Simultaneous and rapid monitoring of biomass and biopolymer production by Sphingomonas paucimobilis using Fourier transform-near infrared spectroscopy

The application of Fourier Transform near infrared spectroscopy (FT-NIRS) to near real-time monitoring of polysaccharide and biomass concentration was investigated using a gellan-producing strain of Sphingomonas paucimobilis grown in a stirred tank reactor. Successful models for both biomass and gellan were constructed despite the physichochemical complexity of the viscous process fluid. Modelling of biomass proved more challenging than for gellan, partly because of the low range of biomass concentration but a model with a good correlation coefficient (0.94) was formulated based on second derivative spectra. The gellan model was highly satisfactory, with an excellent correlation coefficient (0.98), again based on second derivative spectra. No sample pre-treatment was required and all spectral scanning was carried out on whole broth. Additionally, both models should be robust in practice since both were formulated using low numbers of factors. Thus, the near real time simultaneous monitoring of gellan and biomass in this highly complex matrix using FT-NIRS potentially opens the way to greatly improved process control strategies.     

Effect of temperature on bacterial gellan production

The effect of temperature on the production of the polysaccharide gellan by the bacterium Sphingomonas paucimobilis ATCC 31461 was studied in relation to carbon source. When glucose served as the carbon source, gellan formation by the strain was highest after 72 h of growth at an incubation temperature of 30–31 °C. Polysaccharide production by the sphingomonad cells grown on corn syrup for 72 h was maximal at an incubation temperature of 31 °C. The highest cellular productivity in elaborating gellan was observed at 31 °C after 72 h of growth independent of the carbon source utilized.     

Improvement in Production and Quality of Gellan Gum by Sphingomonas paucimobilis Under High Dissolved Oxygen Tension Levels

The effect of agitation rate and dissolved oxygen tension (DOT) on growth and gellan production by Sphingomonas paucimobilis was studied. Higher cell growth of 5.4 g l⁻¹ was␣obtained at 700 rpm but maximum gellan (15 g l⁻¹) was produced at 500 rpm. DOT levels above 20% had no effect on cell growth but gellan yield was increased to 23 g l⁻¹ with increase in DOT level to 100%. Higher DOT levels improved the viscosity and molecular weight of the polymer with change in acetate and glycerate content of the polymer.     

The effect of agitation and aeration on the synthesis and molecular weight of gellan in batch cultures of Sphingomonas paucimobilis

The effects of agitation and aeration upon synthesis and molecular weight of the biopolymer gellan were systematically investigated in batch fermenter cultures of the bacterium, Sphingomonas paucimobilis. High aeration rates and vigorous agitation enhanced growth of S. paucimobilis. Although gellan formation occurred mainly in parallel with cell growth, the increase in cells able to synthesise gellan did not always lead to high gellan production. For example, at very high agitation rates (1000 rpm) growth was stimulated at the expense of biopolymer synthesis.Maximal gellan concentration was obtained at 500 rpm agitation and either 1 or 2 vvm aeration (12.3 and 12.4 g/l gellan, respectively). An increase in aeration (from 1 to 2 vvm) enhanced gellan synthesis only at low agitation rates (250 rpm). However, high aeration or dissolved oxygen was not necessary for high gellan synthesis, in fact oxygen limitation always preceded the phase of maximum gellan production and probably enhanced polysaccharide biosynthesis.Some gellan was formed even after glucose exhaustion. This was attributed to the intracellular accumulation of polyhydroxyalkanoates, (such as polyxydroxybutyrate) which were found in S. paucimobilis cells indicating the existence of a carbon storage system, which may contribute to gellan biosynthesis under glucose-limiting conditions.The autolysis of the culture, which occurred at the late stages of the process, seemed to be triggered mainly by limitations in mass (nutrient) transfer, due to the highly viscous process fluid that gradually develops. Rheological measurements generally gave a very good near real time estimate of maximum biopolymer concentration offering the possibility of improved process control relative to time consuming gravimetric assay methods.While mechanical depolymerisation of gellan did not occur, high aeration rates (2 vvm) led to production of gellan of low molecular weight (at either 250 or 500 rpm). This effect of aeration rate upon gellan molecular weight is reported here for the first time, and is important for the properties and applications of gellan. Mechanisms which may have led to this are discussed, but control of molecular weight of the biopolymers is clearly an area needing further research.     

