Microbial diversity and genomics in aid of bioenergy

In view of the realization that fossil fuels reserves are limited, various options of generating energy are being explored. Biological methods for producing fuels such as ethanol, diesel, hydrogen (H₂), methane, etc. have the potential to provide a sustainable energy system for the society. Biological H₂ production appears to be the most promising as it is non-polluting and can be produced from water and biological wastes. The major limiting factors are low yields, lack of industrially robust organisms, and high cost of feed. Actually, H₂ yields are lower than theoretically possible yields of 4 mol/mol of glucose because of the associated fermentation products such as lactic acid, propionic acid and ethanol. The efficiency of energy production can be improved by screening microbial diversity and easily fermentable feed materials. Biowastes can serve as feed for H₂ production through a set of microbial consortia: (1) hydrolytic bacteria, (2) H₂ producers (dark fermentative and photosynthetic). The efficiency of the bioconversion process may be enhanced further by the production of value added chemicals such as polydroxyalkanoate and anaerobic digestion. Discovery of enormous microbial diversity and sequencing of a wide range of organisms may enable us to realize genetic variability, identify organisms with natural ability to acquire and transmit genes. Such organisms can be exploited through genome shuffling for transgenic expression and efficient generation of clean fuel and other diverse biotechnological applications. JIMB 2008: BioEnergy-Special issue     

Dealing with water stress and microbial preservation

The relevance of preserving microorganisms has been well accepted for several decades. Interest is now shifting towards investigating adequate preservation methods to improve microbial survival rates and to preserve new taxa of previously considered unculturable microorganisms. In addition, a growing interest in preserving fragile microbial consortia or communities with biotechnological interest motivates the improvement of preservation methods. In the present study, we reviewed the effect of water availability in microbial diversity shift. We describe the effect of drought on microorganisms at the molecular level and their molecular responses to this life-threatening challenge focusing on the production of xeroprotectants. We also review the interspecies interactions of those drought-tolerant microorganisms with other sensitive organisms including neighbouring prokaryotes and eukaryotes such as plants, and the potential role of these microorganisms at determining the ecological composition of stressed environments. We emphasize the importance of applying the knowledge derived from the molecular mechanisms used by desiccation-tolerant microorganisms for the improvement of the preservation techniques. An overview of the current and newer techniques for preserving microorganisms and microbial communities is provided. The biotechnological interest in preserving pure cultures, microbial consortia and communities is also discussed.     

人工电活性微生物菌群的设计与应用

包括产电菌群和噬电菌群的人工电活性微生物菌群(synthetic electroactive microbial consortia)通过菌种间的物质能量级联反应介导化学能与(光)电能间的相互转化，其可利用底物来源广泛、双向电子传递速率快、环境稳定性强，在清洁电能开发、废水处理、环境修复、生物固碳固氮以及生物燃料、无机纳米材料、高聚物等高值化学品合成等多个领域具有广泛的应用前景。针对人工电活性微生物菌群设计、构建与应用，本文总结电活性微生物菌群界面电子传递和种间电子传递机制，概括基于“劳力分工”原理设计构建人工电活性微生物菌群物质能量级联反应基本架构，总结菌群关系与菌群生态位优化等人工电活性微生物菌群工程化策略，分类列举人工电活性微生物菌群在利用廉价生物质产电、生物光伏固碳产电，光驱噬电生物菌群固氮等相关应用。最后对人工电活性微生物菌群未来研究方向进行了展望。     

Consortia of cyanobacteria/microalgae and bacteria: biotechnological potential

Microbial metabolites are of huge biotechnological potential and their production can be coupled with detoxification of environmental pollutants and wastewater treatment mediated by the versatile microorganisms. The consortia of cyanobacteria/microalgae and bacteria can be efficient in detoxification of organic and inorganic pollutants, and removal of nutrients from wastewaters, compared to the individual microorganisms. Cyanobacterial/algal photosynthesis provides oxygen, a key electron acceptor to the pollutant-degrading heterotrophic bacteria. In turn, bacteria support photoautotrophic growth of the partners by providing carbon dioxide and other stimulatory means. Competition for resources and cooperation for pollutant abatement between these two guilds of microorganisms will determine the success of consortium engineering while harnessing the biotechnological potential of the partners. Relative to the introduction of gene(s) in a single organism wherein the genes depend on the regulatory- and metabolic network for proper expression, microbial consortium engineering is easier and achievable. The currently available biotechnological tools such as metabolic profiling and functional genomics can aid in the consortium engineering. The present review examines the current status of research on the consortia, and emphasizes the construction of consortia with desired partners to serve a dual mission of pollutant removal and commercial production of microbial metabolites.     

