Beta-Amylase Production by Bacillus polymyxa on a Corn Steep-Starch-Salts Medium

Bacteria were found that are capable of producing good yields of beta-amylase in unrefined media. The culture filtrates are free of alpha-amylase and isoamylase.     

Utilization of Xylan by Yeasts and Its Conversion to Ethanol by Pichia stipitis Strains

Yeasts able to grow on d-xylose were screened for the ability to hydrolyze xylan. Xylanase activity was found to be rare; a total of only 19 of more than 250 strains yielded a positive test result. The activity was localized largely in the genus Cryptococcus and in Pichia stipitis and its anamorph Candida shehatae. The ability to hydrolyze xylan was generally uncoupled from that to hydrolyze cellulose; only three of the xylan-positive strains also yielded a positive test for cellulolytic activity. Of the 19 xylanolytic strains, 2, P. stipitis CBS 5773 and CBS 5775, converted xylan into ethanol, with about 60% of a theoretical yield computed on the basis of the amount of d-xylose present originally that could be released by acid hydrolysis.     

Gavaserin and saltavalin, new peptide antibiotics produced by Bacillus polymyxa

Abstract A strain of Bacillus polymyxa (BP1), isolated from cauliflower seeds, inhibited the growth of microbial phytopathogens. Growth of this strain in liquid medium containing lactose, ammonium sulfate, biotin, and amino acids, resulted in optimal inhibition in vitro. Two new antibacterial substances were isolated and purified from culture broth. Their molecular masses were, respectively, 911 and 903 dallons. The first compound was named gavaserin because it contained glutamic acid, alanine, valine, serine and 2,4-diaminobutyric acid, and octanoic acid. No fatty acid was detected in the second compound, which was named saltavalin because it contained serine, alanine, leucine, threonine, valine, and 2,4-diaminobutyric acid.     

Protoplast formation,regeneration and fusion in Bacillus polymyxa

Two-hour lysozyme treatment (1 mg/ml) of mid-logarithmic phase Bacillus polymyxa cells at 37°C resulted in 99% protoplasting efficiency. Among three typical regeneration media, HCP 1.5 gave the highest regeneration frequency of 1.8 × 10⁻². Protoplast fusion between B. polymyxa B1103 Str⁺ His and B1104 Tyr⁻ produced 39.6% recombinants of the total regenerants, Str^r His⁺ Tyr⁻ and Str^r His⁻ Tyr⁻ being the major recombinant phenotypes.     

Production of 2,3-Butylene Glycol from Citrus Wastes. II. The Bacillus polymyxa Fermentation

The conditions required for production of levo 2,3-butylene glycol by Bacillus polymyxa from citrus molasses were studied. Starter cultures required acclimatization to the substrate prior to inoculation of the fermentation medium. Maximal production of butylene glycol with minimal residual sugar was obtained with a medium consisting of molasses, diluted to 20 degrees Brix, and 0.4% urea. Optimal environmental conditions included aeration at 0.11 volumes of air per volume of medium per minute, maintenance of pH at 6.0 to 6.2, a fermentation temperature of 30 C, and a stirring rate of 420 rev/min. The concentration of butylene glycol obtained in the fermentation beer ranged from 2.3 to 4.4%. The optical rotation of the glycol ranged from [alpha](D) (23 degrees ) = -1.01 degrees to -10.45 degrees . The variation in rotation was probably due to the presence of contaminating substances in the distillate.     

The characteristics of a polygalacturonase produced by Bacillus polymyxa

The bacillus polygalacturonase (BPG) produced by Bacillus polymyxa was studied and characterized. Maximal cell yield and polygalacturonase production were obtained with a mineral medium containing biotin and 3% (w/v) pectin. Growth and BPG production are greatest when the culture is aerated on a rotary shaker.Pectinesterase production was dependent upon pectin concentration up to 4% BPG production is inducible. Pectin is the best inducer, followed by pectic acid and galacturonic acid in that order. The enzyme has an absolute requirement for calcium ion. Sodium, barium, manganese, strontium, zinc, ferric, cupric, and magnesium ions were ineffective in restoring the activity of dialyzed BPG. The pH optimum of BPG is between 8.9 and 9.4, and it is most stable between pH values of 5.0 and 8.0. Rapid loss of activity occurs below pH 5.0. Its temperature optimum is 45 °C. Although two separate enzyme fractions were isolated by cellulose column chromatography, both were similar in that they hydrolyzed pectic acid more rapidly than pectin. In addition it was shown that both fractions hydrolyzed pectic acid randomly.     

