Maßgeschneiderte Mikroorganismen

Tailor‐made microorganisms Microbial diversity provides unlimited resources for the development of novel industrial processes and products. Since the beginning of the 20th century microorganisms have been successfully applied for the large scale production of bio‐based products. In recent years, modern methods of strain development and Synthetic Biology have enabled biotech engineers to design even more sophisticated and tailor‐made microorganisms. These microbes serve industrial processes for the production of bulk chemicals, enzymes, polymers, biofuels as well as plant‐derived ingredients such as Artemisinin in an ecologically and economically sustainable and attractive fashion. In the future, production of advanced biofuels, microbial fuel cells, CO₂ as feedstock and microbial cellulose are research topics as well as challenges of global importance. Continuous efforts in microbiology and biotechnology research will be pivotal for white biotechnology to gain more momentum in transforming the chemical industry towards a knowledge based bio‐economy.     

Enhanced benzaldehyde tolerance in Zymomonas mobilis biofilms and the potential of biofilm applications in fine-chemical production

Biotransformation plays an increasingly important role in the industrial production of fine chemicals due to its high product specificity and low energy requirement. One challenge in biotransformation is the toxicity of substrates and/or products to biocatalytic microorganisms and enzymes. Biofilms are known for their enhanced tolerance of hostile environments compared to planktonic free-living cells. Zymomonas mobilis was used in this study as a model organism to examine the potential of surface-associated biofilms for biotransformation of chemicals into value-added products. Z. mobilis formed a biofilm with a complex three-dimensional architecture comprised of microcolonies with an average thickness of 20 microm, interspersed with water channels. Microscopic analysis and metabolic activity studies revealed that Z. mobilis biofilm cells were more tolerant to the toxic substrate benzaldehyde than planktonic cells were. When exposed to 50 mM benzaldehyde for 1 h, biofilm cells exhibited an average of 45% residual metabolic activity, while planktonic cells were completely inactivated. Three hours of exposure to 30 mM benzaldehyde resulted in sixfold-higher residual metabolic activity in biofilm cells than in planktonic cells. Cells inactivated by benzaldehyde were evenly distributed throughout the biofilm, indicating that the resistance mechanism was different from mass transfer limitation. We also found that enhanced tolerance to benzaldehyde was not due to the conversion of benzaldehyde into less toxic compounds. In the presence of glucose, Z. mobilis biofilms in continuous cultures transformed 10 mM benzaldehyde into benzyl alcohol at a steady rate of 8.11 g (g dry weight)(-1) day(-1) with a 90% molar yield over a 45-h production period.     

Natural products – learning chemistry from plants

Plant natural products (PNPs) are unique in that they represent a vast array of different structural features, ranging from relatively simple molecules to very complex ones. Given the fact that many plant secondary metabolites exhibit profound biological activity, they are frequently used as fragrances and flavors, medicines, as well as industrial chemicals. As the intricate structures of PNPs often cannot be mimicked by chemical synthesis, the original plant providers constitute the sole source for their industrial, large‐scale production. However, sufficient supply is not guaranteed for all molecules of interest, making the development of alternative production systems a priority. Modern techniques, such as genome mining and thorough biochemical analysis, have helped us gain preliminary understanding of the enzymatic formation of the valuable ingredients in planta. Herein, we review recent advances in the application of biocatalytical processes, facilitating generation of complex PNPs through utilization of plant‐derived specific enzymes and combinatorial biochemistry. We further evaluate the options of employing heterologous organisms harboring PNP biosynthetic pathways for the production of secondary metabolites of interest.     

Assessing the impacts of genetically modified microorganisms

The progression towards greater industrial sustainability involves the analysis of biotechnology as a means of achieving clean or cleaner products and processes. Because living systems manage their chemistry more efficiently than man-made factories, and their wastes tend to be recyclable and biodegradable, they can be expected to be more environmentally clean. Industry has begun to use enzymes instead of traditional catalysts in many industrial production processes. The future holds obstacles as well as opportunities for biotechnological applications. A greater ability to manipulate biological materials and processes will have significant impact on manufacturing industries. A growing proportion of biotechnologyderived processes and products is based on the use of genetically modified microorganisms. This extends the analysis from the aspect of cleanliness to the aspect of safety.     

