Optimization of Nutritional Factors for Exopolysaccharide Production by Submerged Cultivation of the Medicinal Mushroom Oudemansiella radicata

Summary Effects of nutritional factors on exopolysaccharide production by submerged cultivation of the medicinal mushroom Oudemansiella radicata were investigated in shake flasks. Sucrose and peptone were optimal carbon and nitrogen sources for cell growth and exopolysaccharide production. The exopolysaccharide production was increased with an increase in initial sucrose concentration within the range of 10–40 g l⁻¹ and initial peptone concentration within the range of 1–3 g l⁻¹. To enhance further exopolysaccharide production, the effect of carbon/nitrogen ratios was studied using central composite design (CCD) and response surface analysis. The maximum exopolysaccharide production of 2.67 ± 0.15 g l⁻¹ was achieved in medium with optimized carbon and nitrogen sources, i.e. 39.3 g sucrose l⁻¹ and 3.16 g peptone l⁻¹ in the same cultivation conditions. The information obtained is helpful for the hyperproduction of exopolysaccharide by submerged cultivation of O. radicata on a large scale.     

Selectively inducing the synthesis of a key structural exopolysaccharide in aerobic granules by enriching for <Emphasis Type="BoldItalic">Candidatus</Emphasis> “<Emphasis Type="BoldItalic">Competibacter phosphatis</Emphasis>”

A gel-forming exopolysaccharide was previously shown to play an important structural role in aerobic granules treating nutrient-rich industrial wastewater. To identify whether this exopolysaccharide performs a similar role in other granular biomass and if conditions favouring its production can be more precisely elucidated, extracellular polymeric substances (EPS) were extracted from granules grown under four different operating conditions. ¹H nuclear magnetic resonance (NMR) spectroscopy of their EPS indicated that the gel-forming exopolysaccharide was expressed in two granular sludges both enriched in Candidatus “Competibacter phosphatis”. In contrast, it was not expressed in granules performing denitrification with methanol as a carbon source and nitrate as the electron acceptor or granules enriched in Candidatus “Accumulibacter phosphatis” performing enhanced biological phosphorus removal from synthetic wastewater. In one of the first two sludges, the exopolysaccharide contained in the seeding granular sludge continued to be a major component of the granule EPS while Competibacter was being enriched. In the second sludge, a floccular sludge not containing the gel-forming exopolysaccharide initially was also enriched for Competibacter. In this sludge, an increase in particle size was detected coinciding with a yield increase of EPS. NMR spectroscopy confirmed its yield increase to be attributable to the production of this structural gel-forming exopolysaccharide. The results show that (1) the particular gel-forming exopolysaccharide previously identified is not necessarily a key structural exopolysaccharide for all granule types, and (2) synthesis of this exopolysaccharide is induced under conditions favouring the selective enrichment of Competibacter. This indicates that Competibacter may be involved in its production.     

The effects of Anabaena spiroides (Cyanophyceae) exopolysaccharide on copper toxicity to Simocephalus serrulatus (Cladocera, Daphnidae)

1. This work investigated the consumption of a microalgal (Anabaena spiroides) exopolysaccharide by the cladoceran Simocephalus serrulatus (Cladocera, Daphnidae) and its effect on copper toxicity. 2. Total organic carbon concentration was used to quantify the microalgal exopolysaccharide. Both copper‐complexed exopolysaccharides and copper‐free exopolysaccharides were taken up by S. serrulatus. 3. A reduction of free copper ions was obtained in the presence of A. spiroides exopolysaccharide. Copper toxicity (EC₅₀) and zooplankton concentrations were inversely correlated to exopolysaccharide concentration in experimental media. 4. An increase of free copper ions in experimental media was obtained after exopolysaccharide consumption, suggesting that S. serrulatus excreted copper to the surrounding environment.     

Cholesterol removal by some lactic acid bacteria that can be used as probiotic

In the present study, the relationship between exopolysaccharide production and cholesterol removal rates of five strains of Lactobacillus delbrueckii subsp. bulgaricus isolated from home‐made yoghurt was studied. Test strains were selected according to their exopolysaccharide production capacity. Influence of different bile concentrations on cholesterol removal was investigated. It was confirmed that B3, ATCC 11842 and G11 strains which produce high amounts of exopolysaccharide (211, 200 and 159 mg/l, respectively) were able to remove more cholesterol from the medium compared to those that produce low amounts of exopolysaccharide (B2, A13). The highest cholesterol removal (31%) was observed by strain L. delbrueckii subsp. bulgaricus B3, producing a high amount of exopolysaccharide, in 3 mg/ml bile concentration. Cholesterol removal by resting and dead cells was investigated and it was found to be 4%–14% and 3%–10%, respectively. Cholesterol removal by immobilized and free cells of the B3 strain was studied and it was determined that immobilized cells are more effective. Influence of cholesterol on exopolysaccharide production has also been tested and it was found that cholesterol increased the production of EPS. The results indicated that: (i) there is a correlation between cholesterol removal and EPS production; and (ii) L. delbrueckii subsp. bulgaricus B3 is regarded as a suitable candidate probiotic and adjunct culture.     

