Biotechnological advantages of laboratory-scale solid-state fermentation with fungi

Despite the increasing number of publications dealing with solid-state (substrate) fermentation (SSF) it is very difficult to draw general conclusion from the data presented. This is due to the lack of proper standardisation that would allow objective comparison with other processes. Research work has so far focused on the general applicability of SSF for the production of enzymes, metabolites and spores, in that many different solid substrates (agricultural waste) have been combined with many different fungi and the productivity of each fermentation reported. On a gram bench-scale SSF appears to be superior to submerged fermentation technology (SmF) in several aspects. However, SSF up-scaling, necessary for use on an industrial scale, raises severe engineering problems due to the build-up of temperature, pH, O₂, substrate and moisture gradients. Hence, most published reviews also focus on progress towards industrial engineering. The role of the physiological and genetic properties of the microorganisms used during growth on solid substrates compared with aqueous solutions has so far been all but neglected, despite the fact that it may be the microbiology that makes SSF advantageous against the SmF biotechnology. This review will focus on research work allowing comparison of the specific biological particulars of enzyme, metabolite and/or spore production in SSF and in SmF. In these respects, SSF appears to possess several biotechnological advantages, though at present on a laboratory scale only, such as higher fermentation productivity, higher end-concentration of products, higher product stability, lower catabolic repression, cultivation of microorganisms specialized for water-insoluble substrates or mixed cultivation of various fungi, and last but not least, lower demand on sterility due to the low water activity used in SSF.     

FLO gene-dependent phenotypes in industrial wine yeast strains

Most commercial yeast strains are nonflocculent. However, controlled flocculation phenotypes could provide significant benefits to many fermentation-based industries. In nonflocculent laboratory strains, it has been demonstrated that it is possible to adjust flocculation and adhesion phenotypes to desired specifications by altering expression of the otherwise silent but dominant flocculation (FLO) genes. However, FLO genes are characterized by high allele heterogeneity and are subjected to epigenetic regulation. Extrapolation of data obtained in laboratory strains to industrial strains may therefore not always be applicable. Here, we assess the adhesion phenotypes that are associated with the expression of a chromosomal copy of the FLO1, FLO5, or FLO11 open reading frame in two nonflocculent commercial wine yeast strains, BM45 and VIN13. The chromosomal promoters of these genes were replaced with stationary phase-inducible promoters of the HSP30 and ADH2 genes. Under standard laboratory and wine making conditions, the strategy resulted in expected and stable expression patterns of these genes in both strains. However, the specific impact of the expression of individual FLO genes showed significant differences between the two wine strains and with corresponding phenotypes in laboratory strains. The data suggest that optimization of the flocculation pattern of individual commercial strains will have to be based on a strain-by-strain approach.     

Biotechnological production of feed nucleotides by microbial strain improvement

Sodium salts of inosine monophosphate (IMP) and guanosine monophosphate (GMP) are potent flavour enhancers. They are widely used as food additives in combination with monosodium glutamate (MSG) to synergistically increase umami flavour. In recent years, both inosine and guanosine derivatives have gained further importance because of their beneficial effects, related to their antioxidant, neuroprotective, cardiotonic and immunomodulatory properties. The industrial production of both IMP and GMP is mainly achieved either by RNA breakdown and nucleotide extraction or by microbial fermentation using different microorganisms such as Corynebacterium, Bacillus, or Escherichia coli. This work reviews the metabolic pathways and regulatory networks of purine synthesis, including both IMP and GMP, and the biotechnological processes applied to the production of these compounds, ranging from classical random mutagenesis to rational design by metabolic engineering. Recent advances of systems biology approaches, along with the rapid development of synthetic biology, may offer a basis for future manipulations to further increase the productivity of the fermentation processes.     

Perspectives on biotechnological applications of archaea

Many archaea colonize extreme environments. They include hyperthermophiles, sulfur-metabolizing thermophiles, extreme halophiles and methanogens. Because extremophilic microorganisms have unusual properties, they are a potentially valuable resource in the development of novel biotechnological processes. Despite extensive research, however, there are few existing industrial applications of either archaeal biomass or archaeal enzymes. This review summarizes current knowledge about the biotechnological uses of archaea and archaeal enzymes with special attention to potential applications that are the subject of current experimental evaluation. Topics covered include cultivation methods, recent achievements in genomics, which are of key importance for the development of new biotechnological tools, and the application of wild-type biomasses, engineered microorganisms, enzymes and specific metabolites in particular bioprocesses of industrial interest.     