Synthetic biology advances and applications in the biotechnology industry: a perspective

Synthetic biology is a logical extension of what has been called recombinant DNA (rDNA) technology or genetic engineering since the 1970s. As rDNA technology has been the driver for the development of a thriving biotechnology industry today, starting with the commercialization of biosynthetic human insulin in the early 1980s, synthetic biology has the potential to take the industry to new heights in the coming years. Synthetic biology advances have been driven by dramatic cost reductions in DNA sequencing and DNA synthesis; by the development of sophisticated tools for genome editing, such as CRISPR/Cas9; and by advances in informatics, computational tools, and infrastructure to facilitate and scale analysis and design. Synthetic biology approaches have already been applied to the metabolic engineering of microorganisms for the production of industrially important chemicals and for the engineering of human cells to treat medical disorders. It also shows great promise to accelerate the discovery and development of novel secondary metabolites from microorganisms through traditional, engineered, and combinatorial biosynthesis. We anticipate that synthetic biology will continue to have broadening impacts on the biotechnology industry to address ongoing issues of human health, world food supply, renewable energy, and industrial chemicals and enzymes.     

Development of applications of industrial enzymes from Malaysian indigenous microbial sources

Malaysian enzyme industry is considered almost non-existence, although the import volume is large. Realizing the importance of enzymes, encompassing a wide range of applications in bioindustry, the development of home grown technologies for enzyme production and applications becomes one of the national priorities in industrial biotechnology. Enzyme production from indigenous microbial isolates was performed either by submerged or solid state fermentation processes. Based on its wide and unique spectrum of properties, enzymes have been developed for wide applications in various industrial processes. The development of the enzyme catalysed applications is based on the modification of the reaction systems to enhance their catalytic activities. Some of the applications of the industrial enzymes include the fine chemicals production, oleochemicals modification, detergent formulation, enzymatic drinking of waste papers, animal feed formulation and effluent treatment processes. Enzymes have also shown to be successfully used as analytical tool in the determination of compounds in body fluids. Although, most of these enzyme catalysed reactions were performed in aqueous phase, the use of enzymes in organic solvents was found to be significant for the production of new chemicals.     

Enhanced Benzaldehyde Tolerance in Zymomonas mobilis Biofilms and the Potential of Biofilm Applications in Fine-Chemical Production

Biotransformation plays an increasingly important role in the industrial production of fine chemicals due to its high product specificity and low energy requirement. One challenge in biotransformation is the toxicity of substrates and/or products to biocatalytic microorganisms and enzymes. Biofilms are known for their enhanced tolerance of hostile environments compared to planktonic free-living cells. Zymomonas mobilis was used in this study as a model organism to examine the potential of surface-associated biofilms for biotransformation of chemicals into value-added products. Z. mobilis formed a biofilm with a complex three-dimensional architecture comprised of microcolonies with an average thickness of 20 μm, interspersed with water channels. Microscopic analysis and metabolic activity studies revealed that Z. mobilis biofilm cells were more tolerant to the toxic substrate benzaldehyde than planktonic cells were. When exposed to 50 mM benzaldehyde for 1 h, biofilm cells exhibited an average of 45% residual metabolic activity, while planktonic cells were completely inactivated. Three hours of exposure to 30 mM benzaldehyde resulted in sixfold-higher residual metabolic activity in biofilm cells than in planktonic cells. Cells inactivated by benzaldehyde were evenly distributed throughout the biofilm, indicating that the resistance mechanism was different from mass transfer limitation. We also found that enhanced tolerance to benzaldehyde was not due to the conversion of benzaldehyde into less toxic compounds. In the presence of glucose, Z. mobilis biofilms in continuous cultures transformed 10 mM benzaldehyde into benzyl alcohol at a steady rate of 8.11 g (g dry weight)⁻¹ day⁻¹ with a 90% molar yield over a 45-h production period.     

