Biofilms最新文献

A microbial fuel cell (MFC) is a renewable energy converter, which transforms organic biomass directly into electricity, using biofilm-electrode metabolic interaction within a bioelectrochemical cell. Efficiency of this transformation can be enhanced through miniaturisation. Miniaturisation of MFCs offers higher surface-area-to-volume ratio and improved mass transfer.

The development of mL-scale; power dense and low cost MFCs, are of great interest in diverse areas of research, ranging from modern bio-robotics, internet-of-things devices, electrical energy generation, remote sensing to wastewater treatment and mineral recovery. The biofilms increased ability in converting organic pollutants into electric power more efficiently, makes mL-sized MFCs attractive for the development of multi-modular stacks and usable off-grid power sources with an ability of enhanced wastewater treatment. This work focuses on small scale MFCs; i) minimising the distance between feeding stream and the biofilm, ii) construction and analysis of a  millilitre scale prototype, using a low cost ceramic separator for higher energy recovery efficiency and sensitivity enhancement to substrates and pollutants. The study aims to test efficient cathode modifications, using graphene ink and magnetite (Fe₃O₄); in order to improve the oxygen reduction reaction (ORR). This in turn is envisioned in an increase of the output, reaching comparable power levels to the larger MFC prototypes tested so far. The additives are chosen such that,  both graphene and iron–based oxides are known from the literature to be catalysts for electrochemical processes, this work focusses on their incorporation into the open-to air cathode in novel, low cost MFC bioreactors.

The miniaturised MFC construction constituted of an in-house fabricated small scale ceramic cylinder of internal volume of 3.88 mL. An anode, made of carbon veil fibre with a coating of activated carbon powder, was placed inside the ceramic cylinder, while the cathode was attached to the outer surface of the structure. Three types of cathodes were tested: i) activated carbon as the control (AC), ii) AC with a graphene ink coating (AC+G) and iii) AC with graphene ink and magnetite powder blend (AC+G+M). Experiments were conducted in triplicate using activated sludge and urine inoculum and thereafter continuously supplemented with 100% real human urine. The results show that the control produced up to 0.85 mW (219 W/m³), while AC+G produced 1.22 mW (312 W/m³), and AC+G+M 1.12 (288 W/m³) which is a 44 % and a 32 % increase respectively in comparison to the control. Comparison of linear sweep voltammetry (LSV) showed superior performance of both modified electrodes against the unmodified AC cathode; further resulting in an enhancement of ORR reaction rate. Power outputs from this work show over 14 times improvement in power density levels in comparison t

In the oil and gas industry, internal corrosion represents one of the major threats to asset lifetime and integrity. Of the types of internal corrosion, microbiologically influenced corrosion (MIC) is the most difficult to predict and monitor due to the unpredictable nature of microbial growth and the minimal metal loss resulting in through wall failure (pitting). MIC results from biofilm communities interacting directly and indirectly with the metal. Due to the structure and nature of these pipelines, directly monitoring sessile growth is impossible. As a result, most MIC monitoring is done through planktonic cells retrieved from fluid samples as a proxy for sessile populations.

Growth curves are one of the most fundamental methods of quantitatively assessing microbial growth. In the lab, pure cultures are measured using optical densities, biomass staining, direct microscopic counting and counting colony forming units (CFU) on specialized media while more advanced techniques involve quantitative PCR (qPCR) of key genes. While PCR technologies are more easily transferred from the field to the lab, CFU counts are impossible in the field. Alternatives to the CFU are colorimetric activity assays such as “bug bottles” or biological activity reaction test (BART) bottles but aren’t sensitive and require long incubation times. More sensitive assays such as ATP measurements are also used but can be misleading as high metabolically active samples will give higher cell count equivalents than a metabolically slow community of an identical size.

To systematically evaluate a best practice, we conducted growth curves in a lab scenario using six pure cultures and techniques predominantly used in the field to determine how these techniques compare and accurately measure microbial growth. The six species used are Acetobacterium woodii, Bacillus subtilis, Desulfovibrio vulgaris, Geoalkalibacter subterraneus, Pseudomonas putida and Thauera aromatica. The techniques used are optical density at 600 nm, ATP activity measurements using a luciferase-based assay, DNA concentration and 16S rRNA copy numbers.

It was found that most lines of data follow the expected sigmoidal growth curve to varying degrees for all species. OD₆₀₀ readings follow the expected sigmoidal curves, exhibiting a lag phase, log growth phase and a stationary phase. ATP peaks during mid log phase and quickly declines, never showing a distinct stationary phase, while DNA concentrations closely follow the OD₆₀₀ readings but decline to death phase more rapidly. qPCR of the 16S rRNA genes revealed this data followed the same trends but was less susceptible to fluctuations.

Assessing microbial biofilms in the environment and on anthropogenic industrial infrastructure is extremely challenging given sampling, storage and transportation to the lab.  This work begins to establish best practices for growth of environmental communities

The membrane aerated biofilm reactor (MABR) is an emerging wastewater treatment technology that can greatly decrease energy requirements for wastewater treatment. It consists of cassettes of air-supplying, hollow-fiber membranes that can retrofit existing activated sludge processes. MABR behavior differs from conventional biofilm processes due to the counter-diffusion of the electron donor (ammonia) and acceptor (oxygen).