The production of gellan exopolysaccharide with Sphingomonas paucimobilis E2 (DSM 6314)

Summary A new screening technique was used to isolate the bacterium Sphingomonas paucimobilis E2 (DSM 6314), which produces the exopolysaccharide gellan. The productivity was found to be about four times higher than that of the industrially used strain Auromonas elodea (ATCC 31461) it was isolated from. The polysaccharide formation was found to be predominantly growth-related. Correspondence to: W.-D. Deckwer     

Cost-effective optimization of gellan gum production by Sphingomonas paucimobilis using corn steep liquor

Abstract

Gellan gum, produced by Sphingomonas paucimobilis, is increasingly used in food and pharmaceutical industries as stabilizing, emulsifying, texturing and gelling agents. However, its high production costs may limit its full commercial potential. Therefore, in this study, we investigated ways to reduce gellan gum production costs and improve yields. We first revealed corn steep liquor (CSL) as a cost-effective nutrient source that can improve gellan gum yields. We then systematically optimized culture conditions even further, and revealed that the addition of Triton X-100 surfactant and selected inorganic nitrogen sources improved gellan gum production. Under our optimized conditions (glucose 33.75?g/L, CSL 10?g/L, urea 2.5?g/L, MgSO₄ 1.08?g/L, KH₂PO₄ 3.24?g/L, K₂SO₄ 1?g/L and Triton X-100 0.75?g/L), we yielded a maximum concentration of 14.41?g/L, which was about 1.5-fold higher than non-optimized CSL-based medium. Our findings highlight the use of CSL as a cost effective and promising nutrient source for industrial production of gellan gum.     

Microbiological degradation of the herbicide dicamba

Summary Pseudomonas paucimobilis was isolated from a consortium which was capable of degrading dicamba (3,6-dichloro-2-methoxybenzoic acid) as the sole source of carbon. The degradation of dicamba byP. paucimobilis and the consortium was examined over a range of substrate concentration, temperature, and pH. In the concentration range of 100–2000 mg dicamba L^–1 (0.5–9.0 mM), the degradation was accompanied by a stoichiometric release of 2 mol of Cl^– per mol of dicamba degraded. The cultures had an optimum pH 6.5–7.0 for dicamba degradation. Growth studies at 10°C, 20°C, and 30°C yielded activation energy values in the range of 19–36 kcal mol^–1 and an average Q₁₀ value of 4.0. Compared with the pure cultureP. paucimobilis, the consortium was more active at the lower temperature.     

Gellan Gum

ABSTRACT:?

For decades microbial exopolysaccharides have been invaluable ingredients in the food industry, as well as having many attractive pharmaceutical and chemical applications. Gellan gum is a comparatively new gum elaborated by the Gram-negative bacterium Sphingomonas paucimobilis. Although its physico-chemical properties have been well characterized, the ecology and physiology of Sphingomonas, and the factors influencing the fermentation process for production of this gum have received much less attention. This review focuses on the metabolism and the enzymic activity of this bacterium, as well as the factors that influence gellan production, including process temperature, pH, stirring rate, oxygen transfer, and composition of the production medium. Potential strategies for improving the production process are discussed in the context of processes for the production of other microbial biopolymers, particularly exopolysaccharides. In addition, the importance and potential utility of gellan lyases in modification of gellan and in other applications is critically evaluated.     

The role of exopolymers in the attachment of sphingomonas paucimobilis

The importance of exopolymers in the adhesion of Sphingomonas paucimobilis was established by studying the attachment to glass of three mutants with defective gellan production. The attachment assays were performed in either phosphate buffered saline (controls) or in the exopolymeric solutions produced by the mutants. The exopolymer was found to have surface active properties, changing the glass surface from hydrophilic to hydrophobic, making adhesion thermodynamically favourable. Only the cells that had a substantial polymeric layer surrounding their walls were able to significantly colonise glass coated with the exopolymer. It is hypothesised that the exopolymer bound to the glass and the exopolymer present at the surface of the bacteria bound together, overcoming the energy barrier created by the negative charge of both surfaces. It is concluded that the exopolymer from S. paucimobilis has a dual role in the process of adhesion by both coating the surface thereby strengthening adhesion and by enhancing adhesion through the establishment of polymeric bridges.     