Biodegradation of Some Organic Flotation Reagents by Bacillus polymyxa

The soil bacterium Bacillus polymyxa is shown to effectively remove organic flotation collectors such as dodecyl amine, diamine, sodium isopropyl xanthate, and sodium oleate from alkaline solutions. Adsorption and bacterial growth studies indicated enhanced surface affinity of the amine collectors toward the bacterial cells. All the above organic reagents could be efficiently removed from alkaline solutions through bacterial interaction. Ultraviolet (UV)-visible and Fourier transform infrared (FTIR) spectra during biodegradation of these reagents revealed the stages in biodegradation. Possible mechanisms are outlined.     

Production of optically active 2,3-butanediol by Bacillus polymyxa

Bacillus polymyxa produces (R, R)-2,3-butanediol from a variety of carbohydrates. Other metabolites are also produced including acetoin, acetate, lactate, and ethanol. The excretion of each metabolite was found to depend on the relative availability of oxygen to the culture. When the relative oxygen uptake rate was high, enhanced yields of acetate and acetoin were noted. At an intermediate oxygen availability, the butanediol yield was maximal. When the availability of oxygen was more restricted, higher yields of lactate and ethanol occurred. The cells appeared to regulate themselves such that energy generation is optimal subject to the constraint that the cells do not produce more reducing equivalents than can be oxidized by the electron transport system. The dependence of each product yield on the relative oxygen availability was determined, and this knowledge was used to carry out a fed-batch fermentation that attained a final butanediol concentration of over 40 g/L in 50 h.     

The degradation of oligogalacturonides by the polygalacturonase of Bacillus polymyxa

Studies were made of the action of the polygalacturonase of Bacillus polymyxa (BPG) on the lower Oligogalacturonides. Both the crude exocellular enzyme and fractions separated by cellulose chromatography were able to hydrolyze trigalacturonic acid but not digalacturonic acid. Hydrolysis of trigalacturonate by BPG yields the altered dimer and monogalacturonate, while hydrolysis of the tetramer yields monomer and an altered trimer and, at a slower rate, normal dimer and an altered dimer. In addition, evidence is presented that the polygalacturonase attacks from the non-reducing end. One of the major end products of the action of BPG on pectic substances appears to be a dimer which is different from normal digalacturonate. This compound has been purified and partially characterized. The carboxyl to aldehyde group ratio is 1.98. Assuming one aldehyde group per molecule, the calculated molecular weight of the free acid is almost identical with that of normal dimer (367 and 370, respectively). The specific rotation [α]_D²² of the altered dimer is +177.8 ° (which is similar to that of normal dimer [α]_D²² = +173 °). This suggests that the altered dimer has retained the α configuration. This compound shows a strong absorption peak between 230 and 235 mμ which in turn indicates that it contains an α,β unsaturated bond. Thus, it was concluded that the enzyme is actually a trans-eliminase rather than a hydrolase. The altered dimer is not hydrolyzed by BPG. However, both this compound and normal dimer are attacked by a digalacturonicidase contained within the cells.     

Acetoin Fermentation by Citrate-Positive Lactococcus lactis subsp. lactis 3022 Grown Aerobically in the Presence of Hemin or Cu

CitrLactococcus lactis subsp. lactis 3022 produced more biomass and converted most of the glucose substrate to diacetyl and acetoin when grown aerobically with hemin and Cu. The activity of diacetyl synthase was greatly stimulated by the addition of hemin or Cu, and the activity of NAD-dependent diacetyl reductase was very high. Hemin did not affect the activities of NADH oxidase and lactate dehydrogenase. These results indicated that the pyruvate formed via glycolysis would be rapidly converted to diacetyl and that the diacetyl would then be converted to acetoin by the NAD-dependent diacetyl reductase to reoxidize NADH when the cells were grown aerobically with hemin or Cu. On the other hand, the Y(Glu) value for the hemincontaining culture was lower than for the culture without hemin, because acetate production was repressed when an excess of glucose was present. However, in the presence of lipoic acid, an essential cofactor of the dihydrolipoamide acetyltransferase part of the pyruvate dehydrogenase complex, hemin or Cu enhanced acetate production and then repressed diacetyl and acetoin production. The activity of diacetyl synthase was lowered by the addition of lipoic acid. These results indicate that hemin or Cu stimulates acetyl coenzyme A (acetyl-CoA) formation from pyruvate and that lipoic acid inhibits the condensation of acetyl-CoA with hydroxyethylthiamine PP(i). In addition, it appears that acetyl-CoA not used for diacetyl synthesis is converted to acetate.     

