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Biotechnological production of amino acids and derivatives: current status and prospects.

For almost 50 years now, biotechnological production processes have been used for industrial production of amino acids. Market development has been particularly dynamic for the flavor-enhancer glutamate and the animal feed amino acids L: -lysine, L: -threonine, and L: -tryptophan, which are produced by fermentation processes using high-performance strains of Corynebacterium glutamicum and Escherichia coli from sugar sources such as molasses, sucrose, or glucose. But the market for amino acids in synthesis is also becoming increasingly important, with annual growth rates of 5-7%. The use of enzymes and whole cell biocatalysts has proven particularly valuable in production of both proteinogenic and nonproteinogenic L: -amino acids, D: -amino acids, and enantiomerically pure amino acid derivatives, which are of great interest as building blocks for active ingredients that are applied as pharmaceuticals, cosmetics, and agricultural products. Nutrition and health will continue to be the driving forces for exploiting the potential of microorganisms, and possibly also of suitable plants, to arrive at even more efficient processes for amino acid production.

Amino Acids↗

Degradation of toxaphene by Bjerkandera sp. strain BOL13 using waste biomass as a cosubstrate.

The white-rot fungus Bjerkandera sp. strain BOL13 was capable of degrading toxaphene when supplied with wood chips, wheat husk or cane molasses as cosubstrates in batch culture experiments. Approximately 85% of toxaphene was removed when wheat husk was the main substrate. The production of lignin peroxidase was only stimulated when wheat husk was present in the liquid medium. Although xylanase was always detected, wheat husk supported the highest xylanase production. A negligible amount of beta-glucosidase and cellulase were found in the batch culture medium. To the best of our knowledge, this is the first reported case of toxaphene degradation by white-rot fungi.

Basidiomycota↗

A microcosm study on bioremediation of p-nitrophenol-contaminated soil using Arthrobacter protophormiae RKJ100.

p-Nitrophenol (PNP), a toxic nitroaromatic compound, can build up in soils due to extensive usage of nitrophenolic pesticides and hence needs to be removed. Arthrobacter protophormiae RKJ100, a PNP-degrading organism, was used in this work to study factors affecting its growth, and then evaluated for its capacity to degrade PNP in soil microcosms. Molasses (10%) treated with 0.1% potassium hexacyanoferrate was found to be a suitable and cheap carbon source for inoculum preparation. Induction studies showed that PNP depletion was quicker when cells were induced by pre-exposure to PNP. The efficiency of PNP degradation in soil by strain RKJ100 was seen to be dependent on pH, temperature, initial PNP concentration and inoculum size. Microcosm studies performed with varying concentrations (1.4-210 ppm) of PNP-spiked soils showed that strain RKJ100 could effectively degrade PNP over the range 1.4-140 ppm. A cell density of 2x10(8) colony forming units/g soil was found to be suitable for PNP degradation over a temperature range of 20-40 degrees C and at a slightly alkaline pH (7.5). Our results indicate that strain RKJ100 has potential for use in in situ bioremediation of PNP-contaminated sites. This is a model study that could be used for decontamination of sites contaminated also with other compounds.

Arthrobacter↗

Conversion of agricultural feedstock and coproducts into poly(hydroxyalkanoates).

Aside from their importance to the survival and general welfare of mankind, agriculture and its related industries produce large quantities of feedstocks and coproducts that can be used as inexpensive substrates for fermentative processes. Successful adoption of these materials into commercial processes could further the realization of a biorefinery industry based on agriculturally derived feedstocks. One potential concept is the production of poly(hydroxyalkanoate) (PHA) polymers, a family of microbial biopolyesters with a myriad of possible monomeric compositions and performance properties. The economics for the fermentative production of PHA could benefit from the use of low-cost agricultural feedstocks and coproducts. This mini-review provides a brief survey of research performed in this area, with specific emphasis on studies describing the utilization of intact triacylglycerols (vegetable oils and animal fats), dairy whey, molasses, and meat-and-bone meal as substrates in the microbial synthesis of PHA polymers.

Crops, Agricultural↗

Chromosomal polymorphism and adaptation to specific industrial environments of Saccharomyces strains.

