Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium

Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by bacteria for carbon and energy storage that also have commercial potential as bioplastics. One promising class of carbon feedstocks for industrial PHA production is plant oils, due to the high carbon content of these compounds. The bacterium Ralstonia eutropha accumulates high levels of PHA and can effectively utilize plant oil. Growth experiments that include plant oil, however, are difficult to conduct in a quantitative and reproducible manner due to the heterogeneity of the two-phase medium. In order to overcome this obstacle, a new culture method was developed in which palm oil was emulsified in growth medium using the glycoprotein gum arabic as the emulsifying agent. Gum arabic did not influence R. eutropha growth and could not be used as a nutrient source by the bacteria. R. eutropha was grown in the emulsified oil medium and PHA production was measured over time. Additionally, an extraction method was developed to monitor oil consumption. The new method described in this study allows quantitative, reproducible R. eutropha experiments to be performed with plant oils. The method may also prove useful for studying growth of different bacteria on plant oils and other hydrophobic carbon sources.     

Identification of Intestinal Bacteria Responsible for Fermentation of Gum Arabic in Pig Model

Acacia spp. produce gum exudates, traditionally called gum arabic or gum acacia, which are widely used in the food industry such as emulsifiers, adhesives, and stabilizers. The traditional gum arabic is highly variable with average molecular weights varying from 300,000–800,000. For this reason a standardized sample was used for the present experiments, based on a specific species of gum arabic (Acacia(sen)SUPER GUM^TM EM2). The literature indicates that gum arabic can be fermented by the intestinal bacteria to short chain fatty acid, particularly propionate. However, the bacteria responsible for the fermentation have not been determined. In this study, we used enrichment culture of pig cecal bacteria from the selected high molecular weight specific gum arabic of (M_W 1.77 × 10⁶). We found Prevotella ruminicola-like bacterium as a predominant bacterium that is most likely to be responsible for fermentation of the gum arabic used to propionate.     

Evaluation of guar gum derivatives as gelling agents for microbial culture media

Guar gum, a galactomannan, has been reported to be an inexpensive substitute of agar for microbial culture media. However, its use is restricted probably because of (1) its highly viscous nature even at high temperatures, making dispensing of the media to Petri plates difficult and (2) lesser clarity of the guar gum gelled media than agar media due to impurities present in guar gum. To overcome these problems, three guar gum derivatives, carboxymethyl guar, carboxymethyl hydroxypropyl guar and hydroxypropyl guar, were tested as gelling agents for microbial growth and differentiation. These were also evaluated for their suitability for other routine microbiological methods, such as, enumeration, use of selective and differential media, and antibiotic sensitivity test. For evaluation purpose, growth and differentiation of eight fungi and eight bacteria grown on the media gelled with agar (1.5%), guar gum (4%) or one of the guar gum derivatives (4%), were compared. All fungi and bacteria exhibited normal growth and differentiation on all these media. Generally, growth of most of the fungi was better on guar gum derivatives gelled medium than on agar medium. The enumeration carried out for Serratia sp. and Pseudomonas aeruginosa by serial dilution and pour plate method yielded similar counts in all the treatments. Likewise, the selective succinate medium, specific for P. aeruginosa, did not allow growth of co-inoculated Bacillus sp. even if gelled with guar gum derivatives. The differential medium, Congo red mannitol agar could not differentiate between Agrobacterium tumefaciens and Rhizobium meliloti on color basis, if gelled with guar gum or any of its derivatives However, for antibiotic sensitivity tests for both Gram-positive and -negative bacteria, guar gum and its derivatives were as effective as agar.     

