The effect of olive mill wastewaters variability on xanthan production

AIMS: Xanthan production by Xanthomonas campestris from several olive mill wastewaters (OMW) was investigated. METHODS AND RESULTS: Maximum xanthan production of 4 g l(-1) was obtained in media with 50% OMW as sole source of nutrients. OMW storage decreased effluent quality for xanthan production. The range of effluent concentration for X. campestris growth and xanthan production varied depending on OMW extraction METHOD: Wastewaters from press and two-phase extraction methods required higher dilution rates (< 10%) than those from the three-phase extraction method (50%). Nitrogen supplementation improved xanthan production in press and two-phase OMW. CONCLUSION: Factors affecting wastewaters composition, namely, waste storage, time of olive harvesting, and method for oil extraction, were found to influence xanthan production in shake-flask cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Conditions for xanthan production from OMW should be optimized in accordance with the nature of the waste material.     

Effects of yeast extract and glucose on xanthan production and cell growth in batch culture of Xanthomonas campestris

Although available kinetic data provide a useful insight into the effects of medium composition on xanthan production by Xanthomonas campestris, they cannot account for the synergetic effects of carbon (glucose) and nitrogen (yeast extract) substrates on cell growth and xanthan production. In this work, we studied the effects of the glucose/yeast-extract ratio (G/YE) in the medium on cell growth and xanthan production in various operating modes, including batch, two-stage batch, and fed-batch fermentations. In general, both the xanthan yield and specific production rate increased with increasing G/YE in the medium, but the cell yield and specific growth rate decreased as G/YE increased. A two-stage batch fermentation with a G/YE shift from an initial low level (2.5% glucose/0.3% yeast extract) to a high level (5.0% glucose/0.3% yeast extract) at the end of the exponential growth phase was found to be preferable for xanthan production. This two-stage fermentation design both provided fast cell growth and gave a high xanthan yield and xanthan production rate. In contrast, fed-batch fermentation with intermittent additions of glucose to the fermentor during the stationary phase was not favorable for xanthan production because of the relatively low G/YE resulting in low xanthan production rate and yield. It is also important to use a moderately high yeast extract concentration in the medium in order to reach a high cell density before the culture enters the stationary phase. A high cell density is also important to the overall xanthan production rate. Received: 30 September 1996 / Received revision: 21 January 1997 / Accepted: 10 February 1997     

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     

Relationship between glucose catabolism and xanthan production in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Two genes involved in central carbon metabolism were inactivated to modulate intracellular glucose 6-phosphate and to evaluate its effects on xanthan production in wild-type Xanthomonas oryzae pv. oryzae. Upon the inactivation of the phosphogluconate dehydratase gene (edd), intracellular glucose 6-phosphate increased from 0.05 to 1.17 mmol/g (dry cell wt). This was accompanied by increased xanthan production of up to 2.55 g/l (culture medium). In contrast, inactivation of 6-phosphogluconate dehydrogenase gene (gndA) did not influence intracellular glucose 6-phosphate nor xanthan production. The intracellular availability of glucose 6-phosphate is proposed as a rate-limiting factor in xanthan production, and it may be possible to increases production of xanthan by modulating the activities of enzymes in central carbon metabolism.     

A novel gene encoding xanthan lyase of Paenibacillus alginolyticus strain XL-1

Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i. e., the xalA gene, encoding pyruvated mannose-specific xanthan lyase of Paenibacillus alginolyticus XL-1. The xalA gene encoded a 100, 823-Da protein, including a 36-amino-acid signal sequence. The 96, 887-Da mature enzyme could be expressed functionally in Escherichia coli. Like the native enzyme, the recombinant enzyme showed no activity on depyruvated xanthan. Compared to production by P. alginolyticus, a 30-fold increase in volumetric productivity of soluble xanthan lyase was achieved by heterologous production in E. coli. The recombinant xanthan lyase was used to produce modified xanthan, which showed a dramatic loss of the capacity to form gels with locust bean gum.     

Nutritional Studies on Xanthan Production by Xanthomonas campestris NRRL B1459

The nutritional requirements of Xanthomonas campestris NRRL B1459 for optimal xanthan production were studied in a chemically defined medium. Of the carbon sources tested, a 4% sucrose or glucose medium yielded the highest xanthan titers. The further addition of certain organic acids, such as succinate, pyruvate, and α-ketoglutarate, stimulated xanthan production; excess concentrations of these organic acids inhibited xanthan formation. Certain amino acids (e.g., glutamate) and nitrate salts were superior to ammonium salts for xanthan production. Concentrations of these nitrogen sources higher than the optimal levels inhibited xanthan production while stimulating growth. Xanthan production was also sensitive to high concentrations of inorganic phosphate. High xanthan potencies, up to 30 g/kg of broth, were achieved in these shake-flask studies, in which completely defined media were used.     