Cytometric monitoring of growth, sporogenesis and spore cell sorting in Paenibacillus polymyxa (formerly Bacillus polymyxa)

AIMS: Formation of bacterial endospores is a basic process in Gram-positive bacteria and has implications for health, industry and the environment. Flow cytometry offers a practical alternative for the rapid detection, enumeration and characterization of bacterial endospores. METHODS AND RESULTS: Paenibacillus polymyxa was chosen for this study because its spores cause sporangium deformation and have thick walls with a star-shaped section. Sporulating populations were analysed with a particle analyser and a flow cytometer after labelling with propidium iodide and Syto-13. Flow cytometric detection of single spores was confirmed by optical and scanning electron microscopy after cell sorting. Four cell sub-populations were cytometrically detected in P. polymyxa cultures grown in liquid sporulation medium. Two sub-populations consisted of vegetative cells differing in both morphology and viability; the other two sub-populations consisted of spores differing in their viability. CONCLUSIONS: This work has shown that flow cytometry is a simple and fast method (less than 15 minutes for sample preparation and analysis) for the study of the sporulation in P. polymyxa. The use of this technique allowed both detection and quantification of sporulation inside a culture, and distinguished cells that differed in viability despite being morphologically identical under microscopic observation. SIGNIFICANCE AND IMPACT OF THE STUDY: Flow cytometry has been proved to be a valuable tool for the analysis of sporulation in P. polymyxa cultures, with the unique capacity of distinguishing between endospores and vegetative cells, and between live and dead cells, in the same analysis. An important percentage of non-viable endospores has been found in aged cultures using this method.     

Degradation of cellulose by nitrogen-fixing strain of Bacillus polymyxa.

During an intensive screening programme, several strains of cellulolytic bacteria were isolated. One nitrogenase-positive strain able to degrade filter paper, Avicel cellulose, carboxymethyl cellulose and cellobiose was selected for further study. On the basis of biochemical characteristics and Mol % of G+C content, the selected strain was identified as Bacillus polymyxa. The highest production of the enzymes degrading filter paper (FP-ase) and carboxymethyl cellulose (CMCase) by B. polymyxa was observed in Park's medium suplemented with Avicel cellulose. The investigated strain of bacteria produced cellulosome-like structures as was shown by transmission electron microscopy.     

Acetoin Catabolism and Acetylbutanediol Formation by Bacillus pumilus in a Chemically Defined Medium

Background

Most low molecular diols are highly water-soluble, hygroscopic, and reactive with many organic compounds. In the past decades, microbial research to produce diols, e.g. 1,3-propanediol and 2,3-butanediol, were considerably expanded due to their versatile usages especially in polymer synthesis and as possible alternatives to fossil based feedstocks from the bioconversion of renewable natural resources. This study aimed to provide a new way for bacterial production of an acetylated diol, i.e. acetylbutanediol (ABD, 3,4-dihydroxy-3-methylpentan-2-one), by acetoin metabolism.

Methodology/Principal Findings

When Bacillus pumilus ATCC 14884 was aerobically cultured in a chemically defined medium with acetoin as the sole carbon and energy source, ABD was produced and identified by gas chromatography – chemical ionization mass spectrometry and NMR spectroscopy.

Conclusions/Significance

Although the key enzyme leading to ABD from acetoin has not been identified yet at this stage, this study proposed a new metabolic pathawy to produce ABD in vivo from using renewable resources – in this case acetoin, which could be reproduced from glucose in this study – making it the first facility in the world to prepare this new bio-based diol product.     

Direct Fermentation of d-Xylose to Ethanol by Kluyveromyces marxianus Strains

Eight strains of Kluyveromyces marxianus were screened, and all of them were found to ferment the aldopentose d-xylose directly to ethanol under aerobic conditions. One of these strains, K. marxianus SUB-80-S, was grown in a medium containing 20 g of d-xylose per liter, and the following results were obtained: maximum ethanol concentration, 5.6 g/liter; ethanol yield, 0.28 g of ethanol per g of d-xylose (55% of theoretical); maximum specific growth rate, 0.12 h; 100% d-xylose utilization was completed in 48 h.