Electroactive biofilms: how microbial electron transfer enables bioelectrochemical applications

Microbial biofilms are ubiquitous. In marine and freshwater ecosystems, microbe–mineral interactions sustain biogeochemical cycles, while biofilms found on plants and animals can range from pathogens to commensals. Moreover, biofouling and biocorrosion represent significant challenges to industry. Bioprocessing is an opportunity to take advantage of biofilms and harness their utility as a chassis for biocommodity production. Electrochemical bioreactors have numerous potential applications, including wastewater treatment and commodity production. The literature examining these applications has demonstrated that the cell–surface interface is vital to facilitating these processes. Therefore, it is necessary to understand the state of knowledge regarding biofilms’ role in bioprocessing. This mini-review discusses bacterial biofilm formation, cell–surface redox interactions, and the role of microbial electron transfer in bioprocesses. It also highlights some current goals and challenges with respect to microbe-mediated bioprocessing and future perspectives.     

Phototrophic biofilms and their potential applications

Phototrophic biofilms occur on surfaces exposed to light in a range of terrestrial and aquatic environments. Oxygenic phototrophs like diatoms, green algae, and cyanobacteria are the major primary producers that generate energy and reduce carbon dioxide, providing the system with organic substrates and oxygen. Photosynthesis fuels processes and conversions in the total biofilm community, including the metabolism of heterotrophic organisms. A matrix of polymeric substances secreted by phototrophs and heterotrophs enhances the attachment of the biofilm community. This review discusses the actual and potential applications of phototrophic biofilms in wastewater treatment, bioremediation, fish-feed production, biohydrogen production, and soil improvement.     

Biofilms and their impact on the food industry

Biofilm could be defined as a complex communities of microorganisms seen affixed to surfaces, they form clusters without sticking to any surface and buried firmly in an extracellular matrix (ECM). This matrix is formed by microorganisms in the formation of either extracellular polymeric substances (EPSS) or extracellular polymer. Many reviews have addressed the negative consequences of biofilm production in the food industry, among which we talk about biofilms being responsible for spoilage microorganisms and foodborne pathogens such as Listeria monocytogenes, Bacillus cereus etc. These contamination could be linked to biofilms presence in the processing plant. Although researches have tried conferring solutions to these challenges in the food industry, however, in this review we have tried to focus on the positive impact of biofilms formed in the food industry. It is critically expedient while trying to find the solution to the challenges of biofilm in the food industry to develop and give a major focus on the advantages and positive impact biofilm has in the food industry, which has been greatly neglected. Hence in this article, we have highlighted some positive impacts of biofilms formed in the food industry, like enhancing plant health and productivity of food products, as an agent of water and wastewater treatment in the food industry, as a tool in reducing the amount of excess sludge in the wastewater treatment plant. The development of edible biofilms, fermented food products and the production of biodegradable food packaging are also part of biofilms beneficial roles in the food industries.     

Phosphorus removal coupled to bioenergy production by three cyanobacterial isolates in a biofilm dynamic growth system

In the present study a closed incubator, designed for biofilm growth on artificial substrata, was used to grow three isolates of biofilm-forming heterocytous cyanobacteria using an artificial wastewater secondary effluent as the culture medium. We evaluated biofilm efficiency in removing phosphorus, by simulating biofilm-based tertiary wastewater treatment and coupled this process with biodiesel production from the developed biomass. The three strains were able to grow in the synthetic medium and remove phosphorus in percentages, between 6 and 43%, which varied between strains and also among each strain according to the biofilm growth phase. Calothrix sp. biofilm turned out to be a good candidate for tertiary treatment, showing phosphorus reducing capacity (during the exponential biofilm growth) at the regulatory level for the treated effluent water being discharged into natural water systems.

Besides phosphorus removal, the three cyanobacterial biofilms produced high quality lipids, whose profile showed promising chemical stability and combustion behavior. Further integration of the proposed processes could include the integration of oil extracted from these cyanobacterial biofilms with microalgal oil known for high monounsaturated fatty acids content, in order to enhance biodiesel cold flow characteristics.     