Several industrial Saccharomyces strains, including bakers', wine, brewers' and distillers' yeasts, have been characterized with regards to their DNA content, chromosomal polymorphism and homologies with the DNA of laboratory strains. Measurement of the DNA contents of cells suggested that most of the industrial yeasts were aneuploids. Polymorphisms in the electrophoretic chromosomal pattern were so large that each strain could be individually identified. However, no specific changes relating to a particular group were observed. Hybridization using different probes from laboratory strains was very strong in all cases, indicating that all industrial strains possess a high degree of DNA homology with laboratory yeasts. Probes URA3, CUP1, LEU2, TRP1, GAL4 or ADC1 demonstrated the presence of one or two bands, two especially in bakers' strains. Also, results indicate that all hybridized genes are located on the same chromosomes both in laboratory and industrial strains. Translocation from chromosome VIII to XVI seems to have occurred in a distillers' strain, judging by the location of the CUP1 probe. Finally, when the SUC2 probe is used, results indicate a very widespread presence of the SUC genes in only bakers' and molasses alcohol distillers' strains. This clearly suggests that amplification of SUC genes is an adaptive mechanism conferring better fitness upon the strains in their specific industrial conditions. The widespread presence of Ty1 and Ty2 elements as well as Y' subtelomeric sequences could account for the inter- and intrachromosomal changes detected.

Adaptation, Physiological↗

Production of the biocontrol agent Pantoea agglomerans strain CPA-2 using commercial products and by-products.

The aim of this paper was to find the nitrogen and carbon sources that provide maximum biomass production of strain CPA-2 of the biocontrol agent Pantoea agglomerans and minimum cost of media, whilst maintaining biocontrol efficacy. To reduce the cost of media, commercial products and by-products were tested. P. agglomerans can be produced using a combination of nitrogen sources such as yeast extract (5 g l(-1)) and dry beer yeast (10 g l(-1)) with inexpensive carbohydrates such as sucrose (10 g l(-1)) and molasses (20 g l(-1)), respectively, maintaining the efficacy of the biocontrol agent against Penicillium digitatum and P. italicum on oranges. The results obtained in this study could be used to provide a reliable basis for a scale-up of this fermentation process to an industrial level.

Biomass↗

Isomaltulose production using free cells: optimisation of a culture medium containing agricultural wastes and conversion in repeated-batch processes.

The enzyme glucosyltransferase is an industrially important enzyme since it produces non-cariogenic isomaltulose (6-O-alpha-D-glucopyronosyl-1-6-D-fructofuranose) from sucrose by intramolecular transglucosylation. The experimental designs and response surface methodology (RSM) were applied for the optimisation of the nutrient concentrations in the culture medium for the production of glucosyltransferase by Erwinia sp. D12 in shaken flasks at 200 rpm and 30 degrees C. A statistical analysis of the results showed that, in the range studied, the factors had a significant effect (P < 0.05) on glucosyltransferase production and the highest enzyme activity (10.84 U/ml) was observed in culture medium containing sugar cane molasses (150 g l(-1)), corn steep liquor (20 g l(-1)), yeast extract Prodex Lac SD (15 g l(-1)) and K2HPO4 (0.5 g l(-1)) after 8 h at 30 degrees C. The production of cell biomass by the strain of Erwinia sp. D12 was carried out in a 6.6-l fermenter with a mixing rate of 200 rpm and an aeration rate of 1 vvm. Fermentation time, cellular growth, medium pH and glucosyltransferase production were observed. The greatest glucosyltransferase activity was 22.49 U/ml, obtained after 8 h of fermentation. The isomaltulose production from sucrose was performed using free Erwinia sp. D12 cells in a batch process using an orbital shaker. The influence of the parameters sucrose concentration, temperature, pH, and cell concentration on the conversion of sucrose into isomaltulose was studied. The free cells showed a high conversion rate of sucrose into isomaltulose using batch fermentation, obtaining an isomaltulose yield of 72.11% from sucrose solution 35% at 35 degrees C.

Biomass↗

Production of poly(3-hydroxybutyrate) by solid-state fermentation with Ralstonia eutropha.

The use of solid-state fermentation is examined as a low-cost technology for the production of poly(hydroxyalkanoates) (PHAs) by Ralstonia eutropha. Two agroindustrial residues (babassu and soy cake) were evaluated as culture media. The maximum poly(hydroxybutyrate) (PHB) yield was 1.2 mg g(-1) medium on soy cake in 36 h, and 0.7 mg g(-1) medium on babassu cake in 84 h. Addition of 2.5% (w/w) sugar cane molasses to soy cake increased PHB production to 4.9 mg g(-1) medium in 60 h. Under these conditions, the PHB content of the dry biomass was 39% (w/w). The present results indicate that solid-state fermentation could be a promising alternative for producing biodegradable polymers at low cost.