Application of whole cell rhodococcal biocatalysts in acrylic polymer manufacture

Rhodococci are ubiquitous in nature and their ability to metabolise a wide range of chemicals, many of which are toxic, has given rise to an increasing number of studies into their diverse use as biocatalysts. Indeed rhodococci have been shown to be especially good at degrading aromatic and aliphatic nitriles and amides and thus they are very useful for waste clean up where these toxic chemicals are present.The use of biocatalysts in the chemical industry has in the main been for the manufacture of high-value fine chemicals, such as pharmaceutical intermediates, though investigations into the use of nitrile hydratase, amidase and nitrilase to convert acrylonitrile into the higher value products acrylamide and acrylic acid have been carried out for a number of years. Acrylamide and acrylic acid are manufactured by chemical processes in vast tonnages annually and they are used to produce polymers for applications such as superabsorbents, dispersants and flocculants. Rhodococci are chosen for use as biocatalysts on an industrial scale for the production of acrylamide and acrylic acid due to their ease of growth to high biomass yields, high specific enzyme activities obtainable, their EFB class 1 status and robustness of the whole cells within chemical reaction systems.Several isolates belonging to the genus Rhodococcus have been shown in our studies to be among the best candidates for acrylic acid preparation from acrylonitrile due to their stability and tolerance to high concentrations of this reactive and disruptive substrate. A critical part of the selection procedure for the best candidates during the screening programme was high purity product with very low residual substrate concentrations, even in the presence of high product concentrations. Additionally the nitrile and amide substrate scavenging ability which enables rhodococci to survive very successfully in the environment leads to the formation of biocatalysts which are suitable for the removal of low concentrations of acrylonitrile and acrylamide in waste streams and for the removal of impurities in manufacturing processes.     

Biobased production of alkanes and alkenes through metabolic engineering of microorganisms

Advancement in metabolic engineering of microorganisms has enabled bio-based production of a range of chemicals, and such engineered microorganism can be used for sustainable production leading to reduced carbon dioxide emission there. One area that has attained much interest is microbial hydrocarbon biosynthesis, and in particular, alkanes and alkenes are important high-value chemicals as they can be utilized for a broad range of industrial purposes as well as ‘drop-in’ biofuels. Some microorganisms have the ability to biosynthesize alkanes and alkenes naturally, but their production level is extremely low. Therefore, there have been various attempts to recruit other microbial cell factories for production of alkanes and alkenes by applying metabolic engineering strategies. Here we review different pathways and involved enzymes for alkane and alkene production and discuss bottlenecks and possible solutions to accomplish industrial level production of these chemicals by microbial fermentation.     

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.     

Integrated operation of continuous chromatography and biotransformations for the generic high yield production of fine chemicals

The rapid progress in biocatalysis in the identification and development of enzymes over the last decade has enormously enlarged the chemical reaction space that can be addressed not only in research applications, but also on industrial scale. This enables us to consider even those groups of reactions that are very promising from a synthetic point of view, but suffer from drawbacks on process level, such as an unfavourable position of the reaction equilibrium. Prominent examples stem from the aldolase-catalyzed enantioselective carbon-carbon bond forming reactions, reactions catalyzed by isomerising enzymes, and reactions that are kinetically controlled. On the other hand, continuous chromatography concepts such as the simulating moving bed technology have matured and are increasingly realized on industrial scale for the efficient separation of difficult compound mixtures - including enantiomers - with unprecedented efficiency. We propose that coupling of enzyme reactor and continuous chromatography is a very suitable and potentially generic process concept to address the thermodynamic limitations of a host of promising biotransformations. This way, it should be possible to establish novel in situ product recovery processes of unprecedented efficiency and selectivity that represent a feasible way to recruit novel biocatalysts to the industrial portfolio.     