A pyruvated mannose-specific xanthan lyase involved in xanthan degradation by Paenibacillus alginolyticus XL-1.

The xanthan-degrading bacterium Paenibacillus alginolyticus XL-1, isolated from soil, degrades approximately 28% of the xanthan molecule and appears to leave the backbone intact. Several xanthan-degrading enzymes were excreted during growth on xanthan, including xanthan lyase. Xanthan lyase production was induced by xanthan and inhibited by glucose and low-molecular-weight enzymatic degradation products from xanthan. A xanthan lyase with a molecular mass of 85 kDa and a pI of 7.9 was purified and characterized. The enzyme is specific for pyruvated mannosyl side chain residues and optimally active at pH 6.0 and 55 degrees C.     

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.     

Xanthan production from olive-mill wastewaters

Olive mill wastewaters (OMW) are a by-product from olive oil manufacture that cause environmental pollution. These wastes have been used as substrate for the production of the extracellular polysaccharide xanthan by Xanthomonas campestris NRRL B1459-S4L41. Growth and xanthan production on dilute OMW as a sole source of nutrients were obtained at OMW concentrations below 60%, yielding a maximal xanthan production of 4.4gl⁻¹ at 30–40% OMW concentration. Addition of nitrogen and/or salts led to significantly increased xanthan yields with a maximum of 7.7gl⁻¹. The N/salts supplements also allowed an increase in the optimal OMW concentration. Inocula pre-grown on OMW can be used. Results suggest that an improved xanthan yield could be obtained with adequate balance between waste concentration and nitrogen or salt supplementation. OMW is proposed as a low-cost substrate for xanthan production with the additional environmental benefit of this use.     

A Novel Gene Encoding Xanthan Lyase of Paenibacillus alginolyticus Strain XL-1

Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i.e., the xalA gene, encoding pyruvated mannose-specific xanthan lyase of Paenibacillus alginolyticus XL-1. The xalA gene encoded a 100,823-Da protein, including a 36-amino-acid signal sequence. The 96,887-Da mature enzyme could be expressed functionally in Escherichia coli. Like the native enzyme, the recombinant enzyme showed no activity on depyruvated xanthan. Compared to production by P. alginolyticus, a 30-fold increase in volumetric productivity of soluble xanthan lyase was achieved by heterologous production in E. coli. The recombinant xanthan lyase was used to produce modified xanthan, which showed a dramatic loss of the capacity to form gels with locust bean gum.     

A Pyruvated Mannose-Specific Xanthan Lyase Involved in Xanthan Degradation by Paenibacillus alginolyticus XL-1

The xanthan-degrading bacterium Paenibacillus alginolyticus XL-1, isolated from soil, degrades approximately 28% of the xanthan molecule and appears to leave the backbone intact. Several xanthan-degrading enzymes were excreted during growth on xanthan, including xanthan lyase. Xanthan lyase production was induced by xanthan and inhibited by glucose and low-molecular-weight enzymatic degradation products from xanthan. A xanthan lyase with a molecular mass of 85 kDa and a pI of 7.9 was purified and characterized. The enzyme is specific for pyruvated mannosyl side chain residues and optimally active at pH 6.0 and 55°C.     

Effect of glucose concentrations on xanthan gum production by xanthomonas campestris

The effect of the glucose concentration on xantham gum production by Xanthomonas campestris ATCC 13951 was studied resulting that the glucose concentration between 30 and 40 g/kg broth was best for xanthan gum production. Controlling the glucose concentration at between 30 and 40 g/kg broth by intermittent addition of glucose prevented the inhibition of cell growth and the cessation of xanthan gum production, which were observation with a higher glucose concentration. By means of a glucose feeding strategy, the xanthan gum concentration reached 43 g/kg broth after 96-h cultivation.     

Valorisation of chicken feathers for xanthan gum production using Xanthomonas campestris MO-03

Xanthan gum is an important commercial polysaccharide produced by Xanthomonas species. In this study, xanthan production was investigated using a local isolate of Xanthomonas campestris MO-03 in medium containing various concentrations of chicken feather peptone (CFP) as an enhancer substrate. CFP was produced with a chemical process and its chemical composition was determined. The addition of CFP (1–8?g/l) increased the conversion of sugar to xanthan gum in comparison with the control medium, which did not contain additional supplements. The highest xanthan production (24.45?g/l) was found at the 6?g/l CFP containing control medium in 54?h. This value was 1.73 fold higher than that of control medium (14.12?g/l). Moreover, addition of CFP improved the composition of xanthan gum; the pyruvate content of xanthan was 3.86% (w/w), higher than that of the control (2.2%, w/w). The xanthan gum yield was also influenced by the type of organic nitrogen sources. As a conclusion, CFP was found to be a suitable substrate for xanthan gum production.     