Biological wastewater treatment model building fits and misfits

The modeling of biological wastewater treatment processes has received much attention over the past ten years. Efforts are underway to develop a unified model for these processes which will greatly aid in the design and operation of wastewater treatment facilities. This paper presents a philosophical discussion of model building strategies augmented by a discussion of statistical problems associated with model parameter estimation and model discrimination. This discussion further illustrates with actual data, that goodness of fit is not a sufficient condition for model acceptance. Numerous rival models are examined to illustrate this point. In order to verify a model or to discriminate between rival models, they must be “Put in jeopardy.” If this attitude is not employed in model building efforts, important discrepancies in the proposed model may go undetected.     

Biofiltration eliminates nuisance chemical odors from industrial air streams

This paper focuses on recent developments of biofiltration technology used in treating nuisance chemical odors from industrial and municipal air streams. In the biofiltration process, odorous chemical constituents in the air are first transported to biofilms by diffusion, solubilization and adsorption processes. Bacteria within the biofilms oxidize odor constituents into harmless and odorless products. Through successful laboratory and pilot research on biofiltration of odorous air-stream constituents, numerous commercial biofilters have been designed and installed across North America. In this paper, case studies related to biofiltration of air emissions from meat rendering plants, municipal wastewater treatment applications, and printed circuit board production are discussed to demonstrate the robustness of this technology in eliminating a wide variety of compounds. Electronic Publication     

Biofilm reactors for industrial bioconversion processes: employing potential of enhanced reaction rates

This article describes the use of biofilm reactors for the production of various chemicals by fermentation and wastewater treatment. Biofilm formation is a natural process where microbial cells attach to the support (adsorbent) or form flocs/aggregates (also called granules) without use of chemicals and form thick layers of cells known as "biofilms." As a result of biofilm formation, cell densities in the reactor increase and cell concentrations as high as 74 gL^-1 can be achieved. The reactor configurations can be as simple as a batch reactor, continuous stirred tank reactor (CSTR), packed bed reactor (PBR), fluidized bed reactor (FBR), airlift reactor (ALR), upflow anaerobic sludge blanket (UASB) reactor, or any other suitable configuration. In UASB granular biofilm particles are used. This article demonstrates that reactor productivities in these reactors have been superior to any other reactor types. This article describes production of ethanol, butanol, lactic acid, acetic acid/vinegar, succinic acid, and fumaric acid in addition to wastewater treatment in the biofilm reactors. As the title suggests, biofilm reactors have high potential to be employed in biotechnology/bioconversion industry for viable economic reasons. In this article, various reactor types have been compared for the above bioconversion processes.     

Pleurotus ostreatus biofilm‐forming ability and ultrastructure are significantly influenced by growth medium and support type

Aims

To investigate the effect of support and growth medium (GM) on Pleurotus ostreatus biofilm production, specific metabolic activity (SMA) and ultrastructure.

Methods and Results

Biofilms were developed on membranes covering a broad range of surface properties and, due to the applicative implications of mixed biofilms, on standard bacterial GM in stationary and shaken culture. Hydrophilic (glass fibre, Duran glass and hydroxyapatite) and mild hydrophobic (polyurethane, stainless steel, polycarbonate, nylon) supports were more adequate for biofilm attachment than the hydrophobic Teflon. Among the GM, sucrose–asparagine (SA) was more conducive to biofilm production than Luria–Bertani and M9. GM was more influential than support type on biofilm ultrastructure, and a high compactness was evident in biofilms developed on SA. Biofilms on Duran glass were more efficient than planktonic cultures in olive‐mill wastewater treatment.

Conclusions

The main effects of support and GM variables and their binary interactions on both biofilm production and SMA were all highly significant (P < 0·001): thus, the magnitude of the effect of each variable strongly depended on the level of the other one.

Significance and Impact of the Study

There is a lack of basic information regarding physiology and ultrastructure of P. ostreatus biofilms. To our knowledge, this is the first attempt to fill this gap, thus representing a basis for future studies.     