An overview of lipid metabolism in yeasts and its impact on biotechnological processes

High energy prices, depletion of crude oil supplies, and price imbalance created by the increasing demand of plant oils or animal fat for biodiesel and specific lipid derivatives such as lubricants, adhesives, and plastics have given rise to heated debates on land-use practices and to environmental concerns about oil production strategies. However, commercialization of microbial oils with similar composition and energy value to plant and animal oils could have many advantages, such as being non-competitive with food, having shorter process cycle and being independent of season and climate factors. This review focuses on the ongoing research on different oleaginous yeasts producing high added value lipids and on the prospects of such microbial oils to be used in different biotechnological processes and applications. It covers the basic biochemical mechanisms of lipid synthesis and accumulation in these organisms, along with the latest insights on the metabolic processes involved. The key elements of lipid accumulation, the mechanisms suspected to confer the oleaginous character of the cell, and the potential metabolic routes enhancing lipid production are also extensively discussed.     

Microbial community composition and dynamics in a semi-industrial-scale facility operating under the MixAlco™ bioconversion platform

Aims:  To monitor microbial community dynamics in a semi‐industrial‐scale lignocellulosic biofuel reactor system and to improve our understanding of the microbial communities involved in the MixAlco? biomass conversion process. Methods and Results:  Reactor microbial communities were characterized at six time points over the course of an 80‐day, mesophilic, semi‐industrial‐scale fermentation using community qPCR and 16S rRNA tag‐pyrosequencing. We found the communities to be dynamic, bacterially dominated consortia capable of changing quickly in response to reactor conditions. Clostridia‐ and Bacteroidetes‐like organisms dominated the reactor communities, but ultimately the communities established consortia containing complementary functional capacities for the degradation of lignocellulosic materials. Eighteen operational taxonomic units were found to share strong correlations with reactor acid concentration and may represent taxa integral to fermentor performance. Conclusions:  The results of this study indicate that the emergence of complementary functional classes within the fermentor consortia may be a trait that is consistent across scales, and they suggest that there may be flexibility with respect to the specific identities of the organisms involved in the fermentor’s degradation and fermentation processes. Significance and Impact of the Study:  This study provides new information regarding the composition, dynamics and potential flexibility of the microbial communities associated with the MixAlco? process and is likely to inform the improvement of this and other applications that employ mixed microbial communities.     

人工微生物混菌系统机制解析中的组学应用及进展

人工微生物混菌系统的生物工程应用价值日益受到重视,使得对于混菌系统中成员菌间的相互作用机制研究也成为近年来的一个热点.其研究结果一方面可以为现有人工混菌系统的进一步优化提供理论依据,另一方面也为全新混菌系统的人工构建提供新的思路和策略,进而促进人工微生物混菌系统未来规模化应用.基因组学、转录组学、蛋白质组学和代谢组学等...     

Engineering microbial consortia: a new frontier in synthetic biology

Microbial consortia are ubiquitous in nature and are implicated in processes of great importance to humans, from environmental remediation and wastewater treatment to assistance in food digestion. Synthetic biologists are honing their ability to program the behavior of individual microbial populations, forcing the microbes to focus on specific applications, such as the production of drugs and fuels. Given that microbial consortia can perform even more complicated tasks and endure more changeable environments than monocultures can, they represent an important new frontier for synthetic biology. Here, we review recent efforts to engineer synthetic microbial consortia, and we suggest future applications.     

Application of gas diffusion electrodes in bioeconomy: An update

The transition of today's fossil fuel based chemical industry toward sustainable production requires improvement of established production processes as well as development of new sustainable and bio-based synthesis routes within a circular economy. Thereby, the combination of electrochemical and biotechnological advantages in such routes represents one important keystone. For the electrochemical generation of reactants from gaseous substrates such as O₂ or CO₂, gas diffusion electrodes (GDE) represent the electrodes of choice since they overcome solubility-based mass transport limitations. Within this article, we illustrate the architecture, function principle and fabrication of GDE. We highlight the application of GDE for conversion of CO₂ using abiotic catalysts for subsequent biosynthesis as well as the application of microbial catalysts at GDE for CO₂ conversion. The reduction of oxygen at GDE is summarized for the application of oxygen depolarized cathodes in microbial fuel cells and generation of H₂O₂ to drive enzymatic reactions. Finally, engineering aspects such as scale-up and the modeling of GDE-based processes are described. This review presents an update on the application of GDE in bio-based production systems and emphasizes their large potential for sustainable development of new pathways in bioeconomy.     

