Technological fundamentals of bacterial nanocellulose production from zero prime-cost feedstock

The concept of manufacturing valuable bacterial nanocellulose (BNC) from plant raw materials having a zero prime cost is substantiated. The process flowsheet involves the chemical transformation of the feedstock to obtain a pulp; enzymatic hydrolysis of the pulp to furnish a solution of reducing sugars, chiefly glucose; preparation of a nutrient broth based on the enzymatic hydrolysate; biosynthesis of nanocellulose microfibrils by the symbiotic Medusomyces gisevii Sa-12 culture; and purification of BNC. BNC has for the first time been synthesized from oat hulls and has a high degree of crystallinity of 88 ± 5% and is composed of 99% Iα-allomorph.     

Improved cellulase production by Botryosphaeria rhodina from OPEFB at low level moisture condition through statistical optimization

The response surface method was applied in this study to improve cellulase production from oil palm empty fruit bunch (OPEFB) by Botryosphaeria rhodina. An experimental design based on a two-level factorial was employed to screen the significant environmental factors for cellulase production. The locally isolated fungus Botryosphaeria rhodina was cultivated on OPEFB under solid-state fermentation (SSF). From the analysis of variance (ANOVA), the initial moisture content, amount of substrate, and initial pH of nutrient supplied in the SSF system significantly influenced cellulase production. Then the optimization of the variables was done using the response surface method according to central composite design (CCD). Botryosphaeria rhodina exhibited its best performance with a high predicted value of FPase enzyme production (17.95 U/g) when the initial moisture content was at 24.32%, initial pH of nutrient was 5.96, and 3.98 g of substrate was present. The statistical optimization from actual experiment resulted in a significant increment of FPase production from 3.26 to 17.91 U/g (5.49-fold). High cellulase production at low moisture content is a very rare condition for fungi cultured in solid-state fermentation.     

Effects of agitator configuration and rotational speed on the production of extracellular polysaccharide by Xanthomonas cucurbitae PCSIR B-52

A strain of Xanthomonas cucurbitae PCSIR B-52, efficiently produced extracellular polysaccharide using partially deproteinized low-acid cheese whey without hydrolysis. The effects of the agitator configuration and rotational speed on the viscosity of the fermented broth and the productivity of extracellular polysaccharide based on bacterial growth were evaluated in a batch process. Agitation was performed by a six-bladed disc turbine impeller and by a similar agitator, equipped with six vertically attached stabilizing fins. Comparatively, the magnitude of the decrease in the DO tension resulting from increased viscosity of the broth was less with the stabilizing-fin agitator due to increased system damping. A drastic increase in the mechanical agitation speed after 30-h fermentation, however, increased the broth viscosity and the accumulation of polysaccharide. Moreover, the volume of the macromixing region decreased with increasing rotational speed of agitator.     

Comparison of productivity and quality of bacterial nanocellulose synthesized using culture media based on seven sugars from biomass

Komagataeibacter xylinus ATCC 23770 was statically cultivated in eight culture media based on different carbon sources, viz. seven biomass-derived sugars and one sugar mixture. The productivity and quality of the bacterial nanocellulose (BNC) produced in the different media were compared. Highest volumetric productivity, yield on consumed sugar, viscometric degree of polymerization (DP_v, 4350–4400) and thermal stability were achieved using media based on glucose or maltose. Growth in media based on xylose, mannose or galactose resulted in lower volumetric productivity and DP_v, but in larger fibril diameter and higher crystallinity (76–78%). Growth in medium based on a synthetic sugar mixture resembling the composition of a lignocellulosic hydrolysate promoted BNC productivity and yield, but decreased fibril diameter, DP_v, crystallinity and thermal stability. This work shows that volumetric productivity, yield and properties of BNC are highly affected by the carbon source, and indicates how industrially relevant sugar mixtures would affect these characteristics.     

Genome sequence and characterization of the bcs clusters for the production of nanocellulose from the low pH resistant strain Komagataeibacter medellinensis ID13488

Komagataeibacter medellinensis ID13488 (formerly Gluconacetobacter medellinensis ID13488) is able to produce crystalline bacterial cellulose (BC) under high acidic growth conditions. These abilities make this strain desirable for industrial BC production from acidic residues (e.g. wastes generated from cider production). To explore the molecular bases of the BC biosynthesis in this bacterium, the genome has been sequenced revealing a sequence of 3.4 Mb containing three putative plasmids of 38.1 kb (pKM01), 4.3 kb (pKM02) and 3.3 Kb (pKM03). Genome comparison analyses of K. medellinensis ID13488 with other cellulose-producing related strains resulted in the identification of the bcs genes involved in the cellulose biosynthesis. Genes arrangement and composition of four bcs clusters (bcs1, bcs2, bcs3 and bcs4) was studied by RT-PCR, and their organization in four operons transcribed as four independent polycistronic mRNAs was determined. qRT-PCR experiments demonstrated that mostly bcs1 and bcs4 are expressed under BC production conditions, suggesting that these operons direct the synthesis of BC. Genomic differences with the close related strain K. medellinensis NBRC 3288 unable to produce BC were also described and discussed.     