Cell immobilization for microbial production of 1,3-propanediol

Cell and enzyme immobilization are often used for industrial production of high-value products. In recent years, immobilization techniques have been applied to the production of value-added chemicals such as 1,3-Propanediol (1,3-PDO). Biotechnological fermentation is an attractive alternative to current 1,3-PDO production methods, which are primarily thermochemical processes, as it generates high volumetric yields of 1,3-PDO, is a much less energy intensive process, and generates lower amounts of environmental organic pollutants. Although several approaches including: batch, fed-batch, continuous-feed and two-step continuous-feed were tested in suspended systems, it has been well demonstrated that cell immobilization techniques can significantly enhance 1,3-PDO production and allow robust continuous production in smaller bioreactors. This review covers various immobilization methods and their application for 1,3-PDO production.     

A discussion of microbial genetics in fermentation process development with particular reference to fungi producing high yields of antibiotics

When trying to improve antibiotic processes that are already high yielding, real industrial problems have to be faced. These include the use of organisms with non-ideal growth and recombination cycles, and problems of scale up from the laboratory to the main production plant. Many of the principles derived from academic studies have to be radically modified before they can be applied in the industrial context. These issues are a challenge to those who genuinely wish to contribute to the solution of industrial problems.     

Impact of Aspergillus oryzae genomics on industrial production of metabolites

Aspergillus oryzae is used extensively for the production of the traditional Japanese fermented foods sake (rice wine), shoyu (soy sauce), and miso (soybean paste). In recent years, recombinant DNA technology has been used to enhance industrial enzyme production by A. oryzae. Recently completed genomic studies using expressed sequence tag (EST) analyses and whole-genome sequencing are quickly expanding the industrial potential of the fungus in biotechnology. Genes that have been newly discovered through genome research can be used for the production of novel valuable enzymes and chemicals, and are important for designing new industrial processes. This article describes recent progress of A . oryzae genomics and its impact on industrial production of enzymes, metabolites, and bioprocesses.     

Metabolic engineering of Saccharomyces cerevisiae for production of carboxylic acids: current status and challenges

To meet the demands of future generations for chemicals and energy and to reduce the environmental footprint of the chemical industry, alternatives for petrochemistry are required. Microbial conversion of renewable feedstocks has a huge potential for cleaner, sustainable industrial production of fuels and chemicals. Microbial production of organic acids is a promising approach for production of chemical building blocks that can replace their petrochemically derived equivalents. Although Saccharomyces cerevisiae does not naturally produce organic acids in large quantities, its robustness, pH tolerance, simple nutrient requirements and long history as an industrial workhorse make it an excellent candidate biocatalyst for such processes. Genetic engineering, along with evolution and selection, has been successfully used to divert carbon from ethanol, the natural endproduct of S. cerevisiae , to pyruvate. Further engineering, which included expression of heterologous enzymes and transporters, yielded strains capable of producing lactate and malate from pyruvate. Besides these metabolic engineering strategies, this review discusses the impact of transport and energetics as well as the tolerance towards these organic acids. In addition to recent progress in engineering S. cerevisiae for organic acid production, the key limitations and challenges are discussed in the context of sustainable industrial production of organic acids from renewable feedstocks.     

Reprogramming <Emphasis Type="Italic">Halomonas</Emphasis> for industrial production of chemicals

Halomonas spp. are able to grow under a high salt concentration at alkali pH, they are able to resist contamination by other microbes. Development of Halomonas spp. as platform production strains for the next-generation industrial biotechnology (NGIB) is intensively studied. Among Halomonas spp., Halomonas bluephagenesis is the best studied one with available engineering tools and methods to reprogram it for production of various polyhydroxyalkanoates, proteins, and chemicals. Due to its contamination resistance, H. bluephagenesis can be grown under open and continuous processes not just in the labs but also in at least 1000 L fermentor scale. It is expected that NGIB based on Halomonas spp. be able to engineer for production of increasing number of products in a competitive manner.     

