Bacteria can attach to essentially all materials and form multicellular biofilms with high-level tolerance to antimicrobials. Detrimental biofilms are responsible for a variety of problems ranging from food and water contamination, bio-corrosion, to drug resistant infections. Besides the challenges in control, biofilms are also difficult to detect due to the lack of biofilm-specific biomarkers and methods for non-destructive imaging. In this article, we present a concise review of recent advancements in this field, with a focus on medical device-associated infections. We also discuss the technologies that have potential for non-destructive detection of bacterial biofilms.

While synthetic nitrogen fuels modern agriculture, its production is energy-intensive, and its application leads to aquatic pollution and greenhouse gas emissions. Sustainable intensification of agriculture to provide both food for humans and feedstocks for bio-based fuels and materials requires alternative options for nitrogen management. For nearly fifty years, nitrogen fixation in cereal crops has been pursued to address this challenge. Efforts to engineer plants for nitrogen fixation have made strides through eukaryotic nitrogenase expression and a deepened understanding of root nodulation pathways, but deployment of transgenic nitrogen fixing cereals may be outpaced by population growth. By contrast, a root-associated bacterium that can fix and supply nitrogen to cereals could offer a sustainable solution for nitrogen management on a shorter timescale.

Microbial consortia are fascinating yet barely understood biological systems with an elusive intrinsic complexity. Studying microbial consortia and the interactions of their members is of major importance for the understanding, engineering and control of synthetic and natural microbial consortia. Microfluidic cultivation and analysis devices are versatile tools for the study of microbial interactions at the single-cell level. While there is a vast amount of literature on microfluidics for the investigation of monocultures only few studies on co-cultures have been conducted in this context. Here we give an overview of different microfluidic single-cell cultivation tools for the analysis of microbial consortia with a focus on their physiology, growth dynamics and cellular interactions. Finally, central challenges and perspectives for the future application of microfluidic tools for microbial consortia investigations will be given.

Phenotypic heterogeneity is a phenomenon in which genetically identical individuals have different characteristics. This behavior can also be found in bacteria, even if they grow as monospecies in well-mixed environments such as bioreactors. Here it is discussed how phenotypic heterogeneity is generated by internal factors and how it is promoted under nutrient-limited growth conditions. A better understanding of the molecular levels that control phenotypic heterogeneity could improve biotechnological production processes.

Some of the oldest and most established industrial biotechnology processes involve the fungal production of organic acids. In these fungi, the transport of metabolites between cellular compartments, and their secretion, is a major factor. In this review we exemplify the importance of both mitochondrial and plasma membrane transporters in the case of itaconic acid production in two very different fungal systems, Aspergillus and Ustilago. Homologous and heterologous overexpression of both types of transporters, and biochemical analysis of mitochondrial transporter function, show that these two fungi produce the same compound through very different pathways. The way these fungi respond to itaconate stress, especially at low pH, also differs, although this is still an open field which clearly needs additional research.

The design of controllable artificial microbial consortia has attracted considerable interest in recent years to capitalize on the inherent advantages in comparison to monocultures such as the distribution of the metabolic burden by division of labor, the modularity and the ability to convert complex substrates. One promising approach to control the consortia composition, function and stability is the provision of defined ecological niches fitted to the specific needs of the consortium members. In this review, we discuss recent examples for the creation of metabolic niches by biological engineering of resource partitioning and syntrophic interactions. Moreover, we introduce a complementing process engineering approach to provide defined spatial niches with differing abiotic conditions (e.g. O2, T, light) in stirred tank reactors harboring biofilms. This enables the co-cultivation of microorganisms with non-overlapping abiotic requirements and the control of the strain ratio in consortia characterized by substrate competition.

Free-living microbes are generally capable of growing on multiple different nutrients. Some of those nutrients are used simultaneously, while others are used sequentially. The pattern of nutrient preferences and co-utilization defines the metabolic strategy of a microorganism. Metabolic strategies can substantially affect ecological interactions between species, but their evolution and distribution across the tree of life remain poorly characterized. We discuss how the confluence of better computational models of genotype-phenotype maps and high-throughput experimental tools can help us fill gaps in our knowledge and incorporate metabolic strategies into quantitative predictive models of microbial consortia.