Enhancement of Anaerobic Digestion Using Particulate Growth Systems

Attached media reactors are used for enhancement of wastewater treatment processes including anaerobic condition. Selection of a suitable biofilm carrier is a compelling method to improve anaerobic digestion systems. This study investigates the performance of four fibrous biofilms installed in batch biogas reactors for treatment of cow manure. BioCords HS1, HS2, LS1, and LS2 are manufactured by Bishop Water Technologies, ON, Canada. Effluents and attached growth media were analyzed after batch experiment; methane production, methane yield, transfer efficiencies, organic and solid removal efficiencies, pH, and attached volatile suspended solid (VSS) were measured; VSS attached to biofilms mainly correlated with the specific surface area of each biofilm. Additionally, SEM (scanning electron microscopy) was used for further understanding of biofilm formation process for BioCords and the dissimilarity in their performance. The results indicated that BioCord LS2 had positive impact on achieving higher methane production and removal efficiencies compared to other support media utilized in batch reactors. It was also demonstrated from the experiment that BioCord LS2 potentially could generate higher methane production than conventional batch bioreactor.     

Biofilm formation and interactions of bacterial strains found in wastewater treatment systems

Biofilm formation and adherence properties of 13 bacterial strains commonly found in wastewater treatment systems were studied in pure and mixed cultures using a crystal violet microtiter plate assay. Four different culture media were used, wastewater, acetate medium, glucose medium and diluted nutrient broth. The medium composition strongly affected biofilm formation. All strains were able to form pure culture biofilms within 24 h in at least one of the tested culture media and three strains were able to form biofilm in all four culture media, namely Acinetobacter calcoaceticus ATCC 23055, Comamonas denitrificans 123 and Pseudomonas aeruginosa MBL 0199. The adherence properties assessed were initial adherence, cell surface hydrophobicity, and production of amyloid fibers and extracellular polymeric substances. The growth of dual-strain biofilms showed that five organisms formed biofilm with all 13 strains while seven formed no or only weak biofilm when cocultured. In dual-strain cultures, strains with different properties were able to complement each other, giving synergistic effects. Strongest biofilm formation was observed when a mixture of all 13 bacteria were grown together. These results on attachment and biofilm formation can serve as a tool for the design of tailored systems for the degradation of municipal and industrial wastewater.     

Spatially resolved abundances of antibiotic resistance genes and intI1 in wastewater treatment biofilms

Attached growth bioprocesses that use biofilms to remove organic matter or nutrients from wastewater are known to harbor antibiotic resistance genes (ARGs). Biofilms in these processes are spatially heterogeneous, but little is known about depth stratification of ARGs in complex, mixed culture biofilms. To address this knowledge gap, we used an experimental approach combining cryosectioning and quantitative polymerase chain reaction to quantify the spatial distribution of three ARGs (sul1, ermB, and qnrS) and the class 1 integron-integrase gene intI1 in biofilms from a lab-scale rotating annular reactor fed with synthetic wastewater. We also used high throughput 16S ribosomal RNA (rRNA) gene sequencing to characterize community structure with depth in biofilms. The ARG sul1 and the integron-integrase gene intI1 were found in higher abundances in upper layers of biofilm near the fluid-biofilm interface than in lower layers and exhibited significant correlations between the distance from substratum and gene abundances. The genes ermB and qnrS were present in comparatively low relative abundances. Microbial community structure varied significantly by date of sampling and distance from the substratum. These findings highlight the genetic and taxonomic heterogeneity with distance from substratum in wastewater treatment biofilms and show that sul1 and intI1 are particularly abundant near fluid-biofilm interfaces where cells are most likely to detach and flow into downstream portions of treatment systems and can ultimately be released into the environment through effluent.     

Biofilm research using calorimetry – a marriage made in heaven?

Aggregated bacteria growing on a surface, so-called biofilms, play an important role in technical processes like wastewater treatment, bioremediation, or bioprocessing. In contrast, problems arise when biofilm growth results in undesired processes that may cause huge financial losses, e.g., clogged pipes, microbially influenced corrosion or pathogenic contamination. For observation purposes and to develop efficient control strategies, real-time monitoring tools for biofilms are required. Among the large variety of tools used in biofilm research, calorimetry is rarely applied, even though many characteristics qualify it for biofilm investigation and monitoring. Calorimetric measurements are non-invasive and non-destructive, and can be applied to nearly any kind of samples (including heterogeneous or turbid solutions) without the need of special sample preparation. Online and real-time data acquisition reduces the labor and facilitates high-throughput measurements. The following article is meant to introduce and promote calorimetry as a future tool in biofilm research. It attempts an assessment of common, existent monitoring tools and specifically addresses the potential of calorimetry in this field.     

