Endospore production of Bacillus spp. for industrial use

The increased occurrence of antibiotic resistance and the harmful use of pesticides are a major problem of modern times. A ban on the use of antibiotics as growth promoters in animal breeding has put a focus on the probiotics market. Probiotic food supplements are versatile and show promising results in animal and human nutrition. Chemical pesticides can be substituted by biopesticides, which are very effective against various pests in plants due to increased research. What these fields have in common is the use of spore-forming bacteria. The endospore-forming Bacillus spp. belonging to this group offer an effective solution to the aforementioned problems. Therefore, the biotechnological production of sufficient qualities of such endospores has become an innovative and financially viable field of research. In this review, the production of different Bacillus spp. endospores will be reviewed. For this purpose, the media compositions, cultivation conditions and bioprocess optimization methods of the last 20 years are presented and reflected.     

On the industrial use of Bacillus licheniformis: a review

We document here that in those rare cases where disease has been related to Bacillus licheniformis, infection was associated with bypassing the normal biological protective barriers or severely debililated patients. No case suggests any invasive properties of this bacterium. B. licheniformis can therefore be considered non-pathogenic to humans in general. Food-borne illness caused by possible B. licheniformis toxins have been reported, but only in a very few cases and only in connection with consumption of inappropriately prepared food. Considerable experience concerning the industrial use of recombinant B. licheniformis strains has now accumulated and authorities in the United States, Europe and Japan have approved production with and products from recombinant B. licheniformis strains. We conclude that B. licheniformis is a safe host for the production of harmless, industrial products. Correspondence to: B. Diderichsen     

Carbon sources affect metabolic capacities of Bacillus species for the production of industrial enzymes: theoretical analyses for serine and neutral proteases and alpha-amylase

The metabolic fluxes through the central carbon pathways were calculated for the genus Bacillus separately for the enzymes serine alkaline protease (SAP), neutral protease (NP) and alpha-amylase (AMY) on five carbon sources that have different reduction degrees (gamma), to determine the theoretical ultimate limits of the production capacities of Bacillus species and to predict the selective substrate for the media design. Glucose (gamma=4.0), acetate (gamma=4.0), and the TCA cycle organic-acids succinate (gamma=3.5), malate (gamma=3.0), and citrate (gamma=3.0) were selected for the theoretical analyses and comparisons. A detailed mass flux balance-based general stoichiometric model based on the proposed metabolic reaction network starting with the alternative five carbon sources for the synthesis of each enzyme in Bacillus licheniformis that simulates the behaviour of the metabolic pathways with 107 metabolites and 150 reaction fluxes is developed. Highest and lowest specific cell growth rates (&mgr;) were calculated as 1.142 and 0.766h(-1), respectively, when glucose that has the highest degree of reduction and citrate that has the lowest degree of reduction were used as the carbon sources. Highest and lowest SAP, NP and AMY synthesis rates were also obtained, respectively, when glucose and citrate were used. Metabolic capacity analyses showed that the maximum SAP, NP, and AMY synthesis rates were, respectively, 0.0483, 0.0215 and 0.0191mmolg(-1)DWh(-1) when glucose uptake rate was 10mmolg(-1)DWh(-1) and specific growth rate was zero. The amino acid compositions and the molecular weights of the enzyme influence the production yield and selectivity. For SAP and NP oxaloacetate and pyruvate, for AMY oxaloacetate appear to be the critical main branch points. Consequently, for SAP and NP syntheses the fluxes towards the alanine group and aspartate group, and for AMY synthesis the flux towards the aspartate group amino acids need to be high. The results encourage the discussion of the potential strategies for improving productions of SAP, NP and AMY.     

Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species

Background

Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products. This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature.

Results

We determined the complete nucleotide sequence of the B. licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes. The B. licheniformis chromosome contains large regions that are colinear with the genomes of B. subtilis and Bacillus halodurans, and approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologs.

Conclusions

Despite the unmistakable organizational similarities between the B. licheniformis and B. subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species. Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B. licheniformis genome. The availability of a completed genome sequence for B. licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.     

Distinct differentiation of closely related species of Bacillus subtilis group with industrial importance

PCR amplification of 16S rRNA gene by universal primers followed by restriction fragment length polymorphism analysis using RsaI, CfoI and HinfI endonucleases, distinctly differentiated closely related Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus pumilus from Bacillus subtilis sensu stricto. This simple, economical, rapid and reliable protocol could be an alternative to misleading phenotype-based grouping of these closely related species.     

Appraisal of a horizontal two-phase flow photobioreactor for industrial production of delicate microalgae species

A closed tubular horizontal photobioreactor with the ability to produce industrially delicate microalgal species was assessed and validated. A co-current gas–liquid flow was chosen for mass transfer along 1,200?m piping which delimited a culture volume of 4,700?L. Mixing was obtained by means of hydrodynamic events accompanying this type of flow, including waves. The constant cross section of the piping allowed the circulation of spherical bodies to wipe the inner wall and avoid the establishment of a biofilm. Two similar reactors were constructed in December 2009 and December 2010, and operated to supply the dermo-beauty care industry with active biomass of three delicate species of microalgae, including the chlorophyte Neochloris oleoabundans and the rhodophyte Porphyridium cruentum. Daily observed production of dry biomass varied between 0.2 and 1.7?kg, depending on the species, the sun exposure and the period of the year. The ability of this production system to reduce the cost of producing algal biomass through scaling-up is discussed.     