Metabolic division of the labour of organic matter decomposition into several steps carried out by different types of microbes is typical for many anoxic - but not oxic environments. An explanation of this well-known pattern is proposed based on the combination of three key insights: (i) well-studied anoxic environments are high flux environments: they are only anoxic because their high organic matter influx leads to oxygen depletion; (ii) shorter, incomplete catabolic pathways provide the capacity for higher flux, but this capacity is only advantageous in high flux environments; (iii) longer, complete catabolic pathways have energetic happy ends but only with high redox potential electron acceptors. Thus, aerobic environments favour longer pathways. Bioreactors, in contrast, are high flux environments and therefore favour division of catabolic labour even if aeration keeps them aerobic; therefore, host strains and feeding strategies must be carefully engineered to resist this pull.

There has been a growing emphasis on understanding the important relationship between human-associated microbial communities and disease development. With technological advancements, we are able to gain further insights into host-microbiome interactions at a deeper level. In order to fully leverage the close associations between microbes and their host, development of therapeutics targeting the microbiome has surged in recent years. In this review, we discuss advances made in engineering gut bacteria to develop novel therapeutic modalities that aim to rewire host-microbiome interactions such as host metabolism and immune functions for prevention and treatment of various diseases. In particular, applications of these engineered bacteria against diseases such as metabolic, immune disorders and cancer are covered.

Bioremediators are cells or non-living subcellular entities of biological origin employed to degrade target pollutants. Rational, mechanistic design can substantially improve the performance of bioremediators for applications, including waste treatment and food safety. We highlight how such improvements can be informed at the cellular level by theoretical observations especially in the context of phenotype plasticity, cell signaling, and community assembly. At the molecular level, we suggest enzyme design using techniques such as Small Angle Neutron Scattering and Density Functional Theory. To provide an example of how these techniques could be synergistically combined, we present the case-study of the interaction of the enzyme laccase with the food contaminant aflatoxin B1. In designing bioremediators, we encourage interdisciplinary, mechanistic research to transition from an observation-oriented approach to a principle-based one.

Microbial genomes encode numerous biosynthetic gene clusters (BGCs) that may produce natural products with diverse applications in medicine, agriculture, the environment, and materials science. With the advent of genome sequencing and bioinformatics, heterologous expression of BGCs is of increasing interest in bioactive natural product (NP) discovery. However, this approach has had limited success because expression of BGCs relies heavily on the physiology of just a few commonly available host chassis. Expanding and diversifying the chassis portfolio for heterologous BGC expression may greatly increase the chances for successful NP production. In this review, we first discuss genetic and genome engineering technologies used to clone, modify, and transform BGCs into multiple strains and to engineer chassis strains. We then highlight studies that employed the multi-chassis approach successfully to optimize NP production, discover previously uncharacterized NPs, and better understand BGC function.

Microbial consortia were designed for the production of small molecules with 'labor' being divided between two or more microorganisms. Examples of linear designs are substrate conversion preceding target molecule production or subdivision of two consecutive steps of target molecule production. Here, we review synthetic biology design approaches for microbial consortia based on ecological principles and microbial interactions that is, mutualism, and commensalism. Besides highlighting the technical challenges regarding industrial application of synthetic microbial consortia, we forecast the extension of the concept from binary linear to ternary linear and more complex microbial consortia in biotechnological applications. Microbial consortia are here reviewed and proposed as a rational solution toward feedstock accessibility as it has been shown for production of l-lysine, l-pipecolic acid and cadaverine from starch or production of fumarate from microcrystalline cellulose and alkaline pre-treated corn, or alternatively to establish new multi-step pathway for the production of rosmarinic acid from xylose and glucose.

The microbial production of natural products has been traditionally accomplished in a single organism engineered to accommodate target biosynthetic pathways. Often times, such approaches result in large metabolic burdens as key cofactors, precursor metabolites and energy are channeled to pathways of structurally complex chemicals. Recently, modular co-culture engineering has emerged as a new approach to efficiently conduct heterologous biosynthesis and greatly enhance the production of natural products. This review highlights recent advances that leverage Escherichia coli-based modular co-culture engineering for making natural products. Potential future perspectives for studies in this promising field are addressed as well.

