Karl Rumbold

Microbial metabolomics: past, present and future methodologies.

Microbial metabolomics has received much attention in recent years mainly because it supports and complements a wide range of microbial research areas from new drug discovery efforts to metabolic engineering. Broadly, the term metabolomics refers to the comprehensive (qualitative and quantitative) analysis of the complete set of all low molecular weight metabolites present in and around growing cells at a given time during their growth or production cycle. This review focuses on the past, current and future development of various experimental protocols in the rapid developing area of metabolomics in the ongoing quest to reliably quantify microbial metabolites formed under defined physiological conditions. These developments range from rapid sample collection, instant quenching of microbial metabolic activity, extraction of the relevant intracellular metabolites as well as quantification of these metabolites using enzyme based and or modern high tech hyphenated analytical protocols, mainly chromatographic techniques coupled to mass spectrometry (LC-MSn, GC-MSn, CE-MSn), where n indicates the number of tandem mass spectrometry, and nuclear magnetic resonance spectroscopy (NMR).

Microbial utilization of crude glycerol for the production of value-added products

Energy fuels for transportation and electricity generation are mainly derived from finite and declining reserves of fossil hydrocarbons. Fossil hydrocarbons are also used to produce a wide range of organic carbon-based chemical products. The current global dependency on fossil hydrocarbons will not be environmentally or economically sustainable in the long term. Given the future pessimistic prospects regarding the complete dependency on fossil fuels, political and economic incentives to develop carbon neutral and sustainable alternatives to fossil fuels have been increasing throughout the world. For example, interest in biodiesel has undergone a revival in recent times. However, the disposal of crude glycerol contaminated with methanol, salts, and free fatty acids as a by-product of biodiesel production presents an environmental and economic challenge. Although pure glycerol can be utilized in the cosmetics, tobacco, pharmaceutical, and food industries (among others), the industrial purification of crude glycerol is not economically viable. However, crude glycerol could be used as an organic carbon substrate for the production of high-value chemicals such as 1,3-propanediol, organic acids, or polyols. Microorganisms have been employed to produce such high-value chemicals and the objective of this article is to provide an overview of studies on the utilization of crude glycerol by microorganisms for the production of economically valuable products. Glycerol as a by-product of biodiesel production could be used a feedstock for the manufacture of many products that are currently produced by the petroleum-based chemical industry.

Microbial production host selection for converting second-generation feedstocks into bioproducts

BackgroundIncreasingly lignocellulosic biomass hydrolysates are used as the feedstock for industrial fermentations. These biomass hydrolysates are complex mixtures of different fermentable sugars, but also inhibitors and salts that affect the performance of the microbial production host. The performance of six industrially relevant microorganisms, i.e. two bacteria (Escherichia coli and Corynebacterium glutamicum), two yeasts (Saccharomyces cerevisiae and Pichia stipitis) and two fungi (Aspergillus niger and Trichoderma reesei) were compared for their (i) ability to utilize monosaccharides present in lignocellulosic hydrolysates, (ii) resistance against inhibitors present in lignocellulosic hydrolysates, (iii) their ability to utilize and grow on different feedstock hydrolysates (corn stover, wheat straw, sugar cane bagasse and willow wood). The feedstock hydrolysates were generated in two manners: (i) thermal pretreatment under mild acid conditions followed by enzymatic hydrolysis and (ii) a non-enzymatic method in which the lignocellulosic biomass is pretreated and hydrolyzed by concentrated sulfuric acid. Moreover, the ability of the selected hosts to utilize waste glycerol from the biodiesel industry was evaluated.ResultsLarge differences in the performance of the six tested microbial production hosts were observed. Carbon source versatility and inhibitor resistance were the major discriminators between the performances of these microorganisms. Surprisingly all 6 organisms performed relatively well on pretreated crude feedstocks. P. stipitis and A. niger were found to give the overall best performance C. glutamicum and S. cerevisiae were shown to be the least adapted to renewable feedstocks.ConclusionBased on the results obtained we conclude that a substrate oriented instead of the more commonly used product oriented approach towards the selection of a microbial production host will avoid the requirement for extensive metabolic engineering. Instead of introducing multiple substrate utilization and detoxification routes to efficiently utilize lignocellulosic hydrolysates only one biosynthesis route forming the product of interest has to be engineered.