嗜碱芽孢杆菌产碱性纤维素酶研究概况

碱性纤维素酶是重要的洗涤剂添加剂,不仅可以去除衣物上的污渍,还可以软化衣物和增加衣物的鲜艳度。嗜碱芽孢杆菌是生产碱性纤维素酶的主要菌种。本文概述了其编码的碱性纤维素酶的性质,编码酶的基因、基因的克隆、表达以及作为洗涤剂添加剂的去污机理,指出了今后的研究方向。     

Bioprocess parameters and oxygen transfer characteristics in beta-lactamase production by Bacillus species

After screening potential beta-lactamase producers in a medium containing penicillin G, an inducible (Bacillus subtilis NRS 1125) and a constitutive (Bacillus licheniformis 749/C ATCC 25972) beta-lactamase producer were selected. As the highest enzyme activity was obtained with B. licheniformis 749/C, the effects of the concentration of carbon sources, i.e., glucose, fructose, sucrose, citric acid, and glycerol, and nitrogen sources, i.e., (NH(4))(2)HPO(4), NH(4)Cl, yeast extract, casamino acids and peptone, pH, and temperature on beta-lactamase production were investigated with B. licheniformis 749/C in laboratory scale bioreactors. Among the investigated media, the highest volumetric activity was obtained as 270 U cm(-)(3) in the medium containing 10.0 kg m(-)(3) glucose, 1.18 kg m(-)(3) (NH(4))(2)HPO(4), 8.0 kg m(-)(3) yeast extract, and the salt solution at 32 degrees C and pH(0) = 6.0. By using the designed medium, fermentation and oxygen transfer characteristics of the bioprocess were investigated at V = 3.0 dm(3) bioreactor systems with a V(R) = 1.65 dm(3) working volume at Q(O)/V(R) = 0.5 vvm and N = 500 min(-1). At the beginning of the process the Damk?hler number was <1, indicating that the process was at biochemical reaction limited condition; at t = 2-5 h both mass-transfer and biochemical reaction resistances were effective; and at t = 6-10 h (Da >1) the bioprocess was at mass transfer limited condition. Overall oxygen transfer coefficients (K(L)a) varied between 0.01 and 0.03 s(-)(1), enhancement factor (K(L)a/K(L)a(O)) varied between 1.2 and 2.3, and volumetric oxygen uptake rate varied between 0.001 and 0.003 mol m(-)(3) s(-)(1) throughout the bioprocess. The specific oxygen uptake and the specific substrate consumption rates were the highest at t = 2 h and then decreased with the cultivation. The maximum yield of cells on substrate and the maximum yield of cells on oxygen values were obtained, respectively, as Y(X/S) = 0.34 and Y(X/O) = 1.40, at t = 5 h, whereas the highest yield of substrate on oxygen was obtained as Y(S/O) = 6.94 at t = 3.5 h. The rate of oxygen consumption for maintenance and the rate of substrate consumption for maintenance values were found, respectively, as m(O) = 0.13 kg kg(-)(1) h(-)(1) and m(S) = 3.02 kg kg(-)(1) h(-)(1).     

Perspectives for the industrial enzymatic production of glycosides

Glycosides are of commercial interest for industry in general and specifically for the pharmaceutical and food industry. Currently chemical preparation of glycosides will not meet EC food regulations, and therefore chemical preparation of glycosides is not applicable in the food industry. Thus, enzyme-catalyzed reactions are a good alternative. However, until now the low yields obtained by enzymatic methods prevent the production of glycosides on a commercial scale. Therefore, high yields should be established by a combination of optimum reaction conditions and continuous removal of the product. Unfortunately, a bioreactor for the commercial scale production of glycosides is not available. The aim of this article is to discuss the literature with respect to enzymatic production of glycosides and the design of an industrially viable bioreactor system.     

Xanthophyllomyces dendrorhous for the industrial production of astaxanthin

Astaxanthin is a red xanthophyll (oxygenated carotenoid) with large importance in the aquaculture, pharmaceutical, and food industries. The green alga Haematococcus pluvialis and the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous are currently known as the main microorganisms useful for astaxanthin production at the industrial scale. The improvement of astaxanthin titer by microbial fermentation is a requirement to be competitive with the synthetic manufacture by chemical procedures, which at present is the major source in the market. In this review, we show how the isolation of new strains of X. dendrorhous from the environment, the selection of mutants by the classical methods of random mutation and screening, and the rational metabolic engineering, have provided improved strains with higher astaxanthin productivity. To reduce production costs and enhance competitiveness from an industrial point of view, low-cost raw materials from industrial and agricultural origin have been adopted to get the maximal astaxanthin productivity. Finally, fermentation parameters have been studied in depth, both at flask and fermenter scales, to get maximal astaxanthin titers of 4.7 mg/g dry cell matter (420 mg/l) when X. dendrorhous was fermented under continuous white light. The industrial scale-up of this biotechnological process will provide a cost-effective method, alternative to synthetic astaxanthin, for the commercial exploitation of the expensive astaxanthin (about $2,500 per kilogram of pure astaxanthin).     