Nearly all microbes rely on other species in their environment to provide nutrients they are unable to produce. Nutritional interactions include not only the exchange of carbon and nitrogen compounds, but also amino acids and cofactors. Interactions involving cross-feeding of cobamides, the vitamin B12 family of cofactors, have been developed as a model for nutritional interactions across species and environments. In addition to experimental studies, new developments in culture-independent methodologies such as genomics and modeling now enable the prediction of nutritional interactions in a broad range of organisms including those that cannot be cultured in the laboratory. New insights into the mechanisms and evolution of microbial nutritional interactions are beginning to emerge by combining experimental, genomic, and modeling approaches.

Microbial communities within anaerobic ecosystems have evolved to degrade and recycle carbon throughout the earth. A number of strains have been isolated from anaerobic microbial communities, which are rich in carbohydrate active enzymes (CAZymes) to liberate fermentable sugars from crude plant biomass (lignocellulose). However, natural anaerobic communities host a wealth of microbial diversity that has yet to be harnessed for biotechnological applications to hydrolyze crude biomass into sugars and value-added products. This review highlights recent advances in 'omics' techniques to sequence anaerobic microbial genomes, decipher microbial membership, and characterize CAZyme diversity in anaerobic microbiomes. With a focus on the herbivore rumen, we further discuss methods to discover new CAZymes, including those found within multi-enzyme fungal cellulosomes. Emerging techniques to characterize the interwoven metabolism and spatial interactions between anaerobes are also reviewed, which will prove critical to developing a predictive understanding of anaerobic communities to guide in microbiome engineering.

Bacterial cells have a limited number of resources that can be allocated for gene expression. The intracellular competition for these resources has an impact on the cell physiology. Bacteria have evolved mechanisms to optimize resource allocation in a variety of scenarios, showing a trade-off between the resources used to maximise growth (e.g. ribosome synthesis) and the rest of cellular functions. Limitations in gene expression also play a role in generating phenotypic diversity, which is advantageous in fluctuating environments, at the expenses of decreasing growth rates. Our current understanding of these trade-offs can be exploited for biotechnological applications benefiting from the selective manipulation of the allocation of resources.

Local manufacturing of protein-based vaccines and therapies in Africa is limited and contributes to a trade deficit, security of supply concerns and poor access to biopharmaceuticals by the poor. Plant molecular farming is a potential technology solution that has received growing adoption by African scientists attracted by the potential for the competitive cost of goods, safety and efficacy. Plant-made pharmaceutical technologies for veterinary and human vaccination and treatment of non-communicable and infectious diseases are available at different stages of development in Africa. There is also growth in the translation of these technologies to commercial operations. Africa is poised to benefit from the real-world impact of molecular farming in the next few years.

Plants are routinely utilized as efficient production platforms for the development of anti-cancer biologics leading to novel anti-cancer vaccines, immunotherapies, and drug-delivery modalities. Various biosimilar/biobetter antibodies and immunogens based on tumor-associated antigens have been produced and optimized for plant expression. Plant virus nanoparticles, including those derived from cowpea mosaic virus or tobacco mosaic virus in particular have shown promise as immunotherapies stimulating tumor-associated immune cells and as drug carriers delivering conjugated chemotherapeutics effectively to tumors. Advancements have also been made toward the development of lectins that can selectively recognize cancer cells. The ease at which plant systems can be utilized for the production of these products presents an opportunity to further develop novel and exciting anti-cancer biologics.

The unknown immune stimulation by nucleic acid nanoparticles (NANPs) has become one of the major impediments to a broad spectrum of clinical developments of this novel technology. Having evolved to defend against bacterial and viral nucleic acids, mammalian cells have established patterns of recognition that are also the pathways through which NANPs can be processed. Explorations into the immune stimulation brought about by a vast diversity of known NANPs have shown that variations in design correlate with variations in immune response. Therefore, as the mechanisms of stimulation are further elucidated, these trends are now being taken into account in the design phase to allow for development of NANPs that are tailored for controlled immune activation or quiescence.

Microorganisms produce extracellular compounds that affect the final product quality in fermentation processes. Selection of overproducing mutants requires coupling of the extracellular product to the producer genotype, which can be achieved by single-cell compartmentalization. Emulsions contain up to billions of microdroplets/mL which significantly increases the screening throughput compared to microtiter-plate-based selections. Factors affecting the success of screening in microdroplets include the nature of the producing organism (robust, no invasive growth), the product (not soluble in oil) and the product assay (preferably fluorescence based). Together these factors determine the required microdroplet production technique and sorting set-up. Because microdroplets allow relatively inexpensive ultrahigh-throughput screening, they are likely to become a standard tool in the strain selection toolbox of the fermentation industry.