Cell Culture Techniques↗

The effect of supplementation on productive performance of Boer goat bucks fed winter veld hay.

Fifteen Boer goat bucks, (6-8 months old), were allocated into two groups and fed ad libitum for a period of 29 days. The WH group (winter hay; n = 8) received a chopped diet consisting of grass hay, predominantly Themeda trianda grass (cut during the winter) from a natural pasture (veld). The WH+S group (winter hay plus supplement; n = 7) received a chopped diet consisting of Themeda trianda veld hay, supplemented with maize meal, molasses meal and urea. Body weights and feed consumption were recorded. Animals were slaughtered and carcass characteristics (weight and percentages of selected carcass cuts) and the carcass chemical composition were determined. The WH+S animals showed a higher live weight and cumulated feed intake. Carcass cuts from WH animals represented a higher percentage of the total carcass, especially cuts where muscle depots are higher (legs, best end chops and prime cuts). There is an attempt by non-supplemented animals to preserve the body's nitrogen reserves under prolonged nutritional stress conditions, and there is consequently a necessity to supplement feeding of small ruminants fed winter veld hay, especially if the animals are to be used in subsequent breeding seasons.

Animal Feed↗

Effects of supplementary feeding and suckling intensity on postpartum reproductive performance of small East African Zebu cows.

Small East African Zebu cows (n = 120) which calved during the dry season were used to determine the effects of postpartum nutritional supplementation and suckling intensity on body weight, body condition score, reproductive performance and on calf mortality and preweaning growth performance. Cows were allocated based on calving body weight to either receive a supplementary feed of molasses blocks containing 10% urea or to serve as controls and to be grazed only. Within nutritional groups, cows were further assigned either to continuous suckling or to restricted suckling twice daily. Intact bulls were introduced 21 days after the first cow had calved, and data were collected for 8 months. Calving body weight and body condition score of dams ranged from 186 to 192 kg and from 3.8 to 4.2 and did not differ among the treatment groups. Supplementary feeding significantly (P<0.05) reduced calf mortality from 40 to 20%, while restricted suckling reduced mortality by 13% from 36 to 23%. Body weight and body condition score did not vary between groups during the supplementation period. Over the 8-month period, 65% of supplemented and 53% of control cows exhibited estrus (P>0.05). A significantly (P<0.05) karger percentage of restricted suckled cows showed estrus than of continuously suckled cows (74 vs 42%). The postpartum estrus interval was shorter by 54 days in supplemented than in control cows and by 13 days in restricted than in continuously suckled cows. The pregnancy rate was higher in supplemented than in control cows (37 vs 33%; P>0.05) and in restricted than in continuously suckled (46 vs 22%; P>0.05) cows.

Journal Article↗

The effect of diet in late pregnancy on progesterone concentration and colostrum yield in ewes.

In the ewe, high level feeding is associated with low levels of circulating progesterone and that progesterone withdrawal is a prerequisite for lactogenesis and the onset of copious milk secretion. A total of 108 ewes was allocated to a 3 x 2 factorial experiment to determine the effect of diet on serum progesterone (P4) concentration and colostrum yield. On D 96 of pregnancy, the ewes were offered either grass silage, molassed sugar beet pulp (beet pulp) silage or grass silage supplemented with beet pulp. All ewes were given ad libitum access to silage. From D 126 of pregnancy, half the diets were supplemented with soybean meal with the aim of achieving a total crude protein intake of 220 g/ewe/day. Blood samples were taken on D 142 of pregnancy and at 1, 10 and 18 h post lambing for P4 determination. Metabolizable energy intakes (Mega Joules/ewe/day) of 9.1, 11.2 and 12.1 (SEM 0.37) and crude protein intakes (g/ewe/day) of 142.8, 167.4 and 162.0 (SEM 3.46) were recorded for grass silage, beet pulp ensiled and beet pulp supplemented silages, respectively, from D 126 to D 147 of pregnancy. Soybean meal supplementation increased the crude protein intake from 97.1 to 217.0 g/ewe/day (SEM 2.86) and Metabolisable Energy intake from 9.0 to 12.6 Mega Joules/ewe/day (SEM 0.31). Supplementation with soybean meal decreased P4 concentration (ng/ml) on D 142 (P < 0.05) and at 1 h (P < 0.01), 10 h (P < 0.01) and 18 h post lambing (P < 0.01). Ewes offered grass silage had higher P4 concentrations on D 142 (P < 0.01) and at 1 h after lambing (P < 0.05) than the beet pulp-supplemented ewes. There were negative linear relationships between P4 concentration at D 142 and colostrum yield at 1 h (P < 0.01) and between P4 concentration at 1 h and colostrum yield at 1 h (P < 0.001) and total yield at 18 h (P < 0.001). In conclusion, ewes which were underfed had higher P4 concentrations at all times, and there were negative linear relationships between colostrum yield and P4 concentrations on D 142 of pregnancy and at 1 h post lambing.