Microbial biofilms in biotechnological processes

This review summarizes the information concerning the following applications of microbial biofilms in biotechnology: the biodegradation of organic substances and other contaminants during wastewater treatment, biosynthesis, and biocatalysis. The main types of reactors for implementing biotechnical processes based on microbial biofilms are discussed. The advantages and drawbacks of biocatalysts in the form of microbial biofilms for the biotransformation of organic substances are examined.     

Potential of biofilm-based biofuel production

Biofilm technology has been extensively applied to wastewater treatment, but its potential application in biofuel production has not been explored. Current technologies of converting lignocellulose materials to biofuel are hampered by costly processing steps in pretreatment, saccharification, and product recovery. Biofilms may have a potential to improve efficiency of these processes. Advantages of biofilms include concentration of cell-associated hydrolytic enzymes at the biofilm–substrate interface to increase reaction rates, a layered microbial structure in which multiple species may sequentially convert complex substrates and coferment hexose and pentose as hydrolysates diffuse outward, and the possibility of fungal–bacterial symbioses that allow simultaneous delignification and saccharification. More importantly, the confined microenvironment within a biofilm selectively rewards cells with better phenotypes conferred from intercellular gene or signal exchange, a process which is absent in suspended cultures. The immobilized property of biofilm, especially when affixed to a membrane, simplifies the separation of biofuel from its producer and promotes retention of biomass for continued reaction in the fermenter. Highly consolidated bioprocessing, including delignification, saccharification, fermentation, and separation in a single reactor, may be possible through the application of biofilm technology. To date, solid-state fermentation is the only biofuel process to which the advantages of biofilms have been applied, even though it has received limited attention and improvements. The transfer of biofilm technology from environmental engineering has the potential to spur great innovations in the optimization of biofuel production.     

Performance Assessment of Full-Scale Wastewater Treatment Plants Based on Seasonal Variability of Microbial Communities via High-Throughput Sequencing

Microbial communities of activated sludge (AS) play a key role in the performance of wastewater treatment processes. However, seasonal variability of microbial population in varying AS-based processes has been poorly correlated with operation of full-scale wastewater treatment systems (WWTSs). In this paper, significant seasonal variability of AS microbial communities in eight WWTSs located in the city of Guangzhou were revealed in terms of 16S rRNA-based Miseq sequencing. Furthermore, variation redundancy analysis (RDA) demonstrated that the microbial community compositions closely correlated with WWTS operation parameters such as temperature, BOD, NH₄⁺-N and TN. Consequently, support vector regression models which reasonably predicted effluent BOD, SS and TN in WWTSs were established based on microbial community compositions. This work provided an alternative tool for rapid assessment on performance of full-scale wastewater treatment plants.     

The evolution of bacteriocin production in bacterial biofilms

Bacteriocin production is a spiteful behavior of bacteria that is central to the competitive dynamics of many human pathogens. Social evolution predicts that bacteriocin production is favored when bacteriocin-producing cells are mixed at intermediate frequency with their competitors and when competitive neighborhoods are localized. Both predictions are supported by biofilm experiments. However, the means by which physical and biological processes interact to produce conditions that favor the evolution of bacteriocin production remain to be investigated. Here we fill this gap using analytical and computational approaches. We identify and collapse key parameters into a single number, the critical bacteriocin range, that measures the threshold distance from a focal bacteriocin-producing cell within which its fitness is higher than that of a sensitive cell. We develop an agent-based model to test our predictions and confirm that bacteriocin production is most favored when relatedness is intermediate and competition is local. We then use invasion analysis to determine evolutionarily stable strategies for bacteriocin production. Finally, we perform long-term evolutionary simulations to analyze how the critical bacteriocin range and genetic lineage segregation affect biodiversity in multistrain biofilms. We find that biodiversity is maintained in highly segregated biofilms for a wide array of critical bacteriocin ranges. However, under conditions of high nutrient penetration leading to well-mixed biofilms, biodiversity rapidly decreases and becomes sensitive to the critical bacteriocin range.     