Partial nitrification (PN), or partial nitrification Anammox (PNA), can further improve MABR energy efficiency and cost effectiveness.  To achieve this, ammonia oxidizing bacteria (AOB) must outcompete nitrite-oxidizing bacteria (NOB).  High temperatures favor AOB, but it is not feasible to heat the wastewater influent.  However, high-temperature compressed air can be supplied to the membrane lumen, increasing temperatures inside the biofilm without increasing the bulk temperatures. No previous research has addressed temperature gradients in biofilms, which can lead to gradients in  biodegradation kinetics, diffusivities, and O₂ solubility.

The objective of this research was to explore the effect of temperature gradients in MABR biofilms, especially with respect to PN. We used a one-dimensional multi-species biofilm model, which considers the MABR physical and biochemical behavior, especially with respect to temperature. The model was implemented using COMSOL Multiphysics. We also used bench-scale experiments to explore the effect of biofilm temperature gradients on MABR nitrification and PN performances and microbial community structure.

Model simulations showed that MABR biofilms exposed to a temperature gradient from 20 ºC (biofilm interior) to 10 ºC (bulk liquid) had a 60% increase in nitrification rates compared with biofilms at 10 ºC. More importantly, the model predicted a complete out competition of NOBs within the biofilm.

Preliminary experimental results confirm a significant (105%) increase in nitrification fluxes with a temperature of 30ºC compared to ambient temperatures (20ºC). Future experiments will validate the model predicted effects of biofilm temperature gradients on nitrification fluxes and microbial community structure.

Escherichia coli biofilms have a great biotechnological potential since this organism has been one of the preferred hosts for recombinant protein production for the past decades and it has been successfully used in metabolic engineering for the production of high-value compounds.

In a previous study, we have demonstrated that the non-induced enhanced green fluorescent protein (eGFP) expression from E. coli biofilm cells was 30-fold higher than in the planktonic state without any optimization of cultivation parameters [1]. The aim of the present work was to evaluate the effect of chemical induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) on the expression of eGFP by planktonic and biofilm cells of E. coli JM109(DE3) transformed with a plasmid containing a T7 promoter.

It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Recombinant protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the recombinant protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of recombinant protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth [2].

It is expected that this work will be of great value to elucidate the mechanisms of induction on recombinant protein production, especially in biofilm cells which have shown potential to be used as protein factories.

References:

[1] Gomes, L.C., & Mergulhão, F.J. (2017) Heterologous protein production in Escherichia coli biofilms: A non-conventional form of high cell density cultivation. Process Biochemistry, 57, 1-8. https://doi.org/10.1016/j.procbio.2017.03.018

[2] Gomes, L., Monteiro, G., & Mergulhão, F. (2020). The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms. International Journal of Molecular Sciences, 21(2), 576. https://doi.org/10.3390/ijms21020576

Background:

The adhesion of planktonic bacteria to a surface (biotic or abiotic), and their subsequent ability to aggregate into multicellular communities called biofilms, is a major driving force of failing antibiotic therapy and persistence in chronic infections caused by a variety of pathogens (e.g., Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus) and plaguing healthcare systems worldwide. Biofilms are estimated to be involved in over 80% of all microbial infections in humans, and commonly exhibit extreme resistance to conventional antimicrobial treatments. Consequently, there is an urgent need for novel antimicrobial agents, which target biofilm residing cells. Here, we present the development and evaluation of a new generation of dual-acting nitroxide functionalised antibiotics with potent biofilm eradication activity.

Methodology:

Synthetic organic chemistry was utilised to produce a new generation of nitroxide functionalised antibiotics with targeted biofilm eradication capabilities. These compounds were tested for biofilm eradication and/or dispersal of several bacterial species using the MBEC^TM device, a reproducible high-throughput static biofilm formation system. Mature biofilms were treated with serial dilutions of the specific test agent(s) and recovered bacterial numbers were quantified by absorbance spectroscopy at 600 nm or plating for viable cell counts. Treated biofilms were also stained with Live/Dead (SYTO-9/PI) bacterial viability kit and analysed by fluorescence and confocal laser scanning microscopy.

Results:

Nitroxide functionalised antibiotics exhibit potent biofilm-eradication activity against a variety of medically important pathogens, including P. aeruginosa, uropathogenic E. coli, and S. aureus. In Minimal Biofilm Eradication Concentration (MBEC) assays nitroxide functionalised antibiotics were 64-fold more potent against S. aureus biofilms, and at least 2-fold more potent against uropathogenic E. coli biofilms than the parent antibiotic ciprofloxacin.

Conclusions:

Currently, antibiotics are often entirely ineffective against biofilm infections. Nitroxide functionalised antibiotics represent a promising new strategy, which could circumvent the resistance of Gram-positive and Gram-negative biofilms to conventional treatments.