Engineered catalytic biofilms for continuous large scale production of n‐octanol and (S)‐styrene oxide

This study evaluates the technical feasibility of biofilm‐based biotransformations at an industrial scale by theoretically designing a process employing membrane fiber modules as being used in the chemical industry and compares the respective process parameters to classical stirred‐tank studies. To our knowledge, catalytic biofilm processes for fine chemicals production have so far not been reported on a technical scale. As model reactions, we applied the previously studied asymmetric styrene epoxidation employing Pseudomonas sp. strain VLB120ΔC biofilms and the here‐described selective alkane hydroxylation. Using the non‐heme iron containing alkane hydroxylase system (AlkBGT) from P. putida Gpo1 in the recombinant P. putida PpS81 pBT10 biofilm, we were able to continuously produce 1‐octanol from octane with a maximal productivity of 1.3 g L day^?1 in a single tube micro reactor. For a possible industrial application, a cylindrical membrane fiber module packed with 84,000 polypropylene fibers is proposed. Based on the here presented calculations, 59 membrane fiber modules (of 0.9 m diameter and 2 m length) would be feasible to realize a production process of 1,000 tons/year for styrene oxide. Moreover, the product yield on carbon can at least be doubled and over 400‐fold less biomass waste would be generated compared to classical stirred‐tank reactor processes. For the octanol process, instead, further intensification in biological activity and/or surface membrane enlargement is required to reach production scale. By taking into consideration challenges such as biomass growth control and maintaining a constant biological activity, this study shows that a biofilm process at an industrial scale for the production of fine chemicals is a sustainable alternative in terms of product yield and biomass waste production. Biotechnol. Bioeng. 2013; 110: 424–436. © 2012 Wiley Periodicals, Inc.     

Interdependence between lignocellulosic biomasses,enzymatic hydrolysis and yeast cell factories in biorefineries

Biorefineries have a pivotal role in the bioeconomy scenario for the transition from fossil-based processes towards more sustainable ones relying on renewable resources. Lignocellulose is a prominent feedstock since its abundance and relatively low cost. Microorganisms are often protagonists of biorefineries, as they contribute both to the enzymatic degradation of lignocellulose complex polymers and to the fermentative conversion of the hydrolyzed biomasses into fine and bulk chemicals. Enzymes have therefore become crucial for the development of sustainable biorefineries, being able to provide nutrients to cells from lignocellulose. Enzymatic hydrolysis can be performed by a portfolio of natural enzymes that degrade lignocellulose, often combined into cocktails. As enzymes can be deployed in different operative settings, such as separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF), their characteristics need to be combined with microbial ones to maximize the process. We therefore reviewed how the optimization of lignocellulose enzymatic hydrolysis can ameliorate bioethanol production when Saccharomyces cerevisiae is used as cell factory. Expanding beyond biofuels, enzymatic cocktail optimization can also be pivotal to unlock the potential of non-Saccharomyces yeasts, which, thanks to broader substrate utilization, inhibitor resistance and peculiar metabolism, can widen the array of feedstocks and products of biorefineries.     

Biofilms and fluidized bed fermentation

The exploitation of biofilms in industrial processes such as wastewater treatment and metabolite production is described. In this way it is intended to highlight the positive aspects of biofilms, and provide a contrast with the negative associations which these microbial aggregations normally have. In particular, the exploitation of adhesive microbes in fluidized bed operation is discussed. A range of processes is outlined, from wastewater treatment to the production of ethanol, enzymes, and antibiotics. These various processes use a range of cell types; which has required the modification of the basic design of FB bioreactor, e.g. for anaerobic or aerobic operation; and for bacteria, actinomycetes, or plant cells. One basic design is illustrated, and reference made to how this is modified for different fermentations.