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Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media.

A strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45 degrees C and in 250 g/l molasses although the optimal growth temperature was 35-37 degrees C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25-35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth-associated process. Factors that normally promote PHB production include high ratios of carbon to nitrogen, and carbon to phosphorus in growth media. Low dissolved oxygen supply resulted in sporulation, which reduced PHB contents and dry weights of the cells. It seems that sporulation induced by reduced supply of nutrients is the reason that PHB content is generally low in the Bacillus strain.

3-Hydroxybutyric Acid↗

Rapid determination of the glucose content of molasses using a biosensor.

A knowledge of the sugar content of molasses is of commercial importance to a number of industrial fermentations. Hence the feasibility of using a glucose oxidase biosensor to determine the glucose content of molasses samples was investigated. This method was compared with standard high-performance liquid chromatographic (HPLC) and gas-liquid chromatographic (GLC) procedures and with the use of a commercially available glucose analyser. A good correlation was obtained between the standard acetic anhydride GLC and glucose oxidase biosensor results (correlation coefficient = 0.98). Rapid and accurate measurements could be carried out using the biosensor without the need to employ the sample preparation step required in standard GLC methods. It was concluded that the use of the biosensor technique for the determination of glucose in molasses samples has distinct advantages over conventional methods.

Biosensing Techniques↗

Enrichment and identification of bacteria capable of reducing chemical oxygen demand of anaerobically treated molasses spent wash.

AIMS: The aim of this study was to isolate and identify bacterial strains capable of using recalcitrant compounds of molasses spent wash as sole carbon source from the soils of abandoned sites of distillery effluent discharge and characterize their ability of reducing the chemical oxygen demand (COD) of the spent wash. METHODS AND RESULTS: The isolates were grouped into six haplotypes by amplified ribosomal DNA restriction analysis (ARDRA) and BOX-PCR. The phylogenetic position of the representatives of the six main haplotypes strains was determined by 16S rDNA sequencing. They showed maximum similarity to six genera viz. Pseudomonas, Enterobacter, Stenotrophomonas, Aeromonas, Acinetobacter and Klebsiella. The extent of COD (44%) reduced collectively by the six strains was equal to that reduced individually by Aeromonas, Acinetobacter, Pseudomonas and Enterobacter. With spent wash as sole carbon source, the COD reducing strains grew faster at 37 degrees C than 30 degrees C. CONCLUSIONS: Bacterial strains capable of degrading some of the recalcitrant compounds of anaerobically digested molasses spent wash can be isolated from the soils of abandoned sites of distillery effluent discharge. Biostimulation of these bacteria, which can degrade 44% of the carbon compounds of anaerobically digested molasses spent wash can be achieved by nitrogen fertilization and relatively higher temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: Supplementation of nitrogen source and controlling the temperature can be used in evolving strategies for in situ bioremediation of anaerobically digested spent wash from distilleries.

Bacteria↗

Production of poly-beta-hydroxyalkanoates from soy molasses oligosaccharides by new, rapidly growing Bacillus species.

AIMS: To isolate and characterize bacteria from nature capable of producing poly-beta-hydroxyalkanoates in high yields from soy molasses oligosaccharides. METHODS AND RESULTS: Several strains of bacteria were obtained from enrichment cultures employing raffinose as major carbon source and inoculated with soybean field soil, lake sediment, or lake water. Many of the isolates were Bacillus species and produced polyhydroxyalkanoates (PHAs) to high yield. The raffinose-degrading isolates produced endospores, were highly saccharolytic, and both respired and fermented a variety of mono-, di-, tri- and tetrasaccharides. Strain CL1 produced 90% of cell dry mass as PHA from various sugars, including raffinose, and did so without requiring a nutrient limitation. CONCLUSIONS: Strain CL1 could be the catalyst for an industrial fermentation converting soy molasses and other waste carbohydrates to PHAs. The properties of this organism that make it ideally suited for such a fermentation include (i) its ability to use a wide variety of plant-associated carbohydrates as PHA feedstocks; (ii) its rapid growth; (iii) its ability to grow under anoxic conditions; and (iv) its ability to produce spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of bacteria capable of making biodegradable plastics to high yield from soy molasses oligosaccharides.

Bacillus↗

Mutagenesis and analysis of mold Aspergillus niger for extracellular glucose oxidase production using sugarcane molasses.