Resistant starch has received a lot of attention for its potential to exert a healthy impact on the gut and certain members of its resident microbiota, particularly through enhanced butyrate production. However, resistant starch is a broad category that encompasses several structurally different starches. While all resist digestion by human enzymes, they differ in their effects on the microbiota. Individual variation in microbiota composition also has a substantial influence on butyrate production. Research on this interaction between resistant starch and the microbiota is using in vitro fermentations, cross-over design clinical trials and mouse studies with isotopically labeled starch. These studies are demonstrating that more personalized approaches are needed for finding resistant starch or other fiber that will promote a healthy gut.

Molecular farming increasingly uses the tobacco relative Nicotiana benthamiana for production of recombinant proteins through transient expression. Several proteins are produced efficiently with this expression platform, but yields for other proteins are often very low. These low yields are frequently due to endogenous proteases. The latest genome annotations indicate that N. benthamiana encodes for at least 1243 putative proteases that probably act redundantly and consecutively on substrates in different subcellular compartments. Here, we discuss the N. benthamiana protease repertoire that may affect recombinant protein production and recent advances in protease depletion strategies to increase recombinant protein production in N. benthamiana.

Interest in applications and benefits that Molecular Pharming might offer to Low and Middle Income Countries has always been a potent driver for the research discipline, and a major reason why many scientists entered the field. Although enthusiasm remains high, the reality is that such a game-changing innovation would always take longer than traditional uptake of new technology in developed countries, and be complicated by external factors beyond technical feasibility. Excitingly, signs of increasing interest by LMICS in Molecular Pharming are now emerging. Here, three case studies from Thailand, South Africa and Brazil are used to identify some of the key issues when a new investment into Molecular Pharming manufacturing capacity is under consideration. At present, academic research is not necessarily addressing these issues. Only by understanding the concerns, can members of the academic community contribute to helping the development of Molecular Pharming for LMICs by focusing their research efforts appropriately.

Methanol and formate are attractive microbial feedstocks as they can be sustainably produced from CO2 and renewable energy, are completely miscible, and are easy to store and transport. Here, we provide a biochemical perspective on microbial growth and bioproduction using these compounds. We show that anaerobic growth of acetogens on methanol and formate is more efficient than on H2/CO2 or CO. We analyze the aerobic C1 assimilation pathways and suggest that new-to-nature routes could outperform their natural counterparts. We further discuss practical bioprocessing aspects related to growth on methanol and formate, including feedstock toxicity. While challenges in realizing sustainable production from methanol and formate still exist, the utilization of these feedstocks paves the way towards a truly circular carbon economy.

Plant-based fermentations and their microbes provide an underexplored source for novel biotechnological applications. Recent advances in DNA sequencing technologies and analyses of sequencing data highlight that a diverse array of lactic acid bacteria (LAB) frequently dominate these plant fermentations. Because of the long history of safe LAB use in fermented foods, we argue here that various novel probiotic, synbiotic and a range of other industrial applications can be produced based on new insights in the functional and genetic potential of these LAB. To aid in this quest, comparative genomics tools are increasingly available enabling a more rational design of wet-lab experiments to screen for the most relevant properties. This is also true for the exploration of useful enzymatic and (secondary) metabolic production capacities of the LAB that can be isolated from these plant-based fermentations, such as the recent discovery of a cellulase enzyme in specific Lactobacillus plantarum group members.

Bacteriophages (phages) can play a useful role as narrow spectrum antimicrobials in food safety and in food production. Consumer attitudes towards traditional additives have led to a search for natural, potentially clean label, alternatives. At the same time, the rise in antimicrobial resistance has created a need for alternative antimicrobials for disease prevention and treatment in animal husbandry. Phages represent a viable option for both of these applications. We highlight important barriers which should be considered to improve the chance of a positive outcome when using phages in food and food production. These include the feasibility of adding high concentrations of phages, the physico-chemical properties of the food or target, how and when phages are applied, and which phages are chosen.