Metabolic engineering of bacteria

Yield and productivity are critical for the economics and viability of a bioprocess. In metabolic engineering the main objective is the increase of a target metabolite production through genetic engineering. Metabolic engineering is the practice of optimizing genetic and regulatory processes within cells to increase the production of a certain substance. In the last years, the development of recombinant DNA technology and other related technologies has provided new tools for approaching yields improvement by means of genetic manipulation of biosynthetic pathway. Industrial microorganisms like Escherichia coli, Actinomycetes, etc. have been developed as biocatalysts to provide new or to optimize existing processes for the biotechnological production of chemicals from renewable plant biomass. The factors like oxygenation, temperature and pH have been traditionally controlled and optimized in industrial fermentation in order to enhance metabolite production. Metabolic engineering of bacteria shows a great scope in industrial application as well as such technique may also have good potential to solve certain metabolic disease and environmental problems in near future.     

Microbial interactions during sugar cane must fermentation for bioethanol production: does quorum sensing play a role?

Microbial interactions represent important modulatory role in the dynamics of biological processes. During bioethanol production from sugar cane must, the presence of lactic acid bacteria (LAB) and wild yeasts is inevitable as they originate from the raw material and industrial environment. Increasing the concentration of ethanol, organic acids, and other extracellular metabolites in the fermentation must are revealed as wise strategies for survival by certain microorganisms. Despite this, the co-existence of LAB and yeasts in the fermentation vat and production of compounds such as organic acids and other extracellular metabolites result in reduction in the final yield of the bioethanol production process. In addition to the competition for nutrients, reduction of cellular viability of yeast strain responsible for fermentation, flocculation, biofilm formation, and changes in cell morphology are listed as important factors for reductions in productivity. Although these consequences are scientifically well established, there is still a gap about the physiological and molecular mechanisms governing these interactions. This review aims to discuss the potential occurrence of quorum sensing mechanisms between bacteria (mainly LAB) and yeasts and to highlight how the understanding of such mechanisms can result in very relevant and useful tools to benefit the biofuels industry and other sectors of biotechnology in which bacteria and yeast may co-exist in fermentation processes.     

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.     

Application of rRNA-targeted oligonucleotide probes in biotechnology

Ribosomal RNA-targeted oligonucleotide probes have become valuable tools for the detection of microorganisms involved in important biotechnological processes. Microorganisms which are of major importance for processes such as wastewater treatment, microbial leaching or methane production can be detected and quantified in situ within a complex microbial community. For certain processes, such as nitrification or biological phosphate removal, new microorganisms have become the focus of interest and have led to an improved understanding of these bioremediation techniques. Hybridization techniques have become fast and reliable alternatives to conventional cultivation techniques in the food industry as a control method for starter cultures for fermentation processes or product control. Recent analytical tools such as flow cytometry and digital image processing have improved the efficiency of these techniques. This review is intended to present a summary of methodological aspects of rRNA-based hybridization techniques and their application in biotechnology.     

Co-Flocculation of Yeast Species,a New Mechanism to Govern Population Dynamics in Microbial Ecosystems

Flocculation has primarily been studied as an important technological property of Saccharomyces cerevisiae yeast strains in fermentation processes such as brewing and winemaking. These studies have led to the identification of a group of closely related genes, referred to as the FLO gene family, which controls the flocculation phenotype. All naturally occurring S. cerevisiae strains assessed thus far possess at least four independent copies of structurally similar FLO genes, namely FLO1, FLO5, FLO9 and FLO10. The genes appear to differ primarily by the degree of flocculation induced by their expression. However, the reason for the existence of a large family of very similar genes, all involved in the same phenotype, has remained unclear. In natural ecosystems, and in wine production, S. cerevisiae growth together and competes with a large number of other Saccharomyces and many more non-Saccharomyces yeast species. Our data show that many strains of such wine-related non-Saccharomyces species, some of which have recently attracted significant biotechnological interest as they contribute positively to fermentation and wine character, were able to flocculate efficiently. The data also show that both flocculent and non-flocculent S. cerevisiae strains formed mixed species flocs (a process hereafter referred to as co-flocculation) with some of these non-Saccharomyces yeasts. This ability of yeast strains to impact flocculation behaviour of other species in mixed inocula has not been described previously. Further investigation into the genetic regulation of co-flocculation revealed that different FLO genes impact differently on such adhesion phenotypes, favouring adhesion with some species while excluding other species from such mixed flocs. The data therefore strongly suggest that FLO genes govern the selective association of S. cerevisiae with specific species of non-Saccharomyces yeasts, and may therefore be drivers of ecosystem organisational patterns. Our data provide, for the first time, insights into the role of the FLO gene family beyond intraspecies cellular association, and suggest a wider evolutionary role for the FLO genes. Such a role would explain the evolutionary persistence of a large multigene family of genes with apparently similar function.     