Bacterial cell disruption: a key unit operation in the recovery of intracellular products

The need for microbial cell disruption has hindered the large scale production of commercial biotechnological products of intracellular derivation. The intracellular nature of many recombinant products and the potential use of the bacterial storage product, PHB as a commodity thermoplastic have renewed interest in the improvement of this unit operation. This paper provides a review of processes of a mechanical, physical, chemical or biological nature used for cell disruption on both the laboratory and large scale. Applicability of the techniques to large scale operation is discussed. Modification of existing processes is suggested for the reduction of energy requirements and improved process economics. The requirements for the liberation of granular intracellular products such as inclusion bodies and virus-like yeast particles are distinguished from those for the liberation of soluble products, mainly proteinaceous in nature. The integrated nature of the process with both upstream and downstream processes is addressed. Finally, the recent approach of selective liberation of soluble products of interest is reviewed.     

Biology and biotechnology of Trichoderma

Fungi of the genus Trichoderma are soilborne, green-spored ascomycetes that can be found all over the world. They have been studied with respect to various characteristics and applications and are known as successful colonizers of their habitats, efficiently fighting their competitors. Once established, they launch their potent degradative machinery for decomposition of the often heterogeneous substrate at hand. Therefore, distribution and phylogeny, defense mechanisms, beneficial as well as deleterious interaction with hosts, enzyme production and secretion, sexual development, and response to environmental conditions such as nutrients and light have been studied in great detail with many species of this genus, thus rendering Trichoderma one of the best studied fungi with the genome of three species currently available. Efficient biocontrol strains of the genus are being developed as promising biological fungicides, and their weaponry for this function also includes secondary metabolites with potential applications as novel antibiotics. The cellulases produced by Trichoderma reesei, the biotechnological workhorse of the genus, are important industrial products, especially with respect to production of second generation biofuels from cellulosic waste. Genetic engineering not only led to significant improvements in industrial processes but also to intriguing insights into the biology of these fungi and is now complemented by the availability of a sexual cycle in T. reesei/Hypocrea jecorina, which significantly facilitates both industrial and basic research. This review aims to give a broad overview on the qualities and versatility of the best studied Trichoderma species and to highlight intriguing findings as well as promising applications.     

Developing symbiotic consortia for lignocellulosic biofuel production

The search for petroleum alternatives has motivated intense research into biological breakdown of lignocellulose to produce liquid fuels such as ethanol. Degradation of lignocellulose for biofuel production is a difficult process which is limited by, among other factors, the recalcitrance of lignocellulose and biological toxicity of the products. Consolidated bioprocessing has been suggested as an efficient and economical method of producing low value products from lignocellulose; however, it is not clear whether this would be accomplished more efficiently with a single organism or community of organisms. This review highlights examples of mixtures of microbes in the context of conceptual models for developing symbiotic consortia for biofuel production from lignocellulose. Engineering a symbiosis within consortia is a putative means of improving both process efficiency and stability relative to monoculture. Because microbes often interact and exist attached to surfaces, quorum sensing and biofilm formation are also discussed in terms of consortia development and stability. An engineered, symbiotic culture of multiple organisms may be a means of assembling a novel combination of metabolic capabilities that can efficiently produce biofuel from lignocellulose.     

Novel approaches to energize microbial biocatalysts

An efficient and cheap energization of microbial biocatalysts is essential in current biotechnological processes. A promising alternative to the use of common organic or inorganic electron donors is the semiconductor nanoparticles (SNs) that absorb light and transfer electrons (photoelectrons) behaving as artificial photosynthetic systems (biohybrid systems). Excited photoelectrons generated by illuminated SNs are highly reductive and readily accepted by membrane-bound proteins and electron shuttles to drive specific cell reduction processes and energy generation in microbes. However, the operational mechanisms of these hybrid systems are still poorly understood, especially at the material–microbe interface, and therefore the design and production of efficient biohybrids are challenging. Some major limitations/challenges and future prospects of SNs as microbial energization systems are discussed.     

Application of hyperthermophiles and their enzymes

Enzymes from hyperthermophiles display extreme (thermo)stability and a wide range of enzymes have been examined to explore their potential for various biotechnological processes. In addition, recent years have witnessed the development of genetic systems in a number of hyperthermophilic archaea. This has provided the means to initiate cell engineering studies in these organisms. Biofuel production is now an important topic in microbial biotechnology, and the hydrogen producing capabilities of (hyper)thermophiles, as well as their thermostable hydrogenases, are now attracting much attention.     