Microalgae are rapidly growing, low-input requiring, sun light-utilizing microorganisms capable of converting carbon dioxide into various natural products, a major portion of which are hydrocarbons. Their cellular compartmentalization and photosynthetic apparatus depend on robust turnover of two hydrocarbon classes, isoprenoids and acyl-lipids. This review summarizes the current understanding of algal hydrocarbon metabolism, including carbon partitioning capacities, the localization and size of precursor pools, environmental effects on flux distribution, and limiting factors towards efficient (heterologous) hydrocarbon production. Questions and challenges regarding our knowledge of algal hydrocarbon metabolism as well as guidelines for systematic engineering are presented. Recent engineering achievements indicate fundamental plasticity in the (heterologous) hydrocarbon metabolism of green algae while highlighting their potential as renewable sources of these products.

Proteases have emerged as a promising class of enzymes to build post-translationally regulated signalling functions in diverse organisms and cell types ranging from simple prokaryotes to higher eukaryotes and in reconstituted systems in vitro. An expanding repertoire of proteases can now be readily configured to build tailored sensors, switches and transducers, and is increasingly facilitating the construction of complex sensory systems for a variety of biotechnological and biomedical applications. This is complemented by an increasing understanding of the fundamental design principles underlying biological signal processing at both protein-level and circuit-level that is now actively probed through synthesis. This review thus aims to summarize and analyse the most promising conceptual and experimental approaches that can be applied to build artificial signalling functions with proteases while highlighting advances, drawbacks and limitations.

Biotechnological exploitation of mosses has several aspects, for example, the use of moss extracts or the whole plant for diverse industrial applications as well as their employment as production platforms for valuable metabolites or pharmaceutical proteins, especially using the genetically and developmentally best-characterised model moss Physcomitrella patens. Whole moss plants, in particular peat mosses (Sphagnum spec.), are useful for environmental approaches, biomonitoring of environmental pollution and CO2-neutral 'farming' on rewetted bogs to combat climate change. In addition, the lifestyle of mosses suggests the evolution of genes necessary to cope with biotic and abiotic stress situations, which could be applied to crop plants, and their structural features bear an inspiring potential for biomimetics approaches.

Because of their biofuel producing capabilities, algae (including microalgae and cyanobacteria) are effective and sustainable tools to attain energy security with a growing world population and for reduction of our current reliance on fossil fuels. Algal metabolic and genetic engineering could provide substantial advancements in producing novel and promising strains for the production of alternative biofuels. In this review, we have highlighted biotechnological strategies for microalgae and cyanobacteria that target the improvement of: (1) biosynthesis of biofuel precursors (fatty acid, TAGs, and lipids etc.), (2) carbon-capture ability to accumulate more lipids, and (3) engineering hydrogenases for augmented production of biohydrogen. Other strategies for improving quality and quantity of algal biofuels are also explored.

Traditional biotechnological applications of microorganisms employ mono-cultivation or co-cultivation in well-mixed vessels disregarding the potential of spatially organized cultures. Metabolic specialization and guided species interactions facilitated through spatial isolation would enable consortia of microbes to accomplish more complex functions than currently possible, for bioproduction as well as biodegradation processes. Here, we review concepts of spatially linked microbial consortia in which spatial arrangement is optimized to increase control and facilitate new species combinations. We highlight that genome-scale metabolic network models can inform the design and tuning of synthetic microbial consortia and suggest that a standardized assembly of such systems allows the combination of 'incompatibles', potentially leading to countless novel applications.

Microbial electrosynthesis (MES) is an electrochemical process used to drive microbial metabolism for bio-production, such as the reduction of CO2 into industrially relevant organic products as an alternative to current fossil-fuel-derived commodities. After a decade of research on MES from CO2, figures of merit have increased significantly but are plateauing yet far from those expected to allow competitiveness for synthesis of commodity chemicals. Here we discuss the substantial technological shortcomings still associated with MES and evoke possible ways to mitigate them. It appears particularly challenging to obtain both relevant production rates (driven by high current densities) and energy conversion efficiency (i.e. low cell voltage) in microbial-compatible electrolytes. More competitive processes could arise by decoupling effective abiotic electroreductions (e.g. CO2 to CO or ethanol; H2 evolution) with subsequent fermentation processes.

The production of biopharmaceuticals in plant-based systems had faced several challenges that hampered broader adoption of this technology. In recent years, various plant production hosts have been improved by genetic engineering approaches to overcome obstacles with regard to post-translational modifications and integrity of target proteins. Together with optimized extraction and purification processes, those advances have put plant molecular farming in a more competitive position compared to established production systems. Certain biopharmaceuticals can be derived from plant systems with unique desired properties, qualifying them as biobetters.