Gellan gum biosynthesis in Sphingomonas paucimobilis ATCC 31461: genes,enzymes and exopolysaccharide production engineering

The commercial gelling agent, gellan, is an extracellular polysaccharide (EPS) produced by Sphingomonas paucimobilis ATCC 31461. In recent years, significant progress in understanding the relationship between gellan structure and properties and elucidation of the biosynthesis and engineering of this recent product of biotechnology has been made. This review focuses on recent advances in this field. Emphasis is given to identification and characterization of genes and enzymes involved, or predicted to be involved, in the gellan biosynthetic pathway, at the level of synthesis of sugar-activated precursors, of the repeat unit assembly and of gellan polymerization and export. Identification of several genes, biochemical characterization of the encoded enzymes and elucidation of crucial steps of the gellan pathway indicate that possibilities now exist for exerting control over gellan production at any of the three levels of its biosynthesis. However, a better knowledge of the poorly understood steps and of the bottlenecks and regulation of the pathway, the characterization of the composition, structure and functional properties of gellan-like polymers produced either by the industrial strain under different culture conditions or by mutants are still required for eventual success of the metabolic engineering of gellan production. Journal of Industrial Microbiology & Biotechnology (2002) 29, 170–176 doi:10.1038/sj.jim.7000266 Received 11 February 2002/ Accepted in revised form 09 April 2002     

Structures and Properties of Gellan Polymers Produced by Sphingomonas paucimobilis ATCC 31461 from Lactose Compared with Those Produced from Glucose and from Cheese Whey

The dairy industry produces large quantities of whey as a by-product of cheese production and is increasingly looking for new ways to utilize this waste product. Gellan gum is reliably produced by Sphingomonas paucimobilis in growth media containing lactose, a significant component of cheese whey, as a carbon source. We studied and compared polysaccharide biosynthesis by S. paucimobilis ATCC 31461 in media containing glucose, lactose (5 to 30 g/liter), and sweet cheese whey. We found that altering the growth medium can markedly affect the polysaccharide yield, acyl substitution level, polymer rheological properties, and susceptibility to degradation. Depression of gellan production from lactose compared with gellan production from glucose (approximately 30%) did not appear to occur at the level of synthesis of sugar nucleotides, which are the donors of monomers used for biosynthesis of the repetitive tetrasaccharide unit of gellan. The lactose-derived biopolymer had the highest total acyl content; the glucose- and whey-derived gellans had similar total acyl contents but differed markedly in their acetate and glycerate levels. Rheological studies revealed how the functionality of a gellan polysaccharide is affected by changes in the acyl substitution.     

Biodegradation of lindane by a native bacterial consortium isolated from contaminated river sediment

The aerobic biodegradation of lindane (γ-hexachlorocyclohexane) by a consortium of acclimated bacteria from sediment at a polluted site on the Suquia River, Cordoba, Argentina, is reported. The bacteria were acclimated for 30 days under aerobic conditions, using a minimal culture medium containing lindane (0.034 mM) as sole carbon source. Growth of the bacterial consortium decreased at a lindane concentration of 1.03 mM and was totally inhibited at 2.41 mM. The consortium showed initial lindane degradation rates of 4.92×10⁻³, 11.0×10⁻³ and 34.8×10⁻³ mM h⁻¹ when exposed to lindane concentrations of 0.069, 0.137 and 0.412 mM, respectively. Chloride concentration increased during aerobic biodegradation, indicating lindane mineralization. A metabolite identified as γ-2,3,4,5,6-pentachlorocyclohexene appeared during the first 24 h of biodegradation. Four different bacteria, identified as Sphingobacterium spiritivorum, Ochrobactrum anthropi, Bosea thiooxidans and Sphingomonas paucimobilis, were isolated. Pure strains of B. thiooxidans and S. paucimobilis degraded lindane after 3 days of aerobic incubation. This is the first report of lindane biodegradation by B. thiooxidans.     