Use of ELISA with Antiexopolysaccharide Antibodies to Evaluate Wheat-Root Colonization by the Rhizobacterium Paenibacillus polymyxa

Enzyme-linked immunosorbent assay with rabbit polyclonal antibodies developed to isolated exopolysaccharide of Paenibacillus polymyxa 1465 was used to evaluate the colonization of wheat-seedling roots by this bacterium. The assay conditions were optimized for detection of the P. polymyxa exopolysaccharide determinants forming part of the samples used (homogenates of inoculated roots). The dynamics of the immunoenzymatic revealing of specific polysaccharidic antigenic determinants in the samples’ composition correlated with an increase in P. polymyxa numbers on the roots found by estimation of colony-forming units.     

Isolation of an Exopolysaccharide-producing Bacterium,Sphingomonas sp. CS101, Which Forms an Unusual Type of Sphingan

An exopolysaccharide-producing Gram negative bacterium was isolated and determined to be a Sphingomonas sp. (CS101). A sugar composition analysis of an exopolysaccharide indicated that the Sphingomonas sp. CS101 secreted an exopolysaccharide composed of glucose, mannose, fucose, and rhamnose in the ratio of 2.1:1.1:1.0:0.1, suggesting that this exoplysaccharide is an unusual type of sphingan family. The mean molecular weight of the exopolysaccharide was determined to be 4.2×10⁵ Da by size exclusion chromatography coupled with multi-angle laser-light scattering (SEC/MALLS) analysis. An exopolysaccharide was produced up to 17 g/l (pH 7; 30 °C) with the optimal medium condition over 4 days of cultivation.     

Influence of Acidic Exopolysaccharide of Xanthomonas campestris IBPM 124 on the Kinetic Parameters of Extracellular Bacteriolytic Enzymes

Interactions of a negatively charged exopolysaccharide of Xanthomonas campestris IBPM 124 with its extracellular enzymes (muramidase, endopeptidase, and neutral phosphatase) and also with egg lysozyme, lysostaphin, muramidase of Streptomyces globisporus, and a bacteriolytic enzyme complex of Streptomyces albus were studied. All these enzymes were positively charged under the conditions of their maximal activity. It was shown that interaction of the acidic exopolysaccharide from X. campestris with these enzymes changed their kinetic parameters. The change was either positive (increase in reaction rate) or negative (decrease in reaction rate) and depended on the enzyme and type of substrate cleaved. Due to such interactions, the acidic exopolysaccharide secreted by X. campestris into the environment not only retained and transported positively charged exoenzymes into the near-cellular space, but also regulated their activity.     

Production of exopolysaccharides by submerged mycelial culture of <Emphasis Type="Italic">Grifola frondosa</Emphasis> TFRI1073 and their antioxidant and antiproliferative activities

The exopolysaccharide (EPS) extract of a newly screened Grifola frondosa TFRI1073 was evaluated antioxidant activity, including superoxide anion scavenging activity and reducing power, and the antiproliferative activities by using lung (A549 cells) and breast (MDA-MD-231 cells) cancer cell line. The exopolysaccharide of G. frondosa plays the important role on antioxidant and antiproliferative activities. Nutritional requirements for mycelial biomass and EPS production by G. frondosa were studied. Flushing the culture medium with carbon sources has a stimulating effect on both mycelial biomass and EPS production. In addition, nitrogen sources appeared to be an important and significant component for mycelial biomass. Vitamins were probably not essential for mycelial biomass and EPS production. Inorganic salts appeared to have a significant effect on both mycelial biomass and EPS productions. The effect of phosphate ion on mycelial biomass was better than on EPS production. The information generated in this study will facilitate physiological research of a newly screened G. frondosa TFRI1073, helping to develop the exopolysaccharide of a new dietary supplement and functional food from G. frondosa.     