Journal Article↗

Construction of a sucrose-fermenting bakers' yeast incapable of hydrolysing fructooligosaccharides.

Fructooligosaccharides stimulate the growth of intestinal bifidobacteria which are related to the favorable health and nutrition of humans and other animals. Since the efficient amount of fructooligosaccharide for an adult human is relatively large (about 5 g per day), its addition to daily foods like bakery goods might be beneficial. However, commercial Bakers' yeast hydrolyses fructooligosaccharides by the action of invertase encoded in SUC genes and ferments the resulting monosaccharides. According to the findings that strains carrying the MAL-constitutive gene and lacking the SUC gene fermented sucrose and not fructooligosaccharide, we constructed a sucrose-fermenting strain, YOY920, incapable of hydrolysing fructooligosaccharide, by cross-breeding a baking strain and a laboratory strain. In a molasses medium, the cell yield of YOY920 was comparable to that of a baking strain FSC6001, and much higher than that of the non-sucrose-fermenting strains. Although fructooligosaccharide inhibited the dough leavening ability of YOY920, white bread containing fructooligosaccharide could be produced in the defined dough formula using the new strain.

Animals↗

Genotoxicity of 'gudakhu', a tobacco preparation. I. In mice in vivo.

'Gudakhu' is a paste-like tobacco preparation used widely in Orissa and neighbouring states of India. During use it is rubbed over the teeth and gum with a finger tip. Besides tobacco, it contains molasses, lime, red soil and water. The genotoxic potential of acetone extract of gudakhu was evaluated in mice in vivo using the chromosome aberration assay, micronucleus test and SCE analysis following single as well as long-term repeated treatment. The animals received an aqueous suspension of the extract via the oral route. Gudakhu extract induced significantly high frequencies, compared to controls, of chromosome aberrations, micronuclei (MN) and SCEs. Single treatment with different doses clearly revealed a distinct dose-dependent increase of the effects in all the assays. Analysis of MN in regenerated hepatocytes also indicated a significant positive correlation between time-course of chronic treatment and frequencies of micronucleated cells. But incidences of chromosome aberrations, MN and SCEs in bone marrow cells following repeated treatment for different periods did not differ greatly from each other; and these repeated treatment data, particularly in the MNT in bone marrow cells and the SCE assay, also did not differ markedly from the respective single treatment data for the same dose. This was probably due to the proliferative nature of the bone marrow cells.

Animals↗

Elimination of adverse effects of ethoxyquin (EQ) by methionine hydroxy analog (MHA). Protective effects of EQ and MHA for bitterweed poisoning in sheep.

Dietary ethoxyquin (EQ) and methionine hydroxy analog (MHA) protected 6-8-month-old wethers from toxic doses of bitterweed (Hymenoxys odorata DC.). The EQ-MHA group received sweet feed (corn, oats, dehydrated alfalfa pellets, cane molasses and minerals), 500 g/day/sheep, supplemented with EQ and MHA (0.5% and 1.0% of feed, respectively) for 9 days prior to the poisoning with bitterweed while the MHA group received the same feed without EQ and controls received the same amount of feed with no additives. Two of 6 MHA-treated and 3 of 7 controls died whereas all 7 EQ-MHA-treated sheep survived after receiving 5 doses of bitterweed (5 X 5.5 g/kg) in 6 days. Coadministration of MHA and EQ eliminated the adverse effect of EQ; dietary EQ lowered the serum albumin, calcium, and alkaline phosphatase content while protecting the animals from bitterweed poisoning. EQ is the most promising protective agent tested for bitterweed poisoning in sheep.

Animals↗

Influence of dietary ingredients on the body retention of strontium, cadmium and mercury in suckling rats.