Amino acids production focusing on fermentation technologies – A review

Amino acids are attractive and promising biochemicals with market capacity requirements constantly increasing. Their applicability ranges from animal feed additives, flavour enhancers and ingredients in cosmetic to specialty nutrients in pharmaceutical and medical fields.This review gives an overview of the processes applied for amino acids production and points out the main advantages and disadvantages of each.Due to the advances made in the genetic engineering techniques, the biotechnological processes, and in particular the fermentation with the aid of strains such as Corynebacterium glutamicum or Escherichia coli, play a significant role in the industrial production of amino acids. Despite the numerous advantages of the fermentative amino acids production, the process still needs significant improvements leading to increased productivity and reduction of the production costs.Although the production processes of amino acids have been extensively investigated in previous studies, a comprehensive overview of the developments in bioprocess technology has not been reported yet. This review states the importance of the fermentation process for industrial amino acids production, underlining the strengths and the weaknesses of the process. Moreover, the potential of innovative approaches utilizing macro and microalgae or bacteria are presented.     

Novel permittivity test for determination of yeast surface charge and flocculation abilities

Yeast flocculation has been found to be important in many biotechnological processes. It has been suggested that flocculation is promoted by decreasing electrostatic repulsion between cells. In this study, we used an unconventional rapid technique—permittivity test—for determination of the flocculation properties and surface charge values of three industrial yeast strains with well-known flocculation characteristics: Saccharomyces cerevisiae NCYC 1017 (brewery, ale), S.?pastorianus NCYC 680 (brewery, lager), and Debaryomyces occidentalis LOCK 0251 (unconventional amylolytic yeast). The measurements of permittivity were compared with the results from two classical methods for determination of surface charge: Alcian blue retention and Sephadex DEAE attachment. The permittivity values for particular strains correlated directly with the results of Alcian blue retention (r?=?0.9). The results also confirmed a strong negative relationship between the capacitance of yeast suspensions and their flocculation abilities. The highest permittivity was noted for the ale strain NCYC 1017, with weak flocculation abilities, and the lowest for the flocculating lager yeast NCYC 680. This paper is the first to describe the possibility of using a rapid permittivity test to evaluate the surface charge of yeast cells and their flocculation abilities. This method is of practical value in various biotechnological industries where flocculation is applied as a major method of cell separation.     

Use of wastes for sophorolipids production as a transition to circular economy: state of the art and perspectives

Chemical processes and petroleum-based chemicals are being substituted by biological processes and bioproducts. Surfactants and biosurfactants are an example of this trend. Among the biosurfactants, sophorolipids (SLs) have excellent surface and interfacial tension properties, which make them ideal to be used in a wide variety of applications. SLs are produced at full scale through submerged fermentation of pure substrates (glucose and oleic acid). However, research trends suggest that there is a lot of interest to produce SLs from waste effluents and other low-cost substrates, both in submerged and solid-state fermentation processes. This study reviews the current research in the production of SLs via fermentation processes, focusing on those using wastes, by-products, or low-cost substrates (liquids or solids). It details the substrates, process variables, microorganisms, and use of supplementary media for batch, fed-batch, and continuous submerged or solid-state fermentation processes. Sophorolipids production based on industrial by-products and waste effluents presents huge potential for its application at an industrial scale in a more economical and environmentally friendly process, boosting the necessary change to circular economy.