Biodegradation of triphenylmethane dye Malachite Green by <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis>

Triphenylmethane dyes belong to the most important group of synthetic colorants and are used extensively in the textile industries for dying cotton, wool, silk, nylon, etc. They are generally considered as the xenobiotic compounds, which are very recalcitrant to biodegradation. Sphingomonas paucimobilis, was isolated from the soil sample collected from contaminated sites of textile industry located in KsarHellal, Tunisia, and it was able to decolorize Malachite Green (MG) dye (50 mg/l) within 4 h under shaking condition (pH 9 and temperature 25°C). The effect of inoculum size, dye concentration, temperature and initial pH of the solution were studied. The results obtained from the batch experiments revealed the ability of the tested bacteria to remove dye. UV–Vis spectroscopy and FTIR analysis of samples before and after decolorization confirmed the ability of the tested strain to decolorize MG. In addition, the phytotoxicity study revealed the degradation of MG into non-toxic product by S. paucimobilis.     

Biosynthesis of a thermostable gellan lyase by newly isolated and characterized strain of Geobacillus stearothermophilus 98

The thermophilic strain able to degrade gellan was isolated from Bulgarian hot spring. According to its morphological and biochemical properties and by partial sequencing of its 16S rDNA, it was classified as Geobacillus stearothermophilus. It grew in a synthetic medium with gellan as the only carbon source with a specific growth rate of 0.69 h⁻¹ and generation time of 60 min. The strain produced thermostable gellan lyase extracellularly during exponential phase. Its synthesis was inducible; the enzyme was not registered in culture liquid without gellan. The enzyme activity was increased tenfold in conditions of continuous cultivation compared to data from batch fermentations and enzyme productivity was almost sixfold higher. The enzyme showed optimal activity at 75°C in a very large pH area 4–8.5. This enzyme is the first reported thermostable gellan lyase, its residual activity was 100% after 24 h incubation at 60°C and its half-life was 60 min at 70°C.     

Identification and organization of genes for diutan polysaccharide synthesis from <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. ATCC 53159

A cluster of genes for diutan polysaccharide synthesis was isolated from a library of Sphingomonas sp. ATCC 53159 genomic DNA by complementation of glucosyl-isoprenylphosphate transferase-deficient mutants of Sphingomonas elodea ATCC 31461 (producing gellan) and Xanthomonas campestris (producing xanthan). The synthesis of polysaccharide in these strains shares a common first step, transfer of glucose-1-phosphate from UDP-glucose to the isoprenylphosphate lipid. The cluster of 24 genes was compared to genes for biosynthesis of gellan, and S-88 sphingan from Sphingomonas sp. ATCC 31554. Diutan, gellan and S-88 sphingan have a common four-sugar backbone but different side chains, one rhamnose for S-88 sphingan, a two-rhamnose side chain for diutan and no side chain for gellan. The genes for biosynthesis of diutan, gellan and S-88 sphingan were similar in general organization but differed in location of some genes, in particular, dpsG (putative polymerase), dpsR (putative lyase) and dpsS (putative repeat unit transporter). An unidentified reading frame urf31, present in the gene clusters for diutan and S-88 sphingan but not gellan, had similarity to glycosyl transferase group 2 proteins, and was detrimental when cloned in Sphingomonas elodea producing gellan that lacks a side chain, but not in Sphingomonas ATCC 31554 producing S-88 sphingan with a rhamnose side chain. Gene urf31 could possibly encode a side-chain rhamnosyl transferase. Another gene urf31.4 was unique to the diutan gene cluster. A plasmid containing 20 of the 24 genes resulted in a slight increase in the amount of diutan produced, but a significant increase in the rheological properties of diutan.     

Rheological properties of concentrated solutions of gellan in an ionic liquid

The rheological properties of solutions of gellan were examined at high concentrations where there is entanglement coupling between gellan chains. An ionic liquid 1-butyl-3-methylimidazolium chloride (BmimCl) was used as a solvent. Concentrated solutions of gellan in BmimCl were obtained by using a hot-molding technique. The concentration of gellan was varied from 1.9 × 10² to 4.8 × 10² kg m⁻³. The measurement temperature ranged from 50 to 100 °C. The master curve of the angular frequency dispersion of the storage modulus for the gellan solutions showed a rubbery plateau at high angular frequency. The molecular weight between entanglements (M_e) for gellan was obtained from the plateau modulus. From the concentration dependence curve of M_e, M_e for gellan in the molten state was determined to be 2.3 × 10³.     