Xanthan production in bubble column and air-lift reactors

A bubble column (0.05 m(3)) and an air-lift fermentor (1.2 m(3)) were used for the production of the exocellular microbial polysaccharide xanthan with Xanthomonas campestris in a synthetic medium. Upon oxygen depletion in the liquid, the xanthan production rate dropped sharply and then became a linear function of the oxygen transfer rate. The volumetric mass transfer coefficients for oxygen conformed to the correlation of Suh et al. Using this correlation in combination with the model for xanthan batch fermentation suggested by Peters et al., the xanthan fermentations in the bubble column were well described. The model also correctly predicted the time course of the molecular weight of the polysaccharide even when a complex medium was used. In the air-lift fermentor, however, the xanthan production rate and the xanthan yields with respect to oxygen and glucose were lower than expected at the overall oxygen transfer rate. The poor performance of the air lift was traced back to the lack of any oxygen supply in the downcomer.     

Sources and methods of manufacturing xanthan by fermentation of various carbon sources

Xanthan gum, an anionic polysaccharide with an exceptionally high molecular weight, is produced by the bacterium Xanthomonas sp. It is a versatile compound that has been utilized in various industries for decades. Xanthan gum was the second exopolysaccharide to be commercially produced, following dextran. In 1969, the US Food and Drug Administration (FDA) approved xanthan gum for use in the food and pharmaceutical industries. The food industry values xanthan gum for its exceptional rheological properties, which make it a popular thickening agent in many products. Meanwhile, the cosmetics industry capitalizes on xanthan gum's ability to form stable emulsions. The industrial production process of xanthan gum involves fermenting Xanthomonas in a medium that contains glucose, sucrose, starch, etc. as a substrate and other necessary nutrients to facilitate growth. This is achieved through batch fermentation under optimal conditions. However, the increasing costs of glucose in recent years have made the production of xanthan economically unviable. Therefore, many researchers have investigated alternative, cost-effective substrates for xanthan production, using various modified and unmodified raw materials. The objective of this analysis is to investigate how utilizing different raw materials can improve the cost-efficient production of xanthan gum.     

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.     

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.     

Variations in xanthan production by antibiotic-resistant mutants of Xanthomonas campestris

Mutants resistant to different antibiotics (streptomycin, tetracycline, ampicillin and penicillin) were obtained from several strains of Xanthomonas campestris and evaluated for xanthan production. Most of the mutants showed alterations in their polysaccharide production, either increasing, decreasing or totally losing their polymer-production capacity. The existence of two types of antibiotic-resistance mechanisms for the assayed drugs is suggested: one that affects xanthan production and another that does not. Differences in outer-membrane protein patterns of mutants that were simultaneously altered in antibiotic resistance and xanthan production were found, in comparison with their parental strains. These findings suggest the existence of a genetic relationship between antibiotic-resistance mechanisms and xanthan production. Some of the mutants obtained showed significant increases in broth viscosity and xanthan concentration. These results suggest that resistance to streptomycin and ampicillin can be used to obtain improved strains in plate screening assays. Received: 8 January 1997 / Received revision: 13 June 1997 / Accepted: 4 July 1997     

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.     

Xanthan production by Xanthomonas campestris using whey permeate medium

Xanthan gum is a polysaccharide that is widely used as stabilizer and thickener with many industrial applications in food industry. Our aim was to estimate the ability of Xanthomonas campestris ATCC 13951 for the production of xanthan gum by using whey as a growth medium, a by-product of dairy industry. X. campestris ATCC 13951 has been studied in batch cultures using a complex medium for the determination of the optimal concentration of glucose, galactose and lactose. In addition, whey was used under various treatment procedures (de-proteinated, partially hydrolyzed by β-lactamase and partially hydrolyzed and de-proteinated) as culture medium, to study the production of xanthan in a 2 l bioreactor with constant stirring and aeration. A production of 28 g/l was obtained when partially hydrolysed β-lactamase was used, which proved to be one of the highest xanthan gum production reported so far. At the same time, an effort has been made for the control and selection of the most appropriate procedure for the preservation of the strain and its use as inoculant in batch cultures, without loss of its viability and its capability of xanthan gum production. The pre-treatment of whey (whey permeate medium hydrolyzed, WPH) was very important for the production of xanthan by the strain X. campestris ATCC 13951 during batch culture conditions in a 2 l bioreactor. Preservation methods such as lyophilization, cryopreservation at various glycerol solution and temperatures have been examined. The results indicated that the best preservation method for the producing strain X. campestris ATCC 13951 was the lyophilization. Taking into account that whey permeate is a low cost by-product of the dairy industry, the production of xanthan achieved under the studied conditions was considered very promising for industrial application.