Current Biotechnology - Volume 3, Issue 1, 2014

The order Halanaerobiales contains a number of well-studied halophiles that possess great potential for biotechnological applications. The unique halophilic adaptations that these organisms utilize, such as “salting-in” mechanisms to increase their intercellular concentration of KCl, combined with their ability to ferment simple sugars, provides an excellent platform for biotechnological development over a wide range of salt levels and possible other extreme conditions, such as alkaline conditions. From fermented foods to oil reservoirs, members of Halanaerobiales are found in many environments. The environmental conditions many of these organisms grow are similar to industrially important processes, such as alkaline pre-treated biomass stocks, treatment of crude glycerol from biodiesel production, salty fermented foods, as well as bioremediation of contaminants under extreme conditions of salinity and in some cases, alkalinity. From salt-stable enzymes to waste fermentations, bioremediation options, bioenergy, and microbially enhanced oil recovery (MEOR), Halanaerobiales can provide a wide spectrum of environmentally friendly solutions to current problems.

Bacteria, Archaea and Eukarya can adapt to saline environments by accumulating compatible solutes in order to maintain an osmotic equilibrium. Compatible solutes are of diverse chemical structure (sugars, polyols, amino acid derivatives) and are beneficial for bacterial cells not only as osmoregulatory solutes, but also as protectants of proteins by mitigating detrimental effects of freezing, drying and high temperatures. The aspartate derivative ectoine is a wide spread compatible solute in Bacteria and possesses additional protective properties compared with other compatible solutes, and stabilizes even whole cells against stresses such as UV radiation or cytotoxins. The protective properties of ectoine for proteins can be explained by its strong (kosmotropic) interaction with water and subsequent exclusion from protein surface, the decrease of the solubility of the peptide backbone and the strengthening of intramolecular hydrogen bonds (secondary structures). The stabilizing and UV-protective properties of ectoine attracted industry, which saw the potential to market ectoine as a novel active component in health care products and cosmetics. In joint efforts of industry and research large-scale fermentation procedures have been developed with the halophilic bacterium Halomonas elongata used as a producer strain. The two key technologies that allow for the annual production of ectoine on a scale of tons are the bacterial milking procedure and the development and application of ectoine-excreting mutants (“leaky” mutant). The details of these two procedures including the strain development and fermentation processes will be introduced and current and future applications of ectoine will be discussed.

Glycoside hydrolases, which are responsible for the degradation of the major fraction of biomass, the polymeric carbohydrates in starch and lignocellulose, are predicted to gain increasing roles as catalysts in biorefining applications in the future bioeconomy. In this context, thermostable variants will be important, as the recalcitrance of these biomass-components to degradation often motivates thermal treatments. The traditional focus on degradation is also predicted to be changed into more versatile roles of the enzymes, also involving specific conversions to defined products. In addition, integration of genes encoding interesting target activities opens the possibilities for whole cell applications, in organisms allowing processing at elevated temperatures for production of defined metabolic products. In this review, we overview the application of glycoside hydrolases related to the biorefining context (for production of food, chemicals, and fuels). Use of thermostable enzymes in processing of biomass is highlighted, moving from the activities required to act on different types of polymers, to specific examples in today’s processing. Examples given involve (i) monosaccharide production for food applications as well as use as carbon source for microbial conversions (to metabolites such as fuels and chemical intermediates), (ii) oligosaccharide production for prebiotics applications (iii) treatment for plant metabolite product release, and (iv) production of surfactants of the alkyl glycoside class. Finally future possibilities in whole cell biorefining are shown.

Enzymes from extreme thermophiles that grow above 70 °C have a number of attractions in industrial applications. They are often highly resistant to denaturing conditions and are stable at elevated temperatures and over a range of pH values. There has been a widespread search for micro-organisms producing novel enzymes and where found, most publications (and research grant applications) promise that their superior properties would be suited to particular industries that operate at elevated temperatures - for example, bleaching of kraft pulp in the pulp and paper industry. Yet examination of the academic and patent literature reveals few of these proteins adopted in industrial enzymology. Most employed successfully have been as laboratory reagents, particularly Thermus aquaticus DNA polymerase, which made the polymerase chain reaction possible and revolutionized gene manipulation at a laboratory level. Extremozymes marketed for the laboratory are lower volume / higher value products and have been cloned and expressed (usually) in Escherichia coli. This review examines the characterization and application of thermophilic enzymes for several activities that have been identified and (usually) produced in recombinant bacteria. DNA polymerases, glycosyl hydrolases, lipases and proteases from extreme thermophiles are described and evaluated for their potential and actual applications in biotechnology. Some of the barriers to widespread industrial acceptance are described. Emphasis is placed on a number of examples illustrated by the anaerobic extreme thermophiles Caldicellulosiruptor sp. and Dictyoglomus sp. with which the authors are familiar. A number of attractive enzymes are not scalable economically in Escherichia coli or the organism from which the gene has been isolated is an obligate anaerobe and enzyme yields from the native organism are low. Consequently, attention has turned to commonly used ‘cell factories’ to provide suitable yields of enzymes used as bulk chemicals. These 'factories' are usually fungi such as Saccharomyces cerevisiae and Trichoderma reesei or bacteria such as Bacillus sp. A number of challenges, such as codon usage incompatibility, must be overcome to achieve economic yields.