Unlocking Streptomyces spp. for use as sustainable industrial production platforms by morphological engineering

Filamentous actinomycetes are commercially widely used as producers of natural products (in particular antibiotics) and of industrial enzymes. However, the mycelial lifestyle of actinomycetes, resulting in highly viscous broths and unfavorable pellet formation, has been a major bottleneck in their commercialization. Here we describe the successful morphological engineering of industrially important streptomycetes through controlled expression of the morphogene ssgA. This led to improved growth of many industrial and reference streptomycetes, with fragmentation of the mycelial clumps resulting in significantly enhanced growth rates in batch fermentations of Streptomyces coelicolor and Streptomyces lividans. Product formation was also stimulated, with a twofold increase in yield of enzyme production by S. lividans. We anticipate that the use of the presented methodology will make actinomycetes significantly more attractive as industrial and sustainable production hosts.     

Developments and perspectives of photobioreactors for biofuel production

The production of biofuels from microalgae requires efficient photobioreactors in order to meet the tight constraints of energy efficiency and economic profitability. Current cultivation systems are designed for high-value products rather than for mass production of cheap energy carriers. Future bioreactors will imply innovative solutions in terms of energy efficiency, light and gas transfer or attainable biomass concentration to lower the energy demand and cut down production costs. A new generation of highly developed reactor designs demonstrates the enormous potential of photobioreactors. However, a net energy production with microalgae remains challenging. Therefore, it is essential to review all aspects and production steps for optimization potential. This includes a custom process design according to production organism, desired product and production site. Moreover, the potential of microalgae to synthesize valuable products additionally to the energetic use can be integrated into a production concept as well as waste streams for carbon supply or temperature control.     

Complete genome sequence of Bacillus amyloliquefaciens TA208, a strain for industrial production of guanosine and ribavirin

Here, we report the complete genome sequence of Bacillus amyloliquefaciens TA208, a strain for industrial production of guanosine and synthesis of ribavirin by assimilation of formamide. Comparison of its genome sequence with those of strains DSM7 and FZB42 revealed horizontal gene transfer represented by unique prophages and restriction-modification systems and indicated significant accumulation of guanosine.     

枯草芽胞杆菌芽胞表面展示研究进展

枯草芽胞杆菌芽胞表面展示技术是把枯草芽胞杆菌作为芽胞表面展示的宿主来展示目的蛋白的一种技术。该技术不仅具备芽胞表面展示技术可展示分子量较大的目的蛋白、目的蛋白无需跨膜及芽胞的极强抗逆性等特点外,同时由于该技术的宿主菌--枯草芽胞杆菌的分子生物学信息研究得比较清楚、安全性高而被广泛应用。介绍了枯草芽胞杆菌表面展示近10年在生产疫苗和固定化酶方面的进展,并对如何提高表面展示目的蛋白的产量做了简要概述。     

Enhanced alpha - amylase production by chromosomal integration of pTVA1 in industrial strain in Bacillus subtilis

Summary Strain Bacillus subtilis MS was constructed with 12–22 fold increase of -amylase production, caused by presence of multiple -amylase gene copies in the chromosome of industrial strain Bacillus subtilis CCM2722, as demonstrated by DNA hybridization. The enhanced production is a result of multiple integration of plasmid pTVA1, carrying a temperature sensitive origin of replication from pE194, and containing the -amylase gene and a modified transposon Tn917.     

Engineering organisms for industrial fuel production

Volatile fuel costs, the need to reduce greenhouse gas emissions and fuel security concerns are driving efforts to produce sustainable renewable fuels and chemicals. Petroleum comes from sunlight, CO(2) and water converted via a biological intermediate into fuel over a several million year timescale. It stands to reason that using biology to short-circuit this time cycle offers an attractive alternative--but only with relevant products at or below market prices. The state of the art of biological engineering over the past five years has progressed to allow for market needs to drive innovation rather than trying to adapt existing approaches to the market. This report describes two innovations using synthetic biology to dis-intermediate fuel production. LS9 is developing a means to convert biological intermediates such as cellulosic hydrolysates into drop-in hydrocarbon product replacements such as diesel. Joule Unlimited is pioneering approaches to eliminate feedstock dependency by efficiently capturing sunlight, CO(2) and water to produce fuels and chemicals. The innovations behind these companies are built with the market in mind, focused on low cost biosynthesis of existing products of the petroleum industry. Through successful deployment of technologies such as those behind LS9 and Joule Unlimited, alternative sources of petroleum products will mitigate many of the issues faced with our petroleum-based economy.