investigation into Rhamnus prinoides as a source of antibacterial and anti-biofilm agents.

Molecules and Mechanisms Underlying the Antimicrobial Activity of Escapin, an l-Amino Acid Oxidase from the Ink of Sea Hares.

Many marine animals use chemicals to defend themselves and their eggs from predators. Beyond their ecologically relevant functions, these chemicals may also have properties that make them beneficial for humans, including biomedical and industrial applications. For example, some chemical defenses are also powerful antimicrobial or antitumor agents with relevance to human health and disease. One such chemical defense, escapin, an l-amino acid oxidase in the defensive ink of the sea hare Aplysia californica, and related proteins have been investigated for their biomedical properties. This review details our current understanding of escapin's antimicrobial activity, including the array of molecules generated by escapin's oxidation of its major substrates, l-lysine and l-arginine, and mechanisms underlying these molecules' bactericidal and bacteriostatic effects on planktonic cells and the prevention of formation and removal of bacterial biofilms. Models of escapin's effects are presented, and future directions are proposed.

Inhibition and Dispersal of Pseudomonas aeruginosa Biofilms by Combination Treatment with Escapin Intermediate Products and Hydrogen Peroxide.

Escapin is an l-amino acid oxidase that acts on lysine to produce hydrogen peroxide (H2O2), ammonia, and equilibrium mixtures of several organic acids collectively called escapin intermediate products (EIP). Previous work showed that the combination of synthetic EIP and H2O2 functions synergistically as an antimicrobial toward diverse planktonic bacteria. We initiated the present study to investigate how the combination of EIP and H2O2 affected bacterial biofilms, using Pseudomonas aeruginosa as a model. Specifically, we examined concentrations of EIP and H2O2 that inhibited biofilm formation or fostered disruption of established biofilms. High-throughput assays of biofilm formation using microtiter plates and crystal violet staining showed a significant effect from pairing EIP and H2O2, resulting in inhibition of biofilm formation relative to biofilm formation in untreated controls or with EIP or H2O2 alone. Similarly, flow cell analysis and confocal laser scanning microscopy revealed that the EIP and H2O2 combination reduced the biomass of established biofilms relative to that of the controls. Area layer analysis of biofilms posttreatment indicated that disruption of biomass occurs down to the substratum. Only nanomolar to micromolar concentrations of EIP and H2O2 were required to impact biofilm formation or disruption, and these concentrations are significantly lower than those causing bactericidal effects on planktonic bacteria. Micromolar concentrations of EIP and H2O2 combined enhanced P. aeruginosa swimming motility compared to the effect of either EIP or H2O2 alone. Collectively, our results suggest that the combination of EIP and H2O2 may affect biofilms by interfering with bacterial attachment and destabilizing the biofilm matrix.

LuxS influences Escherichia coli biofilm formation through autoinducer-2-dependent and autoinducer-2-independent modalities.

Escherichia coli produces biofilms in response to the small molecule autoinducer-2 (AI-2), a product of the LuxS enzyme. LuxS is part of the activated methyl cycle and could also affect biofilm development by AI-2-independent effects on metabolism. A luxS deletion mutant of E. coli W3110 and an inducible plasmid-luxS-complemented strain were used to identify AI-2-independent phenotypes. Differential interference contrast microscopy revealed distinct surface colonization patterns. Confocal microscopy followed by quantitative image analysis determined differences in biofilm topography correlating with luxS expression; deletion mutant biofilms had a 'spreading' phenotype, whereas the complement had a 'climbing' phenotype. Addition of exogenous 4,5-dihydroxy-2,3-pentanedione (DPD), an AI-2 precursor, to the deletion mutant increased biofilm height and biomass, whereas addition of the methyl donor S-adenosyl methionine or aspartate prevented the luxS-complemented strain from producing a thick biofilm. The luxS-complemented strain autoaggregated, indicating that fimbriae production was inhibited, which was confirmed by transmission electron microscopy. DPD could not induce autoaggregation in the deletion mutant, demonstrating that fimbriation was an AI-2-independent phenotype. Carbon utilization was affected by LuxS, potentially contributing to the observed phenotypic differences. Overall, the work demonstrated that LuxS affected E. coli biofilm formation independently of AI-2 and could assist in adapting to diverse conditions.

Constructing multispecies biofilms with defined compositions by sequential deposition of bacteria.

Rationally-assembled multispecies biofilms could benefit applied processes including mixed waste biodegradation and drug biosynthesis by combining complementary metabolic pathways into single functional communities. We hypothesized that the cellular composition of mature multispecies biofilms could be manipulated by controlling the number of each cell type present on newly colonized surfaces. To test this idea, we developed a method for attaching specific numbers of bacteria to a flow cell by recirculating cell suspensions. Initial work revealed a nonlinear relationship between suspension cell density and areal density when two strains of Escherichia coli were simultaneously recirculated; in contrast, sequential recirculation resulted in a predictable deposition of cell numbers. Quantitative analysis of cell distributions in 48-h biofilms comprised of the E. coli strains demonstrated a strong relationship between their distribution at the substratum and their presence in mature biofilms. Sequentially depositing E. coli with either Pseudomonas aeruginosa or Bacillus subtilis determined small but reproducible differences in the areal density of the second microorganism recirculated relative to its areal density when recirculated alone. Overall, the presented method offers a simple and reproducible way to construct multispecies biofilms with defined compositions for biocatalytic processes.

Agarose stabilization of fragile biofilms for quantitative structure analysis.