Partial purification and chemical characterization of a glycoprotein (putative hydrocolloid) emulsifier produced by a marine bacterium Antarctobacter

During screening for novel emulsifiers and surfactants, a marine alphaproteobacterium, Antarctobacter sp. TG22, was isolated and selected for its production of an extracellular emulsifying agent, AE22. This emulsifier was produced optimally in a low-nutrient seawater medium supplemented with glucose and was extractable by cold ethanol precipitation of the high-molecular-weight fraction (>100 kDa). Production of AE22 commenced towards the late exponential phase of growth, with maximum emulsifying activity detected after approximately 4 days of the cells entering the death phase. Chemical, chromatographic and nuclear magnetic resonance spectroscopic analysis confirmed AE22 to be a high-molecular-weight (>2,000 kDa) glycoprotein with high uronic acids content, thus denoting an apparent polyanionic structure. Functional characterization showed this polymer to compare well to xanthan gum and gum arabic as an emulsion-stabilizing agent for a range of different food oils. However, AE22 exhibited better stabilizing than emulsifying properties, which could be conferred by its viscosifying effect in solution or from certain chemical groups found on the polysaccharide or protein moieties of the polymer. This new high-molecular-weight glycoprotein exhibits interesting functional qualities that are comparable to other biopolymers of this type and shows particular promise as an emulsion-stabilizing agent in biotechnological applications.     

Oxidation of gum arabic by soluble colloidal MnO2

In the present work, the oxidative degradation of gum arabic by colloidal manganese dioxide (MnO2) was carried out. Monitoring the disappearance of the MnO2 spectrophotometrically at 375 nm was used to follow the kinetics. The oxidation obeyed fractional-order kinetics with respect to the [gum arabic]. Effect of various experimental parameters such as the initial colloidal [MnO2], [HClO4], temperature, and complexing agents (P2O7(4-), F-, and Mn2+) for the oxidation of gum arabic was studied. The reaction was acid catalyzed. Addition of P2O(7)4-, F-, and Mn2+ ions enhances the rate of oxidation significantly. Gum arabic adsorbs onto the surface of the colloidal MnO2 through the equatorial -OH groups of the rhamnose moiety, and the complex breaks down into products. The Arrhenius equation was valid for the oxidation kinetics between 40 and 60 degrees C. To explain the observed kinetic results, a suitable mechanism and rate law for the reaction taking place at the surface of the colloidal particle has been proposed. The reducing nature of gum arabic is found be due to the presence of -OH group in the skeleton.     

Arabinogalactan proteins improve plant regeneration in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) anther culture

Androgenesis-based methods of doubled haploid (DH) production show considerable variation in efficiency in different barley genotypes. Arabinogalactan proteins (AGPs) have been shown to play a key role in several developmental processes, including embryogenesis, in different plant species. In this study we investigated the effect of exogenous AGPs from gum arabic on androgenesis and the regeneration efficiency in barley anther culture. Supplementation of the induction medium with 10 mg l^?1 gum arabic increased the total plant regeneration rate up to 2.8 times; when exposure to GA was extended to also include the pretreatment step, the regeneration rate was up to 6.6-times higher than in control. The effect of gum arabic was reversed by the Yariv reagent, an AGPs antagonist. This suggests a direct involvement of AGPs in androgenic development from barely microspores. Addition of gum arabic reduced cell mortality, increased the frequency of mitotic divisions of microspores and the number of multicellular structures (MCSs) when compared to control. The positive effect of gum arabic also included reduction in time required for the androgenic induction and substantially improved the quality of formed embryos. Observations made in this study imply a complex role of AGPs during androgenic development and confirmed the usefulness of gum arabic in production of barley androgenic plants.     

Development of Commercially Acceptable Formulations of the Nematophagous FungusVerticillium chlamydosporium

Studies in shaken flasks and a 20-liter bioreactor showed that biomass ofVerticillium chlamydosporiumcould be produced in large quantities in liquid culture. The fungus grew readily in media containing commercially available, low-cost ingredients (e.g., cotton seed meal, soybean meal) and a volumetric productivity of about 0.3 g/h/liter was achieved in the bioreactor. Chlamydospores were not produced in submerged culture, the biomass consisting only of mycelia and conidia. When this biomass was mixed with a carrier (kaolin) and a binder (gum arabic) and the ingredients were granulated and then dried to a moisture content of less than 2%, a biologically active product suitable for application to soil was produced. The fungus grew vigorously from these granules when they were placed on agar and retained its viability when granules were stored in vacuum-sealed bags at 25°C for 12 months. Experiments on tomato in the glasshouse showed that when the formulated product was incorporated into field soil at 10 g granules/liter soil, population densities ofV. chlamydosporiumwere increased to about 10⁴colony-forming units/g soil after 7–14 weeks. Between 37 and 82% of the first generation egg masses produced byMeloidogyne javanicacontained parasitized eggs.     