CRISPR/Cas is in the process of inducing the biggest transformation of plant breeding since the green revolution. Whereas initial efforts focused mainly on changing single traits by error prone non-homologous end joining, the last two years saw a tremendous technical progress achieving more complex genetic, epigenetic and transcriptional changes. The efficiencies of inducing directed changes by homologous recombination have been improved significantly and strategies to break genetic linkages by inducing chromosomal rearrangements have been developed. Cas13 systems have been applied to degrade viral and mRNA in plants. Most importantly, a historical breakthrough was accomplished: By introducing multiple genomic changes simultaneously, domestication of wild species in a single generation has been demonstrated, speeding up breeding dramatically.

The application of site directed nucleases (SDN) for Genome Editing (GE) in plant breeding and research increases exponentially in the last few years. The main research so far was on 'proof of concept' studies or improvement of the precision and delivery of the SDN. Nevertheless, a reasonable amount of research is present on market-oriented applications for cash crops such as rice but also for commercially lesser interesting crops and vegetables. Reported field trials involving GE plants are scarce around the world and almost not existing in Europe. This is due to the regulatory landscape for GE plants, which is quite distinct and especially in the European Union very demanding. By far the most field trials involve GE rice varieties in the Asian area, followed up by tomato and other vegetables and crops.

Drinking water biofiltration processes have evolved over time, moving from unintentional to deliberate, with careful filter media selection, nutrient and trace metal supplementation, oxidant amendment, and bioaugmentation of key microorganisms, to achieve improvements in water quality. Biofiltration is on the precipice of a revolution that aims to customize the microbial community for targeted functional outcomes. These outcomes might be to enhance or introduce target functional activity for contaminant removal, to avoid hydraulic challenges, or to shape beneficially the downstream microbial community. Moving from the foundational molecular techniques that are commonly applied to biofiltration processes, such as amplicon sequencing and quantitative, real-time polymerase chain reaction, the biofiltration revolution will be facilitated by modern biotechnological tools, including metagenomics, metatranscriptomics, and metaproteomics. The application of such tools will provide a rich knowledge base of microbial community structure/function data under various water quality and operational conditions, where this information will be utilized to select biofilter conditions that promote the enrichment and maintenance of microorganisms with the desired functions.

Recent anaerobic digestion studies commonly attribute performance improvements (e.g. increased methane production, enhanced process stability, reduced startup times) to direct interspecies electron transfer (DIET), even though only indirect evidence of DIET is available and DIET alone does not explain enhanced performance in many cases. This review evaluates methods believed to confirm the occurrence of DIET in anaerobic systems. 16S rRNA gene sequencing and meta-omics approaches are necessary to further DIET knowledge but are limited in their ability to confirm the occurrence of DIET. In situ use of cyclic voltammetry should be explored further, as well as microscopy and image analysis procedures to quantify stained cytochromes. Furthermore, linking interspecies distance, interspecies mixing, and cellular activity to a DIET-based electron transfer model is promising but needs further validation for anaerobic digestion systems. In short, a combination of methods is necessary to confirm the occurrence and expand our knowledge of DIET.

Biotreatment technologies offer many advantages for passive stormwater treatment before harvesting, but performance can be variable and sensitive to system design, construction, operation and maintenance. While there is substantial research underpinning pollutant removal, hydraulic function, internal processes and optimal design, specific focus upon stormwater harvesting is relatively limited. Recent advances in system design include testing media amendments for targeted pollutant removal, enhanced pathogen removal using antimicrobial plants, and broadening technology application. However, the production of reliable fit-for-purpose water requires the development of robust validation methodologies to meet public safety expectations. While foundation studies exist, more needs to be done to extend the validation framework, monitor and control system performance and operation in real-time, and apply standards and regulatory checks.

Currently the production of liquid biofuels relies on plant biomass, which in turn depends on the photosynthetic conversion of light and CO2 into chemical energy. As a consequence, the process is renewable on a far shorter time-scale than its fossil counterpart, thus rendering a potential to reduce the environmental impact of the transportation sector. However, the global economy is not intensively pursuing this route, as current generation biofuel production does not meet two key criteria: (1) economic feasibility and (2) long-term sustainability. Herein, we argue that microalgal systems are valuable alternatives to consider, although it is currently technologically immature and therefore not possible to reach criterion 1, nor evaluate criterion 2. In this review we discuss the major limiting factors for this technology and highlight how further research efforts could be deployed to concretize an industrial reality.