Purification and properties of a feruloyl esterase involved in lignocellulose degradation by Aureobasidium pullulans

ABSTRACT The lignocellulolytic fungus Aureobasidium pullulans NRRL Y 2311-1 produces feruloyl esterase activity when grown on birchwood xylan. Feruloyl esterase was purified from culture supernatant by ultrafiltration and anion-exchange, hydrophobic interaction, and gel filtration chromatography. The pure enzyme is a monomer with an estimated molecular mass of 210 kDa in both native and denatured forms and has an apparent degree of glycosylation of 48%. The enzyme has a pI of 6.5, and maximum activity is observed at pH 6.7 and 60°C. Specific activities for methyl ferulate, methyl p-coumarate, methyl sinapate, and methyl caffeate are 21.6, 35.3, 12.9, and 30.4 μmol/min/mg, respectively. The pure feruloyl esterase transforms both 2-O and 5-O arabinofuranosidase-linked ferulate equally well and also shows high activity on the substrates 4-O-trans-feruloyl-xylopyranoside, O-{5-O-[(E)-feruloyl]-α-l-arabinofuranosyl}-(1,3)-O-β-d-xylopyranosyl-(1,4)-d-xylopyranose, and p-nitrophenyl-acetate but reveals only low activity on p-nitrophenyl-butyrate. The catalytic efficiency (kcat/Km) of the enzyme was highest on methyl p-coumarate of all the substrates tested. Sequencing revealed the following eight N-terminal amino acids: AVYTLDGD.

Bioflocculant production by a consortium of Streptomyces and Cellulomonas species and media optimization via surface response model

Species of actinobacteria previously isolated from Tyume River in the Eastern Cape Province of South Africa and identified by 16S rDNA sequence as Cellulomonas and Streptomyces species were evaluated as a consortium for the production of bioflocculant. Sucrose, peptone and magnesium chloride were the nutritional sources which supported optimal production of bioflocculant resulting in flocculation activities of 91%, 82% and 78% respectively. Response surface design revealed sucrose, peptone and magnesium chloride as critical media components following Plackett-Burman design, while the central composite design showed optimum concentration of the critical nutritional source as 16.0 g/L (sucrose), 1.5 g/L (peptone) and 1.6g/L (magnesium chloride) yielding optimal flocculation activity of 98.9% and bioflocculant yield of 4.45 g/L. FTIR spectrometry of the bioflocculant indicated the presence of carboxyl, hydroxyl and amino groups, typical for heteropolysaccharide, while SEM imaging revealed an interwoven clump-like structure. The molecular weight distribution of the constituents of the bioflocculants ranged 494.81-18,300.26 Da thus, an indication of heterogeneity in composition. Additionally, the chemical analyses of the purified bioflocculant revealed the presence of polysaccharides and proteins with neutral sugar, amino sugar and uronic acids in the following concentration: 5.7 mg, 9.3mg and 17.8 mg per 100mg. The high flocculation activity of the bioflocculant suggests commercial potential.

Microbial renewable feedstock utilization: A substrate-oriented approach

Increasingly lignocellulosic biomass hydrolysates are used as the feedstock for industrial fermentations. These biomass hydrolysates consist of complex mixtures of different fermentable sugars, but also contain inhibitors and salts that affect the performance of the product-generating microbes. The performance of six industrially relevant microorganisms, i.e. two bacteria (Escherichia coli and Corynebacterium glutamicum ), two yeasts ( Saccharomyces cerevisiae and Pichia stipitis ) and two fungi ( Aspergillus niger and Trichoderma reesei ) were compared for their ability to utilize and grow on different feedstock hydrolysates (corn stover, wheat straw, sugar cane bagasse and willow wood). Moreover, the ability of the selected hosts to utilize waste glycerol from the biodiesel industry was evaluated. P. stipitis and A. niger were found to be the most versatile and C. glutamicum, and S. cerevisiae were shown to be the least adapted to renewable feedstocks. Clear differences in the utilization of the more abundant carbon sources in these feedstocks were observed between the different species. Moreover, in a species-specific way the production of various metabolites, in particular polyols, alcohols and organic acids was observed during fermentation. Based on the results obtained we conclude that a substrate-oriented instead of the more commonly used product oriented approach towards the selection of a microbial production host will avoid the requirement for extensive metabolic engineering. Instead of introducing multiple substrate utilization and detoxification routes to efficiently utilize lignocellulosic hydrolysates only one biosynthesis route forming the product of interest has to be engineered.