Effect of adsorbants on in vitro biohydrogenation of 22:6n-3 by mixed cultures of rumen microorganisms

Studies on microbial biohydrogenation of fatty acids in the rumen are of importance as this process lowers the availability of nutritionally beneficial unsaturated fatty acids for incorporation into meat and milk but also might result in the accumulation of biologically active intermediates. The impact was studied of adsorption of 22:6n-3 (DHA) to particulate material on its disappearance during 24 h in vitro batch incubations with rumen inoculum. Four adsorbants were used in two doses (1 and 5 mg/ml of mucin, gum arabic, bentonite or silicic acid). In addition, the distribution of 22:6n-3 in the pellet and supernatant of diluted rumen fluid was measured. Bentonite and silicic acid did not alter the distribution of 22:6n-3 between pellet and supernatant nor increased the disappearance of 22:6n-3 during the incubation. Both mucin and gum arabic increased the recovery of 22:6n-3 in the supernatant, indicating that these compounds lowered the adsorption of the fatty acid to ruminal particles. This was associated with an increased disappearance of 22:6n-3, when initial 22:6n-3 was 0.06 or 0.10 mg/ml, and an increased formation of 22:0, when initial 22:6n-3 was 0.02 mg/ml, during the 24 h batch culture experiment. Addition of gum arabic to pure cultures of Butyrivibrio fibrisolvens or Butyrivibrio proteoclasticus did not negate the inhibitory effect of 22:6n-3 on growth. As both mucin and gum arabic provide fermentable substrate for ruminal bacteria, an additional experiment was performed in which mucin and gum arabic were replaced by equal amounts of starch, cellulose or xylan. No differences in disappearance of 22:6n-3 were observed, suggesting that the stimulatory effect of mucin and gum arabic on disappearance of 22:6n-3 most probably is not due to provision of an alternative site of adsorption but related to stimulation of bacterial growth. A relatively high proportion of 22:6n-3 can be reduced to 22:0 provided the initial concentration is low.     

Emulsification using environmental compatible emulsifiers and de-emulsification using D.C. field and immobilized Nocardia amarae

As environmental compatible emulsifiers, various polysaccharides were investigated and de-emulsification methods were studied using filtration, direct current (D.C.) field, and also Nocardia amarae. 0.01% (v/v) of alginic acid, arabic gum, chitosan, and curdlan showed more than about 2 h of emulsion stability in the half-life of emulsion layer by a homogenizer and showed about twice synergic effects with other polysaccharides in emulsification activity at the ratio of 9:1 (v/v). De-emulsification by 110V D.C. field took about 1 h for the separation of oils from 1 l of oil-in-water emulsion in case of 0.01% arabic gum. Oil-water separator was designed using non-woven fabrics in filtration system and Nocardia amarae grown in n-hexadecane or kerosene was immobilized in the non-woven fabrics.     

Cultivation of formerly noncultivable strains ofMycoplasma hyorhinis

Growth on axenic agar medium is one of several characters by which mycoplasmas are defined. In apparent contradiction of the definition, DBS 1050 and other noncultivable strains ofMycoplasma hyorhinis do not grow on axenic medium but grow in cell culture. Our results show that BHK-21 cell extracts support DBS 1050 growth in appropriate medium. An inhibition assay, based on a virus neutralization format, shows that a variety of common medium ingredients inhibit DBS 1050 growth. The most potent activity was found in yeast extract. All other noncultivable strains ofM. hyorhinis tested have a yeast extract sensitivity, while cultivable strains do not. The apparent cell dependence of DBS 1050 can be attributed to growth inhibition due to factors present in a wide variety of peptones and extracts commonly used in medium; preferential growth in cell cultures is due to the absence of effective levels of these factors. Data are not available to determine if cell cultures provide growth factors not found in standard medium. The infraspecific taxon,M. hyorhinis cultivar α, is proposed for formerly noncultivable strains ofM. hyorhinis.     