     

Flocculation, adhesion and biofilm formation in yeasts

Yeast cells possess a remarkable capacity to adhere to abiotic surfaces, cells and tissues. These adhesion properties are of medical and industrial relevance. Pathogenic yeasts such as Candida albicans and Candida glabrata adhere to medical devices and form drug-resistant biofilms. In contrast, cell-cell adhesion (flocculation) is a desirable property of industrial Saccharomyces cerevisiae strains that allows the easy separation of cells from the fermentation product. Adhesion is conferred by a class of special cell wall proteins, called adhesins. Cells carry several different adhesins, each allowing adhesion to specific substrates. Several signalling cascades including the Ras/cAMP/PKA and MAP kinase (MAPK)-dependent filamentous growth pathways tightly control synthesis of the different adhesins. Together, these pathways trigger adhesion in response to stress, nutrient limitation or small molecules produced by the host, such as auxin in plants or NAD in mammals. In addition, adhesins are subject to subtelomeric epigenetic switching, resulting in stochastic expression patterns. Internal tandem repeats within adhesin genes trigger recombination events and the formation of novel adhesins, thereby offering fungi an endless reservoir of adhesion properties. These aspects of fungal adhesion exemplify the impressive phenotypic plasticity of yeasts, allowing them to adapt quickly to stressful environments and exploit new opportunities.     

A perspective on the biotechnological potential of extremophiles.

It is well recognized that many environments considered by man to be extreme are colonized by microorganisms which are specifically adapted to these ecological niches. A diverse range of bacteria, cyanobacteria, algae and yeasts have been isolated from such habitats and it is now widely accepted that these microorganisms provide a valuable resource not only for exploitation in novel biotechnological processes but also as models for investigating how biomolecules are stabilized when subjected to extreme conditions. This short review summarizes our current state of knowledge of this unique group of microorganisms and their enzymes, and attempts to identify their future biotechnological potential.     

果胶酶生产及工业应用进展

果胶酶是水解酶家族成员,也是生物技术领域的重要酶,其在全球工业酶市场中所占份额约为25%。果胶酶在工业生产中应用广泛,如植物纤维的脱胶、茶和咖啡的发酵、废水处理、纸浆漂白和动物饲料生产等。在果胶酶的天然来源中,由于微生物具有独特的理化性质,最常被用以生产果胶酶。然而,与许多其他工业酶一样,果胶酶也存在野生菌株产量低、工业生产率低等制约因素,因此,目前果胶酶的研究重点主要集中在如何提高工业规模的生产水平。主要介绍了果胶酶的天然来源,以及在这些来源的基础上通过基因工程改造以获得果胶酶高效表达的最新策略,并概括总结了果胶酶发酵工艺和工业应用,以期为生产具有高活性的果胶酶,提高工业生产的效益奠定理论基础。     

Biotechnologically relevant enzymes from Thermus thermophilus

. Enzymes produced by Thermus thermophilus are of considerable biotechnological interest. This review covers industrial applications of several protein products of this thermophilic bacterium that are functional under extreme conditions. The purification of proteins from T. thermophilus using either conventional methods or in the light of the cloning of their genes and expression in mesophilic microorganisms is discussed. Enzymes that biodegrade proteins, polysaccharides or key enzymes that are involved in amino acid metabolism, protein folding or in other fundamental biological processes such as DNA replication, DNA repair, and RNA maturation, with potential use in different biotechnological processes are reviewed as well.     

Biotechnological Tools for Enhancing Microbial Solubilization of Insoluble Inorganic Phosphates

During recent years, many studies appeared on microbial solubilization of insoluble phosphates as an alternative of chemically based P-fertilizer production and bearing in mind the progressive increase in P-fertilizer prices based on high global P consumption and the scarcity of rock phosphate reserves. This biotechnological approach is mainly related to microbial production of organic acids such as citric, oxalic, gluconic, itaconic, and lactic acid, which react with the insoluble P-sources. The most applied and studied P-solubilizers are fungal microorganisms cultivated in conditions of submerged and solid-state fermentation systems. Therefore, the aim of this review is to summarize data on the effect of various abiotic factors on the fungal organic acid production. Nutrient medium components, fermentation process parameters, interaction between insoluble P-particles and microbial systems, and mode of fermentation are analyzed for their impact on both organic acid production and P-solubilization. Suggestions for further studies are also discussed.