Agarose was used to stabilize fragile biofilms cultivated in parallel plate flow cells prior to imaging by confocal laser scanning microscopy. An essential element to the success of the procedure was the application of a ceramic heat pad to the flow cell to maintain agarose fluidity until the biofilm was enveloped. Quantitative digital image analysis demonstrated the effectiveness of this technique for generating reproducible measurements of a three-dimensional biofilm structure. The described method will also benefit researchers who transport their flow cell-cultivated biofilms to a core facility for imaging.

Inhibition of quorum sensing in Vibrio harveyi by boronic acids.

Bacterial quorum sensing refers to the ability of bacteria to control gene expression through the detection of a threshold concentration of certain chemicals called autoinducer(s), which are secreted by self and/or other bacteria. Quorum sensing is implicated in the regulation of pathologically relevant events such as biofilm formation, virulence, conjugation, sporulation, and swarming mobility. Inhibitors of bacterial quorum sensing are valuable research tools and potential antimicrobial agents. In this paper, we describe the discovery of several boronic acid inhibitors of bacterial quorum sensing in Vibrio harveyi with IC(50) values in the low to sub-micromolar range in whole cell assays.

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm.

Escherichia coli has been widely used for heterologous protein production (HPP). To determine whether a biofilm environment could benefit E. coli HPP using high copy number plasmids, we compared plasmid maintenance and HPP by E. coli ATCC 33456 containing plasmid pEGFP (a pUC family vector) cultivated in biofilms and in suspended culture. Cells were grown with or without antibiotic selective pressure in flow cells or chemostats for up to 6 days. In biofilms, antibiotic selective pressure increased the plasmid copy number (PCN), but by 144 h, biofilms grown in antibiotic-free media had comparable plasmid concentrations. In the chemostat, the PCN declined steadily, although 100 ppm ampicillin in the medium slowed the rate of plasmid loss. Production of green fluorescent protein (GFP), a representative heterologous protein, was quantified by flow cytometry. In biofilms, at ampicillin concentrations >or=33 ppm, strongly fluorescent cells comprised more than half of the population by 48 h. In the chemostat, more than 50% of the population was non-fluorescent by 48 h in media containing 100 ppm ampicillin, and strongly fluorescent cells were <10% of the population. Biofilm structure was determined by confocal microscopy. Maximum biofilm thickness ranged from 30 to 45 microns, with no significant changes in biofilm structure after 48 h. Plasmid multimer percentages were similar to inocula for cells cultivated in either biofilms or the chemostat. The results indicate that the biofilm environment enhanced both plasmid maintenance and cellular GFP concentrations, and that low levels of antibiotic increased the beneficial effect.

Influences of biofilm structure and antibiotic resistance mechanisms on indirect pathogenicity in a model polymicrobial biofilm.

Indirect pathogenicity (IP), the commensal protection of antibiotic-sensitive pathogens by resistant microorganisms of low intrinsic virulence, can prevent the eradication of polymicrobial infections. The contributions of antibiotic resistance mechanisms and biofilm structure to IP within polymicrobial biofilms were investigated using a model two-member consortium. Escherichia coli ATCC 33456 was transformed with vectors conferring either ampicillin or spectinomycin resistance, creating two distinct populations with different resistance mechanisms. Each strain alone or the consortium was grown as biofilms in flow cells and planktonically in chemostats. Comparisons in survival and activity were made on the basis of MICs and minimum biofilm preventative concentrations, a newly introduced descriptor. In ampicillin-containing medium, commensal interactions were evident during both modes of cultivation, but the sensitive strain experienced a greater benefit in the chemostat, indicating that the biofilm environment limited the commensal interaction between the Amp(r) and Spt(r) strains. In spectinomycin-containing medium, growth of the sensitive strain in chemostats and biofilms was not aided by the resistant strain. However, green fluorescent protein expression by the sensitive strain was greater in mixed-population biofilms (9% +/- 1%) than when the strain was grown alone (2% +/- 0%). No comparable benefit was evident during growth in the chemostat, indicating that the biofilm structure contributed to enhanced activity of the sensitive strain.

Enhanced swarming of bacteria on agar plates containing the surfactant Tween 80.

A widely used method for quantifying swarming motility is the swarm plate assay. A significant increase in the motility halo size formed by Escherichia coli or Azospirillum brasilense was measured on Tween 80-containing agar relative to untreated agar. This improvement could benefit the identification of mutants in swarming motility.

A three-dimensional, stochastic simulation of biofilm growth and transport-related factors that affect structure.

Biofilm structural heterogeneity affects a broad range of microbially catalysed processes. Solute transport limitation and autoinhibitor production, two factors that contribute to heterogeneous biofilm development, were investigated using BacMIST, a computer simulation model. BacMIST combines a cellular automaton algorithm for biofilm growth with Brownian diffusion for solute transport. The simulation represented the growth of microbial unit cells in a three-dimensional domain modelled after a repeating section of a constant depth film fermenter. The simulation was implemented to analyse the effects of various levels of transport limitation on a growing single-species biofilm. In a system with rapid solute diffusion, cells throughout the biofilm grew at their maximum rate, and no solute gradient was formed over the biofilm thickness. In increasingly transport-limited systems, the rapidly growing fraction of the biofilm population decreased, and was found exclusively at the biofilm-liquid interface. Trans-biofilm growth substrate gradients also deepened with increasing transport limitation. Autoinhibitory biofilm growth was simulated for various rates of microbially produced inhibitor transport. Inhibitor transport rates affected both the biofilm population dynamics and the resulting biofilm structures. The formation of networks of void spaces in slow-growing regions of the biofilm and the development of columns in the fast-growing regions suggested a possible mechanism for the microscopically observed evolution of channels in biofilms.