Effects of pH, MES, arabinogalactan-proteins on microspore cultures in white cabbage

The objective of this study was to improve induction of embryogenesis in white cabbage (Brassica oleracea var. capitata) microspore cultures. The effect of NLN-13 liquid medium pH on isolated microspore embryogenesis was investigated in five white cabbage genotypes. Relatively high pH (6.2 or 6.4) was more effective on microspore embryogenesis in most of the white cabbage genotypes than the pH of 5.8, especially for inducing microspore-derived embryos in recalcitrant genotype ??Zhonggan No. 8??. Based on this, 2??(N-Morpholino) ethanesulfonic acid (MES) and the arabinogalactan-protein from gum arabic were tested on four out of five genotypes to see if they could increase embryo yield in microspore cultures. Adding MES or gum arabic alone was effective for these four genotypes, but the frequency of embryos derived from microspores was still low. However, the combination of 10?mg?l^?1 gum arabic and 3?mM MES in NLN-13 at pH 6.4 significantly enhanced microspore embryogenesis efficiency (with embryo production of 4.57?C222.97 embryos per bud), especially with recalcitrant genotype ??Zhonggan No. 8?? for which it was increased by about 35-fold.     

阿拉伯胶酶产生菌株的筛选及其发酵培养基的优化

从土壤中筛选产阿拉伯胶酶的微生物菌株,并通过紫外诱变选育后得到高产突变菌株ZHB05F,依据菌落和孢子形态特征初步鉴定为镰刀茵(Fusarium sp.).通过单因素试验,优化了产酶培养基的主要组分的浓度和pH值,得到最佳的产酶发酵培养基组成为:阿拉伯胶30 g/L,(NH4)2SO4 8 g/L,K2HPO4 1g/...     

Efficacy of crude neem seed kernel oil (NSKO) in controlling the tree locust (Anacridium melanorhodon melanorhodon Walker), a serious pest of the gum arabic producing tree (Acacia senegal L. Willd.) in the Sudan

The present study was carried out at Al Semih (western Sudan) (13° 14′ N: 30° 27′ E: Alt. 550 m). The area is part of the gum arabic belt which is heavily populated with farmers who practice shifting cultivation and/or agroforestry where Acacia senegal L. Willd (the main gum arabic producing tree) is dominant. The tree locust (Anacridium melanorhodon melanorhodon Walker (Orthoptera, Acrididae) is a serious pest of A. senegal. Farmers practice agroforestry within the gum belt where they cultivate their crops e.g. millet, groundnut, sesame and sorghum between A. senegal trees. Gum Arabic is an important source of revenue for farmers. Chemical pesticides have been used to control the tree locust. Although the gum belt (where A. senegal grows) is heavily populated, pesticides are used to control the tree locust in the area. The present study was undertaken with the objective of using alternative environmentally safe pesticides. Several doses of neem seed kernel crude oil (NSKO) were tested against the 4^th, 5^th, 6^th instars and the mature tree locust (A. melanorhodon melanorhodon) in a completely randomized block design. Experiments were carried out in cages placed on part of the gum arabic belt. NSKO at high doses (5 and 10% v/v) significantly reduced feeding, moulting and ovipositing of the tree locust and significantly increased the mortality of all developmental stages tested. However, low doses of NSKO had no significant effect on the tree locust.     

Interaction of gum arabic, maltodextrin and pullulan with lipids in emulsions

The interaction of gum arabic, maltodextrin and pullulan with lipids in emulsion systems was investigated. Interfacial tension and interfacial viscosity measurements revealed that only gum arabic could adsorb and form a viscoelastic film at the oil-water interface. Good emulsifying activity was demonstrated for gum arabic, whereas fine emulsions could not be produced from the other polysaccharide solutions and oil. Frequency-dependent increases in the storage and loss moduli were observed for all the polysaccharide solutions. Such rheological behavior did not substantially change when maltodextrin and pullulan were mixed with oil to form emulsions. However, the frequency-dependence of the dynamic moduli disappeared in the gum arabic-stabilized emulsion, suggesting the formation of a network structure in which oil droplets could form junctions with gum arabic chains. The results on the inhibition of lipid oxidation by polysaccharides suggest that gum arabic protected lipids from the attack of lipoxygenase and free radicals by adsorbing at the oil droplet surface.     