Increasing access to piped water in low-income and middle-income countries combined with the many factors that threaten our drinking water supply infrastructure mean that intermittent water supply (IWS) will remain a common practice around the world. Common features of IWS include water stagnation, pipe drainage, intrusion, backflow, first flush events, and household storage. IWS has been shown to cause degradation as measured by traditional microbial water quality indicators. In this review, we build on new insights into the microbial ecology of continuous water supply systems revealed by sequencing methods to speculate about how intermittent supply conditions may further influence the drinking water microbiome, and identify priorities for future research.

Accumulation of plastic pollution in aquatic ecosystems is the predictable result of high demand for plastic functionalities, optimized production with economies of scale, and recalcitrance. Strategies are needed for end-of-life conversion of recalcitrant plastics into useful feedstocks and for transition to materials that are biodegradable, non-bioaccumulative, and non-toxic. Promising alternatives are the polyhydroxyalkanoates (PHAs), a vast family of polymers amenable to decentralized production from renewable feedstocks. Establishment of a global-scale PHA-based industry will require identification of PHAs with tailored properties for use as 'drop-in' replacements for existing plastics; use of low-cost renewable/waste-derived feedstocks; high productivity cultures that may be genetically modified microorganisms or non-axenic mixed cultures maintained by selection pressures that favor high PHA-producing strains; and low-cost extraction/purification schemes.

Microbial immigration from sewers to wastewater treatment systems is attracting increasing attention for understanding community assembly mechanisms, and improving process modeling and operation. While there is no consensus on approaches to analyze immigration, we suggest to classify them as relevant to either rare (non-observable) diffusive immigration or to time-continuous high-rate mass flow immigration (i.e. mass effects). When analyzed by a mass flow approach, heterotrophs appear to be strongly influenced by deterministic selection, suggesting that the heterotrophs should be subdivided into several functional guilds when assessing their assembly mechanisms. Conversely, nitrifiers appear to transfer neutrally from sewer to activated sludge, and this immigration can restore full nitrification in otherwise non-nitrifying reactors. With further refinement, these findings could be included in predictive process models with various objectives.

Wastewater infrastructure is designed, in part, to remove microorganisms. However, many microorganisms are able to colonize infrastructure and resist treatment, resulting in an enormous flux of microorganisms to urban adjacent waters. These urban-associated microorganisms are discharged through three primary routes 1) failing infrastructure, 2) stormwater, and 3) treated wastewater effluent. Bacterial load estimates indicate failing infrastructure should be considered an equivalent source of microbial pollution as the other routes, but overall discharges are not well parameterized. More sophisticated methods, such as machine learning algorithms and microbiome characterization, are now used to track urban-derived microorganisms, including targets beyond fecal indicators, but development of methods to quantify the impact of these microbes/genes on human and ecosystem health is needed.

Mixed culture anaerobic processes are important to environmental systems, including the global carbon cycle, and industrial and environmental biotechnology. Mixed culture metabolic modelling (MM) is an essential tool to analyse these systems. MM predicts microbial function based on knowledge or assumption of cellular metabolism. It may be developed based on observations at the process level - biochemical process modelling (BPM) or fundamental knowledge of the cell being modelled - cellular level modelling (CLM). There is a substantial gap between these two fields, with BPM not considering genetic constraints, particularly where this may be important to interspecies interactions (e.g. amino acid transfer), and CLM commonly not considering mass transfer principles, such as advection/diffusion/migration. No unified approach is useful for all applications, but there is an increasing need to consider genetic information and constraints in developing BPM, and translate BPM principles (including mass-transfer and inorganic chemistry) for application to CLM.

Nontuberculous mycobacteria (NTM) pulmonary infections are a growing concern worldwide, with a disproportionate incidence in persons with pre-existing health conditions. NTM have frequently been found in municipally-treated drinking water and building plumbing, leading to the hypothesis that an important source of NTM exposure is drinking water. The identification and quantification of NTM in environmental samples are complicated by genetic variability among NTM species, making it challenging to determine if clinically relevant NTM are present. Additionally, their unique cellular features and lifestyles make NTM and their nucleic acids difficult to recover. This review highlights a recent work focused on quantification and characterization of NTM and on understanding the influence of source water, treatment plants, distribution systems, and building plumbing on the abundance of NTM in drinking water.