Aspergillus niger ORS-4.410, a mutant of A. niger ORS-4, was generated by repeated ultraviolet (UV) irradiation. Analysis of the UV treatment dose on wild-type (WT) A. niger ORS-4, conidial survival, and frequency of mutation showed that the maximum frequency of positive mutants (25.5%) was obtained with a 57% conidial survival rate after the second stage of UV irradiation. The level of glucose oxidase (GOX) production from mutant A. niger ORS-4.410 thus obtained was 149% higher than that for WT strain A. niger ORS-4 under liquid culture conditions using hexacyanoferrate (HCF)-treated sugarcane molasses (TM) as a cheaper carbohydrate source. When subcultured monthly for 24 mo, the mutant strain had consistent levels of GOX production (2.62 +/- 0.51 U/mL). Mutant A. niger ORS-4.410 was markedly different from the parent strain morphologically and was found to grow abundantly on sugarcane molasses. The mutant strain showed 3.43-fold increases in GOX levels (2.62 +/- 0.51 U/mL) using HCF-TM compared with the crude form of cane molasses (0.762 +/- 0.158 U/mL).

Aspergillus niger↗

[Estimation of chemical composition of molasses for leucine biosynthesis by the methods of statistical analysis].

Chemical composition of molasses, used as sources of carbohydrates for leucine biosynthesis has been studied. Influence of molasses parameters on biomass growth and leucine synthesis in the process of cultivation of the producer of Corynebacterium sp. genus is proved. The results are processed by the personal computer with the use of applied statistical programs. The limits of changes of molasses parameters, allowing one to obtain 19.0-21.2 g/l leucine at the level of carbohydrate conversion of 19.4-21.8 %, have been determined.

Analysis of Variance↗

Changes in development and ultrastructure of Aspergillus niger mycelium, strain with increased tolerance to toxic substances from beet molasses.

Effects of a defoamer and toxic molasses compounds on development and ultrastructure of A. niger mycelium, strain Z, characterized by high tolerance to these substances and producing citric acid in surface fermentation on proper molasses media with 70% yield were presented. Spumol BJ in concentration of 5 microliters/100 cm3 as well as toxic molasses compounds stimulated the process of swelling and germinating of conidia. Moreover, giant conidia, unable to germinate, appeared. Developing mycelium with dispersed hyphae became mucilaginous after 17-20 h culture, which indicated the process of sinking but after 24 h some part of the mycelium developed normally. Electron microscopic observations of mycelium developing in the presence of the toxic substances showed along with electron-transparent cytoplasm in a consequence of decrease in ribosome number, changes in ultrastructure of mitochondria. It may be assumed that one of the reasons of the above described abnormalities in development and ultrastructure of mycelium was a disturbance of respiration processes. The appearance of deposits of electron-dense material in mitochondria suggested the existence of a defence mechanism, eliminating toxic substances.

Aspergillus niger↗

Molasses induced changes in Saccharomyces cerevisiae: alterations in plasma membrane structure and function and metallothionein level.

Growth of yeast cells in molasses caused changes in plasma membrane properties. The polypeptide profile of plasma membrane prepared by sucrose density gradient centrifugation showed a range of new polypeptides above 55 kD which were not present in malt extract-glucose-yeast extract-peptone medium grown cells. Isolated new bands appeared in the range of 25-50 kD. Transport of glucose was markedly enhanced in molasses trained cells. Among the amino acids, L-lysine uptake was decreased while L-aspartate uptake was enhanced considerably. Finally the metallothionein levels in the cells grown in molasses was found to be increased significantly, possibly to protect the cells from the harmful effect of heavy metal ions.

Alanine↗

Lysosome injury by perfusion of the isolated rat liver with ethanol or a molasses distillate (cachaça).

An isolated rat liver perfusion model was used to study the effects of acute exposure of the organ to either ethanol or a molasses distillate (cachaça). When ethanol (72 mM) or a molasses distillate (68 mM ethanol) was added to the perfusion fluid, lysosomal injury was indicated by the increased release of tartrate-inhibited acid phosphatase activity at the end of a 3 h period of perfusion. Other cellular compartments were not significantly damaged in these acute experiments, as judged by the release of aspartate and alanine aminotransferases, lactate dehydrogenase and alkaline phosphatase. The behavior of both ethanol itself and the alcoholic beverage was similar as far as enzyme release is concerned but only the molasses distillate caused significant acidosis (a decrease in perfusate pH) at the end of a 3 h period of perfusion. These data may be of importance for a better understanding of the hepatic damage caused by alcohol abuse and useful for laboratory investigation of alcohol intoxication.