Recent advances in plasmid-based tools for establishing novel microbial chassis

A key challenge for domesticating alternative cultivable microorganisms with biotechnological potential lies in the development of innovative technologies. Within this framework, a myriad of genetic tools has flourished, allowing the design and manipulation of complex synthetic circuits and genomes to become the general rule in many laboratories rather than the exception. More recently, with the development of novel technologies such as DNA automated synthesis/sequencing and powerful computational tools, molecular biology has entered the synthetic biology era. In the beginning, most of these technologies were established in traditional microbial models (known as chassis in the synthetic biology framework) such as Escherichia coli and Saccharomyces cerevisiae, enabling fast advances in the field and the validation of fundamental proofs of concept. However, it soon became clear that these organisms, although extremely useful for prototyping many genetic tools, were not ideal for a wide range of biotechnological tasks due to intrinsic limitations in their molecular/physiological properties. Over the last decade, researchers have been facing the great challenge of shifting from these model systems to non-conventional chassis with endogenous capacities for dealing with specific tasks. The key to address these issues includes the generation of narrow and broad host plasmid-based molecular tools and the development of novel methods for engineering genomes through homologous recombination systems, CRISPR/Cas9 and other alternative methods. Here, we address the most recent advances in plasmid-based tools for the construction of novel cell factories, including a guide for helping with “build-your-own” microbial host.     

Oligopolyphenylenevinylene-Conjugated Oligoelectrolyte Membrane Insertion Molecules Selectively Disrupt Cell Envelopes of Gram-Positive Bacteria

The modification of microbial membranes to achieve biotechnological strain improvement with exogenous small molecules, such as oligopolyphenylenevinylene-conjugated oligoelectrolyte (OPV-COE) membrane insertion molecules (MIMs), is an emerging biotechnological field. Little is known about the interactions of OPV-COEs with their target, the bacterial envelope. We studied the toxicity of three previously reported OPV-COEs with a selection of Gram-negative and Gram-positive organisms and demonstrated that Gram-positive bacteria are more sensitive to OPV-COEs than Gram-negative bacteria. Transmission electron microscopy demonstrated that these MIMs disrupt microbial membranes and that this occurred to a much greater degree in Gram-positive organisms. We used a number of mutants to probe the nature of MIM interactions with the microbial envelope but were unable to align the membrane perturbation effects of these compounds to previously reported membrane disruption mechanisms of, for example, cationic antimicrobial peptides. Instead, the data support the notion that OPV-COEs disrupt microbial membranes through a suspected interaction with diphosphatidylglycerol (DPG), a major component of Gram-positive membranes. The integrity of model membranes containing elevated amounts of DPG was disrupted to a greater extent by MIMs than those prepared from Escherichia coli total lipid extracts alone.     

Streamlining of a synthetic co-culture towards an individually controllable one-pot process for polyhydroxyalkanoate production from light and CO2

Rationally designed synthetic microbial consortia carry a vast potential for biotechnological applications. The application of such a consortium in a bioprocess, however, requires tight and individual controllability of the involved microbes. Here, we present the streamlining of a co-cultivation process consisting of Synechococcus elongatus cscB and Pseudomonas putida for the production of polyhydroxyalkanoates (PHA) from light and CO₂. First, the process was improved by employing P. putida cscRABY, a strain with a higher metabolic activity towards sucrose. Next, the individual controllability of the co-culture partners was addressed by providing different nitrogen sources, each exclusively available for one strain. By this, the growth rate of the co-culture partners could be regulated individually, and defined conditions could be set. The molC/molN ratio, a key value for PHA accumulation, was estimated from the experimental data, and the necessary feeding rates to obtain a specific ratio could be predicted. This information was then implemented in the co-cultivation process, following the concept of a DBTL-cycle. In total, the streamlining of the process resulted in an increased maximal PHA titer of 393 mg/L and a PHA production rate of 42.1 mg/(L•day).     

Relevance of microbial coculture fermentations in biotechnology

The purpose of this article is to review coculture fermentations in industrial biotechnology. Examples for the advantageous utilization of cocultures instead of single cultivations include the production of bulk chemicals, enzymes, food additives, antimicrobial substances and microbial fuel cells. Coculture fermentations may result in increased yield, improved control of product qualities and the possibility of utilizing cheaper substrates. Cocultivation of different micro‐organisms may also help to identify and develop new biotechnological substances. The relevance of coculture fermentations and the potential of improving existing processes as well as the production of new chemical compounds in industrial biotechnology are pointed out here by means of more than 35 examples.