Xanthan gum production by wild-type isolates of Xanthomonas campestris

Five newly-isolated strains of Xanthomonas campestris when compared with the standard strain, NRRL B-1459, showed higher broth viscosity and xanthan gum production. Evaluation of polysaccharide rheology is a very important determinant for selecting new xanthan-producing isolates.     

High production of xanthan gum by a glycerol-tolerant strain Xanthomonas campestris WXLB-006

The superior properties of xanthan gum make it an industrial aginomoto used in many industries, especially in oil recovery. In the present work, xanthan production from glycerol by a mutant strain Xanthomonas campestris WXLB-006 reached as high as 17.8?g/L in flask culture. With the adoption of pH control, varied aeration and agitation, and varied glycerol feeding strategy, xanthan production reached 33.9?g/L in a 7-L fermenter and fermentation time decreased to 60?hr. Instead of difficultly and costly purifying glycerol, this research provides a very good case for glycerol utilization. At the same time, this is the first report on a high glycerol-tolerant strain for microbial polysaccharide production and 33.9?g/L is the highest production of xanthan gum produced from glycerol so far.     

Impact of the extrusion process on xanthan gum behaviour

Processing xanthan gum by extrusion and subsequent drying produces a biopolymer showing particulate, rather than molecular behaviour in aqueous solution. This form of xanthan disperses very readily to give a viscosity that is strongly dependent on salt concentration. On heating above the temperature of the order-disorder transition as determined by calorimetry, there is a viscosity transition that is indicative of the irreversible loss of the particulate structure. It is suggested that the extrusion process melts and aligns xanthan macromolecules. On cooling reordering will occur but in the highly concentrated environment in the extruder ( approximately 45% water w/w), inter-molecular association between neighbouring macromolecules cannot proceed to completion due to kinetic trapping. As a consequence a network structure is created maintained by associations involving ordered regions. A xanthan solution can be prepared from this particulate material by dispersing and subsequent heating far more readily than can be achieved with non-processed xanthan.     

Production of xanthan gum by Sphingomonas bacteria carrying genes from Xanthomonas campestris

Twelve genes coding for assembly, acetylation, pyruvylation, polymerization, and secretion of the polysaccharide xanthan gum are clustered together on the chromosome of the bacterium Xanthomonas campestris. These genes (gumBCDEFGHIJKLM) are sufficient for synthesis of xanthan gum when placed in bacteria from a different genus, Sphingomonas. The polysaccharide from the recombinant microorganism is largely indistinguishable, structurally and functionally, from native xanthan gum. These results demonstrate that a complex pathway for biosynthesis of a specific polysaccharide can be acquired by a single inter-generic transfer of genes between bacteria. This suggests the biological and commercial feasibility of synthesizing xanthan gum or other polysaccharides in non-native hosts. Received 23 October 1996/ Accepted in revised form 14 April 1997     

Bioproduction of low-pigment xanthan gum by a cell-wall deficient mutant of Xanthomonas campestris

Abstract

This work aims to enhance the bioproduction of xanthan gum by screening a hyper-yield producer from the wild-type Xanthomonas campestris during a long-term continuous subculture. We reported a cell-wall deficient mutant, which performed a shift of cell morphology from rod-shaped to round-shaped. Both the yield of xanthan gum and the conversion rate of feedstock were assessed using sucrose as a carbon source with the supplement of yeast extract powder, l-glutamic acid, and other raw materials. After 96?h aerobic fermentation, the yield of xanthan gum of the mutant reached up to 32?g/L, which was 3.4 times of that of the wild-type strain. The conversion rate of feedstock in the mutant was up to 92.1%, which was 3 times of that of the wild-type (31.2%). Furthermore, pigments generated were determined and compared. As a result, the fermentation broth of the wild-type performed an OD_560nm of 0.296, which was 5.8 times of that (OD_560nm?=?0.051) of the mutant. Microscopy analysis showed that the percentage of free-living cells in broth affected the color of the final product. Moreover, the robustness of the fermentation performance of the cell-wall deficient mutant at a pilot scale showed potential for industrial application.     

Direct utilization of lactose in clarified cheese whey for xanthan gum synthesis byXanthomonas campestris

Summary A derivative ofXanthomonas campestris B1459 was constructed that utilizes lactose in clarified cheese whey for xanthan gum synthesis. Genes conferring lactose utilization carried by transposon Tn951 were inserted into the bacterial chromosome. The ability to use lactose for xanthan gum synthesis was stably inherited and the amount of xanthan produced suggested carbohydrate conversion efficiencies similar to wild-typeX. campestris growing in the presence of glucose. Bench-scale fermentation of this organism and identification of the optimal whey sources and pretreatments can now proceed.     

Genetic construction of Xanthomonas campestris and xanthan gum production from whey

Summary Plasmids pUR291 and pNZ521 containing lacZ gene, maturation protein and proteinase P genes, were transferred into X. campestris either by conjugation or by transformation. Plasmid pNZ521 was also conjugally transferred into X. campestris XMT1 a transformant carrying plasmid pUR291. All the constructed strains were evaluated for xanthan gum production in either a medium of 50% whey or the same medium supplemented with 1.5% lactose or 1.5% glucose. Mixed cultures either with transconjugants or with transformants were tested for xanthan gum production as well.     

Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey

Xanthomonas campestris pv. campestris possesses a low level of beta-galactosidase and therefore is not able to grow and produce significant amounts of xanthan gum in a medium containing lactose as the sole carbon source. In this study, a beta-galactosidase expression plasmid was constructed by ligating an X. campestris phage phi LO promoter with pKM005, a ColE1 replicon containing Escherichia coli lacZY genes and the lpp ribosome-binding site. It was then inserted into an IncP1 broad-host-range plasmid, pLT, and subsequently transferred by conjugation to X. campestris 17, where it was stably maintained. The lacZ gene under the control of the phage promoter was expressed at a high level, enabling the cells to grow in a medium containing lactose. Production of xanthan gum in lactose or diluted whey by the engineered strain was evaluated, and it was found to produce as much xanthan gum in these substrates as the cells did in a medium containing glucose.     

AFM studies on gelation mechanism of xanthan gum hydrogels

The effect of annealing on xanthan gum molecules was investigated using atomic force microscopy (AFM). The values of height and width of xanthan gum molecules in AFM images are ca. 1 nm, which strongly indicates that xanthan gum molecules extended on the mica surface are in mono- or double layers. When xanthan gum aqueous solution was annealed, a network structure was observed. In contrast, a network structure was not observed for non-annealed solution. AFM images provide direct information concerning oscillational change of the network structure. It is concluded that xanthan gum molecular chains in aqueous solution aggregate and dissociate in an oscillational manner with increasing annealing time and that a homogeneous network structure was formed by annealing at 40 °C for 24 h.     

Development of a phenomenological modeling approach for prediction of growth and xanthan gum production using Xanthomonas campestris

An unstructured kinetic model for xanthan production is described and fitted to experimental data obtained in a stirred batch reactor. The culture medium was composed of several nitrogen sources (soybean hydrolysates, ammonium and nitrate salts) consumed sequentially. The model proposed is able to describe this sequential consumption of nitrogen sources, the consumption of inorganic phosphate and carbon, the evolution of biomass, and production of xanthan. The parameter estimation has been performed by fitting the kinetic model in differential form to experimental data. Runs of the model for simulating xanthan gum production as a function of the initial concentration of inorganic phosphate have shown the positive effect of phosphate limitation on xanthan yield, though diminishing rates of production. The model was used to predict the kinetic parameters for a medium containing a 2-fold lower initial phosphate concentration. When tested experimentally, the measured fermentation parameters were in close agreement with the predicted model values, demonstrating the validity of the model.     

Chemical modification of xanthan gum to increase dissolution rate

Xanthan gum is modified with formaldehyde to improve the dissolution rate. The FT-IR spectra and the X-ray diffraction spectra both show that chemical modification reduces intermolecular interactions and crystallinity. Viscosity measurements show that the chemically modified gum dissolves more rapidly than before.     

Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey.

Xanthomonas campestris pv. campestris possesses a low level of beta-galactosidase and therefore is not able to grow and produce significant amounts of xanthan gum in a medium containing lactose as the sole carbon source. In this study, a beta-galactosidase expression plasmid was constructed by ligating an X. campestris phage phi LO promoter with pKM005, a ColE1 replicon containing Escherichia coli lacZY genes and the lpp ribosome-binding site. It was then inserted into an IncP1 broad-host-range plasmid, pLT, and subsequently transferred by conjugation to X. campestris 17, where it was stably maintained. The lacZ gene under the control of the phage promoter was expressed at a high level, enabling the cells to grow in a medium containing lactose. Production of xanthan gum in lactose or diluted whey by the engineered strain was evaluated, and it was found to produce as much xanthan gum in these substrates as the cells did in a medium containing glucose.     

Stimulation of xanthan production by Xanthomonas campestris using citric acid

The biosynthesis of xanthan by Xanthomonas campestris was found to be affected by the addition of citric acid in fed batch mode. Under oxygen-limiting conditions, the addition of up to 2.6 g citric acid per litre improved cell viability as well as increasing xanthan yield by up to 80%. Comparative xanthan formation profiles at different operating conditions indicate that at higher aeration (when there was no oxygen limitation), citric acid addition did not improve xanthan production.     

Nutritional study of Xanthomonas campestris in xanthan gum production by factorial design of experiments

A nutritional study of Xanthomonas campestris in the production of xanthan gum was carried out by factorial design and analysis of experiments. The concentrations of nutrients such as nitrogen, phosphorus, magnesium, and sulfur were studied by means of a factorial design at two levels, reduced to a half with four central points for error estimation. Three responses were considered, corresponding to biomass, sucrose, and xanthan concentrations, at four different times: 12, 16, 20, and 24 h. The influence of nitrogen, phosphorus, and magnesium on the biomass, and the influence of nitrogen, phosphorus, and sulfur on xanthan production, were significant, although any variable influenced sucrose consumption. An optimized medium was proposed for xanthan production.     