Improved production of heterologous proteins by a glucose repression-defective mutant of Kluyveromyces lactis

The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1beta compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.     

Improved ethanol production from xylose with glucose isomerase and Saccharomyces cerevisiae using the respiratory inhibitor azide

Summary Ethanol was produced from xylose, using the enzyme glucose isomerase (xylose isomerase) and Saccharomyces cerevisiae. The influence of aeration, pH, enzyme concentration, cell mass and the concentration of the respiratory inhibitor sodium azide on the production of ethanol and the formation of by-products was investigated. Anaerobic conditions at pH 6.0, 10 g/l enzyme, 75 g/l dry weight cell mass and 4.6 mM sodium azide were found to be optimal. Under these conditions theoretical yields of ethanol were obtained from 42 g/l xylose within 24 hours.In a fed-batch culture, 62 g/l ethanol was produced from 127 g/l xylose with a yield of 0.49 and a productivity of 1.35 g/l·h.     

Ethanol production from glucose by free and agar-entrapped batch cultures ofSaccharomyces cerevisiae at different oxygenation levels

Summary The influence of the oxygenation level on glucose alcoholic fermentation by free- and immobilized-cell batch cultures ofSaccharomyces cerevisiae (CBS 1200) was investigated. Ethanol production by suspended cultures was stimulated by moderate oxygen flow rates (OFRs) but inhibited by higher OFRs (1.25 mol O₂ h^–1 dm^–3). On the contrary, agar-entrapped yeasts displayed the best fermentation performance under strict anaerobiosis and were less affected than free cultures by high OFRs. These results are dicussed by referring to the substrate mass transfer limitations inherent in immobilized-cell systems.     

Continuous production of non-alcohol beer by immobilized yeast at low temperature

Summary A system for production of non-alcohol beer is described. A limited fermentation is carried out with immobilized cells ofSaccharomyces cerevisiae in a packed bed reactor. In the reactor, combined stress factors such as low temperature (2–4°C) and anaerobic conditions limit cell metabolism. Of the available sugars only a small amount of glucose is metabolized, resulting in low concentrations of ethanol (<0.08%). The absence of oxygen affects the redox balance of the yeast cell, and thus stimulates formation of esters and higher alcohols. Products are formed by reduction of wort aldehydes, as well as reduction of intracellular metabolites. Despite the stress conditions, biomass increases during prolonged production periods. In batch experiments,S. cerevisiae strain W34 grows at low temperatures and a mininum growth temperature of –2 °C was found, indicating that a further reduction of temperature during production will not inhibit growth. The characteristics of the system allow its use in very different applications. Potential applications of the immobilized system are discussed.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Improved protocol for the extraction of bacterial mRNA from soils

An improved protocol for extraction of prokaryotic mRNA from soil samples was developed by modifying the extraction procedure to obtain higher yields of mRNA and to reduce co-extraction of humic acids. The modified protocol was found to be more robust and efficient compared to the original protocol by Griffiths et al. (2000) without compromising with the quality and quantity of RNA.     

Galactose inhibition of citric acid production from glucose by Aspergillus niger

Summary Previous work in this laboratory has demonstrated that although Aspergillus niger can readily utilize galactose, no citric acid is produced from this carbon source (Hossain et al. 1984). Experiments were now conducted where galactose was added at various concentrations to synthetic growth medium containing glucose as carbon source, so that the effect of galactose on citric acid production from glucose could be observed. The results showed that the presence of galactose or a product of galactose metabolism caused inhibition of citric acid production, and also reduced the rate of glucose utilization. Enzyme analyses using mycelial cell-free extracts indicated that galactose interfered with the glucose-repression of the key enzyme 2-oxoglutarate dehydrogenase.     

Improved arachidonic acids production from the fungus Mortierella alpina by glutamate supplementation

The effect of various concentrations of glutamate on arachidonic acid (AA) production from Mortierella alpina in shaker flask culture was studied. Glutamate supplementation promoted Mortierella growth, accelerated substrate metabolism, and increased AA production, and a concentration of 0.8 g/l glutamate resulted in the greatest AA yield (1.41 g/l). In 10 l airlift stirred fermenter culture, AA yield in the cultures exposed to 0.8 g/l glutamate was also greater than that in the control (0.56 g/l).