Alcoholic Intoxication↗

High performance liquid chromatographic determination of fructose, glucose, and sucrose in molasses.

Fructose, glucose, and sucrose were determined quantitatively in molasses by high performance liquid chromatography (HPLC) using maltose as an internal standard. After a simple cleanup procedure, chromatographic separation required less than 20 min for the 17 molasses samples. HPLC gave lower results for reducing sugars (fructose plus glucose) and for sucrose by an average of 3.9 and 1.2%, respectively, when compared to results obtained by classical methods. The lower values are believed to result from elimination of interfering substances by this method. The values probably reflect more accurately the true composition of the molasses. Repetitive injections of a standard solution indicated a coefficient of variation of 2.0% for fructose and glucose and of 2.2% for sucrose.

Chromatography, High Pressure Liquid↗

Changes in dehydrogenases activity, number and ultrastructure of mitochondria in Aspergillus niger mycelium growing on molasses media.

Effects of toxic molasses compounds and of the antifoamer (Spumol BJ) on dehydrogenases activity, on the number and ultrastructure of mitochondria in A. niger mycelium of two strains characterized by different tolerance to toxic agents, were observed. In spite of significantly higher dehydrogenases activity in the intolerant strain (R-16) mycelia developing both on productive molasses in the presence of the defoamer and on non-productive molasses are characterized by marked reduction in the activity of these enzymes. Changes in respiratory enzymes activity are partly correlated with the number of mitochondria but mostly with abnormalities in their ultrastructure.

Aspergillus niger↗

Batch decolorization of molasses by suspended and immobilized fungus of Geotrichum candidum Dec 1.

Geotrichum candidum Dec 1, which exhibits a broad dye-decolorizing spectrum, was used to decolorize molasses during semi-batch cultivation. An eighty % decolorization of molasses and a stable peroxidase activity were maintained for approximately four weeks, after which, both activities deteriorated significantly. Subsequently, repeated batch cultivation of fungal cells immobilized on polyurethane foam was employed to solve the aforementioned problems and stable decolorization of molasses as well as stable peroxidase activity was realized for more than eight weeks.

Journal Article↗

Continuous production of lactic acid from molasses by perfusion culture of Lactococcus lactis using a stirred ceramic membrane reactor.

A perfusion culture system was used for continuous production of lactic acid by retaining cells at a high density of Lactococcus lactis in a stirred ceramic membrane reactor (SCMR). After the cell concentration increased to 248 g/l, half of the culture broth volume was replaced with the fermentation medium. Subsequently, a substrate solution containing glucose (run 1) or molasses (run 2) was continuously supplied to the cells retained in the SCMR. Simultaneously, the culture supernatant was extracted using a ceramic filter with a pore size of 0.2 mum. The dilution rate was initially set at 0.4 h(-1) and gradually decreased to 0.2 h(-1) due to reduction in the permeability of the filter. The concentration of glucose in the substrate solution was adjusted to 60 g/l for the transition and the first period until 240 h, 90 g/l for the second period from 240 h to 440 h, and 70 g/l for the third period from 440 h to 643 h. The average concentration of lactic acid in the filtrate reached 46 g/l in the first period, 43 g/l in the second period, and 33 g/l for the third period. The productivity obtained for the first period reached 15.8 g.l(-1).h(-1), twice as much as that achieved in repeated batch fermentations. Based on the results obtained in run 1, the substrate solution containing 120 g/l of molasses was continuously supplied for 240 h in run 2. The concentration and productivity of lactic acid reached 40 g/l and 10.6 g.l(-1).h(-1), respectively, by continuously replenishing the culture medium at a dilution rate of 0.26 h(-1). These results demonstrated that the filtration capacity of the SCMR was sufficient for a continuous and rapid replenishment of molasses solution from the dense cell culture and, therefore, the perfusion culture system is considered to provide a low-cost process for continuous production of lactic acid from cheap resources.

Journal Article↗

Supercritical fluid extraction of limonoid glucosides from grapefruit molasses.

Limonoid glucosides (primarily limonin 17-beta-D-glucopyranoside, LG) were extracted from grapefruit molasses by supercritical fluid extraction using a supercritical carbon dioxide-ethanol (SC CO(2)-ethanol) system. Extraction conditions to maximize the yield of LG were determined by varying pressure, temperature, ethanol concentration, and extraction time. The highest yield of LG at 0.61 mg/g molasses was obtained at a pressure 48.3 MPa, a temperature of 50 degrees C, 10% ethanol (X(Eth) = 0.1), and 40 min of extraction time at a flow rate of 5.0 L/min. The results demonstrated that SC CO(2) extraction of limonoid glucosides from grapefruit molasses has practical significance for commercial production.