Co-production of ice nuclei and xanthan gum by transformed Xanthomonas campestris grown in sugar beet molasses

Two recombinant plasmids, expressing ice nucleation activity, were constructed and named pCPP30inaZ and pCPP38inaZ. They were transferred to the ice-negative, xanthan-producing Xanthomonas campestris pv. campestris by electroporation. The transformants were used for co-production of xanthan gum and ice nuclei from sugar beet molasses. The highest values obtained were 20 g l^–1 and 10¹⁸ ice nuclei ml^–1, respectively. The above values fulfil the criteria for industrial manipulation. This is the first report on co-formation of two products by a transformed X. campestris strain.     

Potential application of a low-viscosity and high-transparency xanthan gum produced from Xanthomonas campestris CCTCC M2015714 in foods

Xanthan gum is commonly used as a thickener in food industry, while the usage of xanthan gum as a dietary fiber is restricted for its low additive volume. Herein, the potential use of a low-viscosity and high-transparency xanthan gum as a dietary fiber was evaluated in vitro. This new xanthan shows better transparency and faster dissolution rate than most commercial products, and its viscosity increases along with the treatment of freeze–thaw cycles at ?20°C. Moreover, this new xanthan can absorb heavy metals (Pb, Cd, Cu) and retard starch digestion by glucoamylase. In summary, this new xanthan could be potentially used as a dietary fiber or fiber ingredient for preventing and treating diabetes, hyperlipemia, heavy metal poisoning, and cardiovascular diseases effectively.     

Use of agricultural wastes for xanthan production by Xanthomonas campestris

Four different acid-hydrolyzed wastes, from melon, watermelon, cucumber and tomato were compared for xanthan production. Growth of Xanthomonas campestris, xanthan biosynthesis, kinetics and chemical composition were investigated. Both growth and xanthan production were dependent on the acid hydrolysate concentrations and available nitrogen. Melon acid hydrolyzed waste was the best substrate for xanthan production. Exopolysaccharide obtained throughout this study was compared to commercial xanthan, showing a very similar chemical composition. Acid hydrolyzed wastes are proposed as a new carbon source for xanthan production. Received 16 July 1998/ Accepted in revised form 8 October 1998     

Kinetic analysis of growth and xanthan gum production with Xanthomonas campestris on sucrose, using sequentially consumed nitrogen sources

A batch fermentation strategy using Xanthomonas campestris ATCC 13951 for xanthan gum production has been established in which all essential medium components are supplied at the onset. This has been achieved using sucrose as sole sugar feedstock. Sequential consumption of nitrogen sources (soybean hydrolysates, ammonium and nitrate salts) was observed to facilitate the further optimisation of the medium. Biomass accumulation was limited by phosphate availability. Xanthan yields of more than 60% (grams of xanthan per gram of sugar) have been obtained with constant acetyl content. However, pyruvyl substitution decreased as the growth rate declined, due to the metabolic constraints specific to phosphate depletion. High rates of carbon conversion into xanthan were observed throughout the culture and the ATP/ADP ratio was not affected by the decline in the specific growth rate.     

Xanthan gum and ethanol production by Xanthomonas campestris and Zymomonas mobilis from peach pulp

The wild type of Xanthomonas campestris and a mutant strain of Zymomonas mobilis CP4, tolerant to sucrose up to 40% (w/v), were used to produce either xanthan gum or ethanol, respectively, from peach pulp supplemented with different salts. Both bacteria grew well (2.7 mg/ml for X. campestris and 1.45 mg/ml for Z. mobilis) in fine peach pulp and the production of xanthan gum or ethanol was 0.1–0.2 g/l or 110 g/l, respectively.     

Genetic engineering of polysaccharide structure: production of variants of xanthan gum in Xanthomonas campestris.

Xanthan gum is an extracellular heteropolysaccharide produced by the bacterium Xanthomonas campestris. Xanthan has wide commercial application as a viscosifier of aqueous solutions. Previously, through genetic engineering, a set of mutants defective in the xanthan biosynthetic pathway has been obtained. Certain mutants were shown to synthesize and polymerize structural variants of the xanthan repeating unit and thus produce "variant xanthans". Initial studies of solution viscosities of these polymers, presented here, indicate that the variants have rheological properties similar to, but not identical with, xanthan. These results indicate that acetylation and pyruvylation can affect the viscometric properties of xanthan. Specifically, the presence of pyruvate increases viscosity, whereas acetate decreases viscosity. In addition, the elimination of sugar residues from xanthan side chains also has a major effect on viscosity. Compared to wild-type xanthan, polymer lacking the terminal mannose (polytetramer) is a poor viscosifier. In contrast, polymer lacking both the terminal mannose and glucuronic acid (polytrimer) is a superior viscosifier, on a weight basis. There is a negative effect of acetylation on the viscosity of polytetramer xanthan, but there is seemingly no effect of acetylation on polytrimer xanthan viscosity. The further study of these materials should provide insight into the relationship between xanthan structure and rheological behavior.