The effect of light on growth and sporulation of certain fungi

Summary The response of the fungi investigated to duration of exposure to light varies with the variation in the light intensity employed. Using relatively low intensity light (160 and/or 300 foot candles/inch²), the growth ofMyrothecium verrucaria &Pestalotia gracilis was not affected by the increase in the time of light exposure, while that ofPleurotus ostreatus was checked. Fruiting under the same conditions was hastened on exposure to light. Under higher light intensity (950 foot candles/inch²), growth ofMyrothecium was not affected, while that ofPestalotia andPleurotus decreased as the daily period of exposure to light increased. Pleurotus cultures exposed continuously to light showed practically no growth, and combined addition of malt and yeast extracts had a noticeable growth promoting effect on cultures exposed continuously to light, but not significantly on those kept in the dark. This was explained by assuming the presence in malt and yeast extracts of light sensitive growth promoting substances. The effect of light on growth ofPleurotus was found to be concerned with both cell mechanism and medium: light probably inhibits inside the cell the synthesis of one or more substances essential for growth and at the same time it favors the breakdown in the medium of one or more substances required for growth.     

Polysaccharide-hydrolyzing enzymes ofFrankia (Actinomycetales)

Studies were made of the polysaccharide-hydrolyzing activity inFrankia (Actinomycetales) grown in synthetic media using modifications of three standard assay procedures. In screening five different strains ofFrankia for cellulase activity, based on the method of utilization of cellulose in liquid culture, only one strain, CcI3, degraded filter paper cellulose to complete disintegration and only under very specific conditions of pH and primary carbon source. When carboxymethylcellulose (CMC) at 1% was used as substrate, all five strains showed the capacity to produce reducing sugars as hydrolytic products. Microcystalline cellulose, xylans and gum arabic were hydrolyzed to a lesser extent. Optimum activity depended upon pH and primary carbon source with pH 5.0 and pyruvate or propionate producing highest activities. In fractionation studies of culturedFrankia, assays for hydrolysis of 1% CMC in liquid medium showed that highest activity was in the enzyme preparation supernatant with lesser activity in the cell-free extract and cell wall fractions.Frankia strain CpI1 showed the greatest total hydrolytic activity against CMC after 2 weeks of culture. Strains ArI3 and CcI3 also showed good activity. The agar plate method for direct dye-polysaccharide interaction proved to be the least sensitive assay method with only ArI3 showing significant activity using CMC as substrate. It appears that theFranka strains grown in synthetic media all showed hydrolytic activity but the degree of hydrolysis of polysaccharides to reducing sugars depends upon strain of bacteria and very specific cultural conditions.     

Effects of carbon sources on extracellular lipase production and lipA transcription in Acinetobacter calcoaceticus

  The effects of lactic acid, oleic acid, gum arabic and their mutual interactions, on the production of extracellular lipase and the regulation of the expression of the lipase encoding gene (lipA) in Acinetobacter calcoaceticus were investigated. Formation of extracellular lipase was measured in culture supernatants of wild-type strain BD413 and expression of the lipA gene was monitored in vivo with a chromosomal fusion of lipA to lacZ. At the level of lipA expression only oleic acid had a significant effect; it lowered expression. Neither gum arabic nor lactic acid had any effect on lipA expression. On the other hand, the yield of extracellular lipase increased 2–5 times by the addition of gum arabic, possibly due to the release of cell surface-bound lipase. An interaction between oleic acid and lactic acid was also detected. Journal of Industrial Microbiology & Biotechnology (2000) 24, 25–30. Received 03 May 1999/ Accepted in revised form 04 September 1999     

Development and assessment of fusarium oxysporum-based mycoherbicide for control of Striga Hermonthica in sorghum