Chromatography, Supercritical Fluid↗

MOL1, a Saccharomyces cerevisiae gene that is highly expressed in early stationary phase during growth on molasses.

We have isolated a new Saccharomyces cerevisiae gene, MOL1, that is transiently expressed at high levels in the early stationary phase of batch cultures growing on industrial molasses medium. The DNA sequence of the MOL1 gene (for MOLasses-inducible) with its flanking regions was determined (EMBL accession number X61669). It encodes a polypeptide of M(r) 35 kDa that is closely related to stress-inducible proteins of similar size from two Fusarium species. Unlike ST135 of Fusarium, MOL1 is not induced by ethanol or heat shock. MOL1 expression is absent in rich (YP) medium, and only very low levels of expression are detectable in minimal (YNB) medium. The gene is not essential, and a MOL1 disruption strain showed no apparent phenotype under a variety of growth conditions. The 5' region of MOL1 contains the complete sequence previously determined for the SUF4 locus, encoding a tRNA-gly (UCC) gene, which has been mapped to chromosome VII.

Amino Acid Sequence↗

Microbial production of propionic acid and vitamin B12 using molasses or sugar.

With a cell concentration of 125 g dry biomass l-1 and a dilution rate of 0.1 h-1, Propionibacterium acidipropionici produces 30 g propionic acid l-1 from sugar with a productivity of 3 g l-1 h-1. The yield of propionic acid is approx. 0.36-0.45 g propionic acid g-1 sucrose and is independent of the dilution rate and cell concentration. Acetic acid is an unwanted by-product in the production of propionic acid. The concentration of acetic acid only increases slightly when the cell concentration is increased. A two-stage fermentation process was developed for the conversion of sugar or molasses of various types to propionic acid and vitamin B12. By fermentation of blackstrap molasses (from sugar beet and sugar cane) in the first fermentation stage 17.7 g propionic acid l-1 with a yield of 0.5 g propionic acid g-1 carbohydrate was produced with a dilution rate of 0.25 h-1. In the second stage 49 mg vitamin B12 1-1 was produced at a dilution rate of 0.03 h-1.

Carbohydrate Metabolism↗

The promotion of molasse alcoholic fermentation using Saccharomyces cerevisiae in the presence of gamma-alumina.

Using gamma-alumina pellets, more than threefold increase of the ethanol productivity in the fermentation molasse has been obtained in the present work. Also, molasse fermentation in the presence of gamma-alumina gave 78.4 g/L ethanol, ethanol yield factor 0.44 g/g, and conversion 89.4% at initial sugar concentration (ISC) 179.5 g/L, compared to 53.9 g/L, 0.30 g/g and 62.7% in its absence, respectively. Furthermore, it was found that gamma-alumina reduces the activation energy Ea of fermentation. This inorganic material does not promote the fermentation of raisin extract.

Aluminum Oxide↗

Constitutive expression of the Trichoderma reesei beta-1,4-xylanase gene (xyn2) and the beta-1,4-endoglucanase gene (egl) in Aspergillus niger in molasses and defined glucose media.

The xylanase II (xyn2)- and endoglucanase I (egl)-encoding regions of Trichoderma reesei QM6a were successfully expressed in Aspergillus niger D15 under the transcriptional control of the glyceraldehyde-6-phosphate dehydrogenase (gpd) promoter from A. niger and the glaA terminator of Aspergillus awamori. A stable xyn2 transformant produced beta-xylanase activity of 8,000 nkat/ml and 5,000 nkat/ml in shake-flask cultures containing defined or 20% (v/v) molasses medium, respectively. The recombinant Xyn2 enzyme expressed highest activity at pH 5-6 and 50-60 degrees C and retained more than 75% of its activity after 3 h of incubation at 50 degrees C. A stable egl transformant produced endo-P-1,4-glucanase activity of 2,300 nkat/ml in shake-flask cultures containing defined media and about half the activity in 20% molasses medium. Maximum endoglucanase activity was obtained at pH 5 and 60 degrees C. Both Xyn2 and EgI retained >80% activity after incubation at 50 degrees C for 3 h. The heterologous Xyn2 and EgI represent a significant portion of the total extracellular proteins produced.

Aspergillus niger↗