Six-day-old suckling rats were artificially fed over 8 h with cow's milk or a mixture of rat food ingredients (fish meal, sunflower meal, alfalfa, cane molasses and premix) labelled with 85Sr, 115mCd or 203Hg. Whole-body radioactivity was determined in a double crystal scintillation counter every 24 h over a 6-day period. Carcass, gut, liver and kidney retention were determined at the end of the experiment. Relative to pups that were fed on cow's milk, administration of the mixture of the solid dietary ingredients caused a significant reduction in whole body, carcass and organ retention of all metals and was highest for 203Hg. This indicates that high absorption of toxic metals in sucklings can be reduced by dietary means. Rat food ingredients deserve further consideration because they are non-toxic dietary constituents and might be useful for decreasing the body burden of some metals in conditions of increased environmental exposure.

Animal Population Groups↗

Fuels and industrial chemicals through biotechnology. 2.

An overview of research on the biotechnical production of fuels and industrial chemicals during the two-year period of 1983-1984 is presented. Ethanol fermentation has continued to be the subject of major interest. A considerable amount of work has been directed to alternative feedstocks such as pentose sugars and lactose, and to bacterial fermentations. Reports on extrusion cooking as a continuous pretreatment method for subsequent ethanol fermentation, and on novel alternative downstream processing techniques have been published. In addition to ethanol fermentation, much attention has been paid to the biotechnical production of 2,3-butanediol, and of a number of organic and amino acids. In general, there appears to be a growing interest in the application of biocatalysis for the production of specialty chemicals, although only a few examples will be discussed in this paper. The construction of a demonstration plant to produce ethanol from molasses by a two 10 kL bed-volume immobilized yeast bioreactors at the Kyowa Hakko Kogyo Company Hofu plant, the announcement by Nitto Chemical Industries Company to begin the biotechnical production of acrylamide, and the French decision to construct pilot plants for the biotechnical production of acetone-butanol-ethanol cosolvent and of ethanol from renewable resources represent major scale-up developments.

Journal Article↗

Production of a novel polygalacturonic acid bioflocculant REA-11 by Corynebacterium glutamicum.

The production of a novel polygalacturonic acid bioflocculant REA-11 from a newly isolated strain, Corynebacterium glutamicum CCTCC M201005, was investigated. Sucrose was chosen as a carbon source for REA-11 production. Complex nitrogen sources containing urea and an organic nitrogen compound enhanced both bacterial growth and REA-11 production, among which urea plus corn steep liquor was shown to be the most efficient combination. A cost-effective medium for REA-11 production mainly comprised 17 g/l sucrose, 0.45 g/l urea, and 5 ml/l corn steep liquor, under which conditions the flocculating activity reached 390 U/ml. The molar ratio of carbon to nitrogen (C/N) significantly affected REA-11 production, where a C/N ratio of 20:1 was shown to be the best. Interestingly, by simultaneously feeding sucrose and urea at a C/N ratio of 20:1 at 24 h of fermentation, REA-11 production (458 U/ml) was enhanced by 17% compared to the control. In a 10 l jar fermentor, lower dissolved oxygen tension was favorable for REA-11 production: a flocculating activity of 520 U/ml was achieved at a kappaLa of 100 h(-1). REA-11 raw product is relatively thermo-stable at acidic pH ranges of 3.0-6.5. Preliminary application studies showed that REA-11 had stronger flocculating activity to Kaolin clay suspension compared to chemical flocculants. In addition, the capability of decolorizing molasses wastewater indicates the industrial potential of this novel bioflocculant.

Carbon↗

Isolation of Salmonella spp. from liquid and solid excreta prior to and following ensilage in ten swine farms located in central Mexico.

A study was carried out to define selected bacteriological characteristics of residues from 10 swine farms, 5 with or without prior clinical enteric disease (PCED) and to determine the effect of ensilage on the bacteria present in the solid fraction. At each farm, samples were taken from the sedimentation basin (SB), the solid fraction (SF), and the liquid fraction (LF). For each sample, CFU/g for enteric bacteria were quantified; Salmonella spp. were isolated and typified. Solid phase samples from each farm were used to prepare the ensilage, with a mixture of solids (80%), sorghum (12%) and molasses (8%). The quantity of enteric bacteria was significantly greater in farms without PCED (P < 0.05). Salmonella enterica were isolated from 8/10 of the farms with and without PCED; in 8 from SB; in 6 from LF; and in 5 from SF. Enteric bacteria were not isolated from silage, therefore, ensilage may be an alternative treatment for excreta that allows the elimination of pathogens such as Salmonella spp.

Agriculture↗