Structure and role in symbiosis of the exoB gene of Rhizobium leguminosarum bv trifolii

The Rhizobium leguminosarum bv trifolii exoB gene has been isolated by heterologous complementation of an exoB mutant of R. meliloti. We have cloned a chromosomal DNA fragment from the R. leguminosarum bv trifolii genome that contains an open reading frame of 981 bp showing 80% identity at the amino acid level to the UDP-glucose 4-epimerase of R. meliloti. This enzyme produces UDP-galactose, the donor of galactosyl residues for the lipid-linked oligosaccharide repeat units of various heteropolysaccharides of rhizobia. An R. leguminosarum bv trifoliiexoB disruption mutant differed from the wild type in the structure of both the acidic exopolysaccharide and the lipopolysaccharide. The acidic exopolysaccharide made by our wild-type strain is similar to the Type 2 exopolysaccharide made by other R. leguminosarum bv trifolii wild types. The exopolysaccharide made by the exoB mutant lacked the galactose residue and the substitutions attached to it. The exoB mutant induced the development of abnormal root nodules and was almost completely unable to invade plant cells. Our results stress the importance of exoB in the Rhizobium-plant interaction. Received: 31 May 1996 / Accepted: 18 December 1996     

Energy requirements for microbial exopolysaccharide synthesis

Calculations indicate that at the high specific rates of exopolysaccharide synthesis obtained in continuous cultures of Xanthomonas campestris and mucoid Pseudomonas aeruginosa strains the ATP demand for this synthesis is a significant proportion of total cellular ATP demand. However, depending upon the proportion of polymer substituents more oxidised than the carbohydrate substrate, some, and in certain cases all of this energy can be provided via NAD(P)H₂ produced during exopolysaccharide synthesis. For xanthan production by X. campestris a P/O ratio of 2.2 to 2.6, depending on the content of pyruvyl substituents, would be necessary for energy generation as a direct result of xanthan synthesis to support the ATP demand for this synthesis. In sulphur-limited cultures of X. campestris, however, energy metabolism is shown to be inefficient, the organism exhibiting either a low P/O ratio or low Y _ATP. In such cultures the yield of exopolysaccharide from glucose was 0.62/g glucose compared with maximum theoretical yields of 0.81 to 0.87/g glucose for P/O ratio of 1 to 3.     

Extracellular DNA and lipoteichoic acids interact with exopolysaccharides in the extracellular matrix of Streptococcus mutans biofilms

Streptococcus mutans-derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA – ?lytS and ?lytT; LTA – ?dltA and ?dltD; and insoluble exopolysaccharide – ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms.     

Respiration in Organic Acid-Forming Molds

Mycelia of Rhizopus oryzae, a lactate forming fungus, were used to study respiration and glucose degradation in this organism.

The respiration of the mycelia obtained during an early stage of shake culture growth, when only a little lactate was produced, was completely inhibited by KCN, while the respiration of the mycelia obtained from older shake cultures, or from surface cultures, regardless of the age, was not inhibited by KCN. These results suggest that energy for growth of Rhizopus oryzae is predominantly obtained by a respiratory system involving cytochromes and that a loss of this system occurs in older shake cultures which then begin to accumulate lactate. This observation was confirmed by the enzymatic studies on cell-free extracts.     

Sulfated exopolysaccharide production by the halophilic cyanobacterium Aphanocapsa halophytia

Aphanocapsa halophytia MN-11 isolated from a hypersaline environment was found to produce large quantities of exopolysaccharide. We describe here production of exopolysaccharide and the characterization of its properties. The effects of medium composition, particularly NaCl concentration, were tested. Maximum exopolysaccharide production was obtained with nitrogen and phosphorus concentrations in the medium exceeding 100 and 40 mg · L^-1 respectively. In addition, when 30 g · L^-1 NaCl was added to the medium, exopolysaccharide could be recovered from the medium supernatant. Exopolysaccharide from this strain was made up of at least six mono-oses and did not contain uronic derivatives or osamines. Proteins represented about 10% of total weight and, interestingly, 12% (wt/wt) sulfated residues, which is unusual for photosynthetic prokaryotes.     

Shifts in reclamation management strategies shape the role of exopolysaccharide and lipopolysaccharide-producing bacteria during soil formation

Polymeric substances produced by microbes play a key role for the development of soil aggregates. Here, we investigated the dynamics of bacterial families contributing to the formation of exopolysaccharides and lipopolysaccharides, major constituents of polymeric substances, at a managed land reclamation site of a post-mining area. We collected soil samples from the initial and the agricultural management phase and expected a peak in the abundance of bacteria capable for exopolysaccharide and lipopolysaccharide production at the points of the biggest disturbances. We used shotgun metagenomic sequencing in combination with measurements of exopolysaccharide concentrations. Our results underline the importance of exopolysaccharide and lipopolysaccharide-producing bacteria after nutrient input combined with structural disturbance events, caused here by the initial planting of alfalfa and the introduction of a tillage regime together with organic fertilization in the agricultural management phase. Moreover, the changes in management caused a shift in the exopolysaccharide/lipopolysaccharide-producing community. The initial phase was dominated by typical colonizers of oligotrophic environments, specifically nitrogen fixers (Rhizobiaceae, Comamonadaceae, Hyphomicrobiaceae), while bacteria common in agricultural soils, such as Sphingomonadaceae, Oxalobacteraceae and Nitrospiraceae, prevailed in the agricultural management phase.     