Microflora from leaf debris is suitable for treatment of starch industry wastewater

Biological treatment of industrial waste is a widely practiced technique that generates comparatively less environmentally hazardous waste than other chemical treatment processes. Wet milling of maize generates huge amount of wastewater (5 m³/ton) of low pH with organic matter and nutrients. Anaerobic methanogenic and aerobic bacteria are mostly highly sensitive to low pH. The treatment of wastewater causes huge cost of chemical neutralization or hydraulic recirculation for maintaining neutral pH. In the present study, different microbial consortia isolated from cow dung, active sludge from an anaerobic reactor for treatment of industrial wastewater, and leaf debris from benthic soil were screened for tolerance against low pH and for potential of chemical oxygen demand (COD) removal in order to find out an alternative microbial population for industrial water treatment at low pH. The most effective consortia found from leaf debris were further investigated for optimal operation. The microscopic analysis of leaf debris sludge showed abundance of Gram‐negative methanococci, which was found tolerant to low pH in plate culture method. On further investigation for COD removal from starch industry effluent, they were found to be most effective at pH 5 with highest COD removal rate of 70% and lowest biomass generation of 81%. Hence, it was concluded that the low pH‐tolerant methanogen bacteria, enriched from leaf debris sludge, is highly beneficial for anaerobic treatment of wastewater from several industries including corn starch industry by reducing cost of operation for neutralization to neutral pH and through reducing excess waste sludge production by the treatment system.     

Microbial composition and structure of aerobic granular sewage biofilms

Aerobic activated sludge granules are dense, spherical biofilms which can strongly improve purification efficiency and sludge settling in wastewater treatment processes. In this study, the structure and development of different granule types were analyzed. Biofilm samples originated from lab-scale sequencing batch reactors which were operated with malthouse, brewery, and artificial wastewater. Scanning electron microscopy, light microscopy, and confocal laser scanning microscopy together with fluorescence in situ hybridization (FISH) allowed insights into the structure of these biofilms. Microscopic observation revealed that granules consist of bacteria, extracellular polymeric substances (EPS), protozoa and, in some cases, fungi. The biofilm development, starting from an activated sludge floc up to a mature granule, follows three phases. During phase 1, stalked ciliated protozoa of the subclass Peritrichia, e.g., Epistylis spp., settle on activated sludge flocs and build tree-like colonies. The stalks are subsequently colonized by bacteria. During phase 2, the ciliates become completely overgrown by bacteria and die. Thereby, the cellular remnants of ciliates act like a backbone for granule formation. During phase 3, smooth, compact granules are formed which serve as a new substratum for unstalked ciliate swarmers settling on granule surfaces. These mature granules comprise a dense core zone containing bacterial cells and EPS and a loosely structured fringe zone consisting of either ciliates and bacteria or fungi and bacteria. Since granules can grow to a size of up to several millimeters in diameter, we developed and applied a modified FISH protocol for the study of cryosectioned biofilms. This protocol allows the simultaneous detection of bacteria, ciliates, and fungi in and on granules.     

Unmasking photogranulation in decreasing glacial albedo and net autotrophic wastewater treatment

In both natural and built environments, microbes on occasions manifest in spherical aggregates instead of substratum-affixed biofilms. These microbial aggregates are conventionally referred to as granules. Cryoconites are mineral rich granules that appear on glacier surfaces and are linked with expanding surface darkening, thus decreasing albedo, and enhanced melt. The oxygenic photogranules (OPGs) are organic rich granules that grow in wastewater, which enables wastewater treatment with photosynthetically produced oxygen and which presents potential for net autotrophic wastewater treatment in a compact system. Despite obvious differences inherent in the two, cryoconite and OPG pose striking resemblance. In both, the order Oscillatoriales in Cyanobacteria envelope inner materials and develop dense spheroidal aggregates. We explore the mechanism of photogranulation on account of high similarity between cryoconites and OPGs. We contend that there is no universal external cause for photogranulation. However, cryoconites and OPGs, as well as their intravariations, which are all under different stress fields, are the outcome of universal physiological processes of the Oscillatoriales interfacing with goldilocks interactions of stresses. Finding the rules of photogranulation may enhance engineering of glacier and wastewater systems to manipulate their ecosystem impacts.