Use of lysozyme for treatment of bacterial contamination in <Emphasis Type="Italic">in vitro</Emphasis> shoot cultures of fruit plants

Summary Use of lysozyme was tested for treatment of bacterial contaminations in in vitro shoot cultures of quince (Cydonia oblonga) ‘BA 29’ and the hybrid (Prunus persica × P. amygdalus) rootstock ‘GF 677’. Shoots which had been contaminated for about 1 yr by Bacillus circulans and Sphingomonas paucimobilis were treated in liquid culture, at pH 4.5, with 9–36 mg ml⁻¹ egg white lysozyme (EWL), and compared to each other and to untreated cultures for their growth, proliferation, and number of bacterial colony-forming units in the tissues. EWL did not negatively affect shoot growth up to 18 mg ml⁻¹; furthermore, the proliferation rates of EWL-treated shoots were sometimes higher than those of controls. In contrast, the concentration of 36 mg ml⁻¹ had some deleterious effect on the regrowth capacity and shoot production of ‘GF 677’ at the first subculture to solid medium after EWL, treatments. EWL had a simple bacteriostatic effect against Sphingomonas paucimobilis; in contrast, it was effective at 18 mg ml⁻¹ in eliminating Bacillus circulans in both ‘BA 29’ and ‘GF 677’ cultures, after optimal treatment duration.     

Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium × formolongi

An efficient system for Agrobacterium-mediated transformation of Lilium × formolongi was established by preventing the drastic drop of pH in the co-cultivation medium with MES. Meristematic nodular calli were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransfease II (NPTII) genes. After three days of co-cultivation on 2 g/l gellan gum-solidified MS medium containing 100 μM acetosyringone, 30 g/l sucrose, 1 mg/l picloram and different concentrations of MES, they were cultured on the same medium containing 12.5 mg/l meropenem to eliminate Agrobacterium for 2 weeks and then transferred onto medium containing the same concentration of meropenem and 25 mg/l hygromycin for selecting putative transgenic calli. Transient GUS expression was only observed by adding MES to co-cultivation medium. Hygromycin-resistant transgenic calli were obtained only when MES was added to the co-cultivation medium especially at 10 mM. The hygromycin-resistant calli were successfully regenerated into plantlets after transferring onto picloram-free medium. Transformation of plants was confirmed by histochemical GUS assay, PCR analysis and Southern blot analysis.     

Isolation and characterization of phenanthrene-degrading <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis> strain ZX4

Phenanthrene-degrading bacterium strain ZX4 was isolated from an oil-contaminated soil, and identified as Sphingomonas paucimobilis based on 16S rDNA sequence, cellular fatty acid composition, mol% G + C and Biolog-GN tests. Besides phenanthrene, strain ZX4 could also utilize naphthalene, fluorene and other aromatic compounds. The growth on salicylic acid and catechol showed that the strain degraded phenanthrene via salicylate pathway, while the assay of catechol 2, 3-dioxygenase revealed catechol could be metabolized through meta-cleavage pathway. Three genes, including two of meta-cleavage operon genes and one of GST encoding gene were obtained. The order of genes arrangement was similar to S-type meta-pathway operons. The phylogenetic trees based on 16S rDNA sequence and meta-pathway gene both revealed that strain ZX4 is clustered with strains from genus Sphingomonas.     

Effect of gelling agents and antibiotics on adventitious bud regeneration from In vitro leaves of pear

Summary The effect of the type of gelling agent and of several antibiotics on the adventitious bud regeneration from in vitro leaves was tested on eight pear genotypes. The use of gellan gum (Phytagel™) in the medium instead of agar had a very strong positive effect on the rate of adventitious bud regeneration for all pear genotypes tested in this study. This gelling agent induced faster cell divisions than agar, thus more callus was produced on wound sites and subsequently more buds regenerated. Incubation on gellan gum medium during the first 20 d of bud induction was sufficient to induce a stimulatory effect on regeneration and limited the production of hyperhydric buds. In the prospect of Agrobacterium transformation, the effect of several antibiotics was tested. Cefotaxime (200 mg/l) plus ticarcillin/clavulanic acid (100 mg/l) could be used in the culture medium without affecting the frequency of bud regeneration. The inhibition of bud regeneration was obtained with different kanamycin concentrations according to the gelling agent in the medium. On gellan gum medium, a concentration of 100 mg/l of kanamycin was suitable. These conditions can be recommended for experiments on Agrobacterium-mediated transformation of pear, where bacterial inoculation and presence of antibiotics generally reduce and delay bud regeneration.