Abstract

Experiments were carried out from 2002 to 2003 to determine the most suitable form of fungal delivery for possible use by farmers in biological control of Striga hermonthica. Six mycoherbicides were developed, based on Fusarium oxysporum isolated from wilted S. hermonthica. In mycoherbicide formulation, rock phosphate powder, sorghum bran and gum arabic powder were used as carriers. Besides its role as a carrier, gum arabic powder was used as a sticker. There were three carriers with two formulations each, making six treatments altogether. Living propagule studies were based on colony, mycelium and conidium number of F. oxysporum. In greenhouse evaluation of mycoherbicides, each kg sorghum seed was coated with 10 g mycoherbicide before sowing. Carrier rock phosphate powder with gum arabic powder as a sticking agent was the most suitable form of its delivery for use by peasant farmers.     

Chlamydospore Production,Inoculation Methods and Pathogenicity of Fusarium oxysporum M12-4A,a Biocontrol for Striga hermonthica

Fusarium oxysporum isolate M12-4A is currently being evaluated for the biological control of Striga hermonthica . Inoculum production, inoculum delivery to the target, chlamydospore germination, and weed growth suppression of this weed-pathogen system were investigated. Liquid fermentation systems using organic material were evaluated for the production of large numbers of chlamydospores. A 1% sorghum straw powder (< 1 mm) substrate, exposed to black light at 21°C for 21 days, yielded 3.23 X 10 8 colony forming units (CFU) l -1 medium. A two-stage fermentation system using 5% w/v straw substrate under black light at 30°C for 14 days yielded 3.5 X 10 8 CFU l -1 medium. In vitro variations in chlamydospore germination were governed by the presence of exogenous carbon, nitrogen, and sorghum root exudates. Ammonium-nitrogen compounds and urea, in combination with glucose had a stronger stimulatory effect on chlamydospore germ tube growth than did potassium nitrate. Maximal germ tube elongation occurred when chlamydospores were exposed to urea at a C/N ratio of 10. Some mineral solutions and sorghum root exudates inhibited chlamydospore germ tube elongation; however, arabic gum, a complex polysaccharide, stimulated chlamydospore germ tube elongation and the production of secondary chlamydospores. In field trials, chlamydospore powder harvested from small-scale fermenters reduced S. hermonthica emergence by 92%. Complete inhibition of S. hermonthica emergence occurred when the chlamydospore powder was added to the soil at sowing and when sorghum seeds coated with chlamydospores were sown. Effective biological control of S. hermonthica was achieved using a simple fermentation system with sorghum straw as the inoculum growth substrate. For inoculum delivery to the farmers' fields, sorghum seeds were coated with the inoculum using arabic gum as the adhesive. This simple delivery system permits a uniform inoculation of the field as well as the proper positioning of the inoculum in the immediate environment of sorghum roots, where S. hermonthica attaches to its host. To facilitate a broad usage of F. oxysporum M12-4A for the biocontrol of S. hermonthica , we propose an inoculum production strategy based on a cottage industry model that utilizes a liquid fermentation process and inexpensive locally-available substrates including sorghum straw and arabic gum.     

Arabinogalactans and arabinogalactan-proteins induce embryogenesis in wheat (Triticum aestivum L.) microspore culture

The objective of this study was to improve induction of embryogenesis in wheat microspore culture in order to obtain a high number of regenerable embryos. The arabinogalactan (AG) Larcoll and the arabinogalactan-protein (AGP) from gum arabic were tested on two spring genotypes to see if they could increase microspore viability and induce embryogenesis in the microspore culture. Adding Larcoll significantly decreased microspore mortality in both genotypes regardless of the presence or absence of ovaries in the culture. Similarly, gum arabic had a strong effect on the number of embryos produced and regenerated green plants. In fact, by using only gum arabic we were able to obtain green plants from wheat microspore cultures without the presence of ovaries. In addition to preventing a high mortality rate of the cells, our results show that the induction of embryogenesis in wheat microspore cultures is strongly affected by the use of both AG or AGP.An erratum to this article can be found at