Influence of nutritional conditions on exopolysaccharide production by submerged cultivation of the medicinal fungus <Emphasis Type="Italic">Shiraia bambusicola</Emphasis>

Maltose and yeast extract were the most favourable carbon and nitrogen sources for exopolysaccharide production by submerged culture of Shiraia bambusicola WZ-003, and initial maltose and yeast extract concentrations were at 30 and 3 g l⁻¹, respectively. Plant oils could increase the mycelial growth and exopolysaccharide production in tested concentration. K⁺ and Mg²⁺ could enhance the mycelial growth and exopolysaccharide biosynthesis. The optimal cultivation temperature and initial pH were found to be 26°C and 6.0, respectively. Exopolysaccharide concentration reached 0.53 g l⁻¹ in 15-l fermenter under optimal nutritional conditions.     

Structural studies of the exopolysaccharide consisting of a nonasaccharide repeating unit isolated from Lactobacillus rhamnosus KL37B

A novel structure of exopolysaccharide from the lactic acid bacteria (LAB) Lactobacillus rhamnosus KL37B, from the human intestinal flora, is described. During the structural investigation of the exopolysaccharide it was found that the repeating unit is a nonasaccharide, which is the largest repeating unit found in LAB exopolysaccharides to date. The polysaccharide material was prepared by TCA extraction of a bacterial cell mass, purified by anion-exchange and gel permeation chromatography and characterized using chemical and enzymatic methods. On the basis of monosaccharide and methylation analysis and also 1D and 2D ¹H and ¹³C NMR spectroscopy the exopolysaccharide was shown to be composed of the following nonasaccharide repeating unit:The physicochemical cell surface study and adhesive properties indicated distinct surface properties of Lactobacillus rhamnosus strain KL37B with high adhesive abilities to Caco-2 cells, hydrophobicity and slime production, in comparison to other Lactobacillus strains used as controls.     

茎瘤固氮根瘤菌环二鸟苷酸代谢相关基因的功能鉴定

【目的】考察茎瘤固氮根瘤菌ORS571中c-di-GMP合成酶AZC-2412的编码基因缺失的突变表型,初步探究其功能机理。【方法】本实验构建基于cre-loxp重组酶系统的根瘤菌基因敲除系统,以及采用三亲接合技术构建突变株。测定野生型和突变株的生长速率、趋化能力、胞外多糖产量、生物膜形成等表型。【结果】突变株与野生型生长速率几乎相同。与野生型相比突变株由于细胞内c-di-GMP水平降低,胞外多糖、生物膜产量等均有所下降。【结论】实验表明,环二鸟苷酸合成酶AZC-2412缺失,使得c-di-GMP水平降低,对胞外多糖生成、细菌的运动能力、生物膜的形成、细胞絮凝、与植物的互作等均有调控作用。     

Influence of nutritional conditions on the mycelial growth and exopolysaccharide production in Paecilomyces sinclairii

AIMS: The objective of the study was to optimize the submerged culture conditions for the production of exopolysaccharide from Paecilomyces sinclairii. METHODS AND RESULTS: The optimal temperature and initial pH for exopolysaccharide production by Paecilomyces sinclairii in shake flask culture were found to be 30 degrees C and 6.0, respectively. Sucrose (60 g l(-1)) and corn steep powder (10 g l(-1)) were the most suitable carbon and nitrogen source for exopolysaccharide production. CONCLUSIONS: Under optimal culture medium, the maximum exopolysaccharide concentration in a 5-l stirred-tank fermenter indicated 7.4 g l(-1), which was approximately three times higher than that in basal medium. The maximum specific growth rates (micro max) and yield coefficient (Y(P/S)) in the optimal culture medium was 0.16 h(-1) and 0.19, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimal culture conditions reported in this article can be widely applied to the processes for submerged cultures of other mushrooms.     

New culture media to suppress exopolysaccharide production by Rhizobium japonicum

Summary Secretion of exopolysaccharide by Rhizobium japonicum strain USDA 110 ws measured during growth on media comprising 12.5 mM MES [2-(N-morpholino)ethanesulfonic acid] buffer, pH 5.9; 5 mM NH₄Cl; trace elements; and 25 or 100 mM of one of 23 different carbon sources. Significant amounts of exopolysaccharide were produced on all aldopentoses, polyols, organic acids, and sugar acids tested, but little or none was secreted during growth on aldohexoses or amino acids. Media suitable for routine slime-free culture of several. R. japonicum strains were made by using 100 mM d-galactose or 25 mM l-aspartate as the carbon source.