This review starts with a brief introduction on bacterial endophytes. Only small fractions of rhizosphere and phyllosphere bacteria are able to live inside the plant. Endophytes are bacteria and fungi that can be detected at a particular moment within the tissues of apparently healthy plant hosts without producing symptoms in or on the plant. Possible traits required by these bacteria to enter the plant and live inside will be discussed. Furthermore, we will focus on possible biotechnological applications of bacterial endophytes and present case studies as examples. Endophytes can promote plant growth, for example by the production of hormones or by making nutrients (such as nitrogen, phosphate and ferric ions) available to the plant. Endophytes can also promote plant growth indirectly, for example by suppression of plant diseases, by inactivating environmental pollutants, and by alleviating stresses of the plant caused by excess of the hormone ethylene, by heavy metals, by draught and by salinated soil. Some endophytic bacteria can produce nanoparticles which have numerous applications. At the end of the review we will discuss aspects involved in the commercialization of microbes.

A novel and non-ubiquitous thermostable DNA polymerase in Thermus antranikianii was expressed in E. coli, isolated and biochemically characterized. The enzyme here referred to as Thermophi, has a C-terminal polymerase domain and a proofreading 3′→5′ exonuclease domain, but lack the 5′→3′ exonuclease domain. The corresponding gene is apparently only found in some but not all Thermus strains. The initial rate of specific activity of this polymerase on nicked DNA was about 360,000 U/mg protein. The optimum activity was found at 55 °C, pH 8.5 and 1.5 mM Mg+2. The polymerase was stable at 70 °C and lost 50% of its activity after 5 min at 85 °C, but could be stabilized above 80 °C by addition of 0.5 M L-proline. A pronounced strand-displacement activity was indicated by the large amount of DNA produced by the enzyme after an overnight, isothermal incubation in presence of hexamer primers. Both single and double stranded DNA was isothermally amplified by the enzyme. The amplified DNA was large and apparently highly branched material and composed of both single and double stranded DNA. The produced material could be partly digested by T7 enonuclease I but it was difficult to cut with common restriction enzymes. Amplification of selected genes from dilute samples was successfully demonstrated with the human β-actin gene. Good amplification was also found with 5 microsatellite markers from salmon DNA. Thermophi amplifies DNA by orders of magnitude but upon extended reaction time the DNA becomes very large and highly branched. It is composed of both single and double strands and then correctly amplified sequences only represent about 10-20% of the total DNA, and long stretches of TATATA repeats frequently occur in the amplified DNA.

Improvements in genomic sequencing technology have accelerated the accumulation of gene information, leading to the emergence of proteomics as a powerful tool to study the functional genome. Specifically, the emergence of two technologies, two-dimensional gel electrophoresis (2-DE) and high-throughput protein identification using mass spectrometry (MS), was a milestone in the development of proteomics. Due to bacteria having a simple genomic system and ease of sample preparation, these organisms were rapidly subjected to proteomic analyses. Bacterial proteomics is considered complementary to genomic analyses, including full-genome sequencing and transcriptomics. Proteomics has revealed novel and valuable information on gene products (i.e., proteins), including their translation level, posttranslational modifications, turnover, and localization. Recently, the proteomic approach was applied to the study of extremophiles. In this review, we briefly summarize recent proteomic technologies applicable to bacteria and archea extremophiles and review the literature describing proteomic research in these organisms. Finally, we discuss future perspectives on the use of proteomics to study extremophiles.

The earth is a cold biosphere with a major part (> 85.0%) permanently exposed to temperatures below 5 oC. Antarctica, Arctic, high altitude mountains, glaciers and deep-sea are the major constituents of the cold biosphere. The cold biosphere provides habitats such as snow, permafrost, sea-ice, glaciers, oceanic waters and sediments, cold water lakes, soil and caves which are conducive for the survival and reproduction of life forms. Diverse microorganisms such as bacteria, archaea, yeast, fungi and algae are known to survive, divide and colonize these cold habitats and are referred to as psychrophiles. Therefore, studies on the diversity, physiology and molecular biology of psychrophilic bacteria would provide important inputs on their distribution, survival strategies and molecular basis of their adaptation to low temperature. In this review, we focused on microbial biodiversity of psychrophilic microorganisms from Antarctica, Arctic and Himalayan glaciers, on their adaptation and biotechnological applications.

Proteins and enzymes are the basic regulatory units in living systems. Radiation-induced damages in protein and enzyme structure can lose their regulatory and biocatalytic properties lead to radiation induced structural and thus functional impairment. In the present study we report in vitro radioprotective efficacy of semiquinone glucoside derivative (SQGD) isolated from a radioresistant bacterium Bacillus sp. INM-01. Radiation induced damage in Bovine Serum Albumin (BSA) and its protection by SQGD pre-treatment was evaluated by UV-Vis, Circular Dichroism (CD) spectroscopic and SDS-PAGE analysis. Protection to functional activity of restriction enzyme (Hind III) by SQGD was analysed through agarose gel electrophoresis. While antioxidant activities of SQGD was analyzed by fluorescence spectro-photometeric analysis using fluorescein as oxidized adduct and 2,2'-azobis(2-amidino-propane) dihydrochloride (AAPH) as oxidizing agent. Significant increase in absorption maxima represented oxidation of BSA with irradiated (600- 1200Gy) samples was observed. Whereas, no such increase in absorption maxima in the irradiated BSA pretreated with SQGD was observed. Circular Dichroism (CD) spectroscopic analysis showed dose dependent upward shift in the spectra of BSA upon irradiation. While, pre-treatment by SQGD, shift the BSA spectra towards control. NATIVE-PAGE analysis showed a clear smearing in irradiated BSA, which was found to be abolished upon SQGD pre-treatment. Further, significant loss of functional activity of Hind III endonulease was observed upon irradiation, while SQGD pretreatment helps to protect catalytic activity of irradiated Hind III. Thus, in view of above, present study was suggested that SQGD carries excellent radioprotective activities and protects enzymes and proteins against ionizing radiation-induced oxidative damage. In conclusion, SQGD can be used as protector of vital proteins, vitamins, enzymes and other important radiosensitive food supplements during radiosterilization process of food and meat industries.