The Bioethanol Industry in Sub-Saharan Africa: History, Challenges, and Prospects

Recently, interest in using bioethanol as an alternative to petroleum fuel has been escalating due to decrease in the availability of crude oil. The application of bioethanol in the motor-fuel industry can contribute to reduction in the use of fossil fuels and in turn to decreased carbon emissions and stress of the rapid decline in crude oil availability. Bioethanol production methods are numerous and vary with the types of feedstock used. Feedstocks can be cereal grains (first generation feedstock), lignocellulose (second generation feedstock), or algae (third generation feedstock) feedstocks. To date, USA and Brazil are the leading contributors to global bioethanol production. In sub-Saharan Africa, bioethanol production is stagnant. During the 1980s, bioethanol production has been successful in several countries including Zimbabwe, Malawi, and Kenya. However, because of numerous challenges such as food security, land availability, and government policies, achieving sustainability was a major hurdle. This paper examines the history and challenges of bioethanol production in sub-Saharan Africa (SSA) and demonstrates the bioethanol production potential in SSA with a focus on using bitter sorghum and cashew apple juice as unconventional feedstocks for bioethanol production.

Characterization of an Exopolymeric Flocculant Produced by a Brachybacterium sp.

We evaluated the bioflocculant production potential of an Actinobacteria, which was isolated from a freshwater environment in the Eastern Cape province of South Africa. 16S rDNA nucleotide sequencing analyses revealed that the actinobacteria belongs to the Brachybacterium genus, and the sequences were deposited in the GenBank as Brachybacterium sp. UFH, with accession number HQ537131. Optimum fermentation conditions for bioflocculant production by the bacteria include an initial medium pH of 7.2, incubation temperature of 30 °C, agitation speed of 160 rpm and an inoculum size of 2% (vol/vol) of cell density 3.0 × 108 CFU/mL. The carbon, nitrogen and cation sources for optimum bioflocculant production were maltose (83% flocculating activity), urea (91.17% flocculating activity) and MgCl2 (91.16% flocculating activity). Optimum bioflocculant production coincided with the logarithmic growth phase of the bacteria, and chemical analyses of the bioflocculant showed 39.4% carbohydrate and 43.7% protein (wt/wt). The mass ratio of neutral sugar, amino sugar and uronic acids was 1.3:0.7:2.2. Fourier transform infrared spectroscopy (FTIR) indicated the presence of carboxyl, hydroxyl and amino groups, amongst others, typical for heteropolysaccharide and glycosaminoglycan polysaccharides. Bioflocculant pyrolysis showed thermal stability at over 600 °C, while scanning electron microscope (SEM) imaging revealed a maze-like structure of interlaced flakes. Its high flocculation activity suggests its suitability for industrial applicability.

Draft Genome Sequence of Streptomyces albulus Strain CCRC 11814, an -Poly-L-Lysine-Producing Actinomycete

ABSTRACT Here, we report the draft genome sequence of Streptomyces albulus strain CCRC 11814, a soil-dwelling, Gram-positive bacterium. S. albulus produces ε-poly-l-lysine, which has diverse antimicrobial activity. The genome is 9.43 Mb in size, with a G+C content of 72.2%, and contains 9,177 protein-coding sequences.

Characterization and flocculation efficiency of a bioflocculant produced by a marine Halobacillus

We reported earlier on the bioflocculant production potential of Halobacillus sp. Mvuyo, a marine bacteria isolated from Algoa Bay sediment samples. In this paper we report on the detailed characterization of the purified bioflocculant composed of polysaccharide and protein. The optimum dose of the purified bioflocculant for the clarification of 4 g l−1 kaolin clay suspension was 0.2 mg ml−1 at neutral pH. Scanning electron micrograph (SEM) revealed the bioflocculant to have an amorphous structure. The Fourier transform infrared (FTIR) spectrum exhibited the presence of hydroxyl, carboxyl and amino groups in its structure. The bioflocculant was thermostable with relative bioflocculant activity residue of 74.4% after heat treatment at 100°C. Moreover thermogravimetric analysis (TGA) exhibited a degradation temperature (Td) of ∼ 140°C. The flocculation efficiency of the bioflocculant was 86.2% compared with 82.6%, 74.5% and 70.9% for polyethylimine, ferric chloride and alum, respectively. This bioflocculant has immense promise as a substitute to inorganic and synthetic flocculants in view of their hazard implications.