Customized Modification of Welan Gum Properties Through Controllable Grafting of Acrylamide.

Welan gum (WG) has a wide range of applications, but it is not yet suitable for applications such as oil recovery profile control that have complex requirements for viscosity, gelation properties, and so forth. Grafting modification is an important strategy for improving the property of WG, but there are few reports on controllable modification of WG to customize it for specific application. Acrylamide (AM) dosage was identified as the key factor affecting the grafting ratio of AM onto WG by a uniform experimental design. The grafting ratio can be directly adjusted between 99% and 378% based on the positive correlation with dosage of AM, and viscosity can be adjusted between 206 and 327 mPa s based on the negative correlation with grafting ratio. The 50% weight loss temperature of W11 with a grafting ratio of 110% raised from 314 to 336°C after grafting. The viscosity of the hydrogel formed with WG11 reached 15,654 mPa s, nearly nine times higher than that of unmodified WG. In addition, the gelation time can be controlled within 5 days, so that it can be injected to the optimal area in oilfield profile, avoiding pipeline blockage. This study enables adjusting viscosity of WG grafted with AM by controlling the grafting rate, and enhances gelation performance and thermal stability of WG, which will expand the application of WG in oil recovery and other fields.

Optimization of welan gum extraction and purification using lysozyme and alkaline protease.

Welan gum, a natural polysaccharide produced by Sphingomonas sp. ATCC 31555, has attracted considerable attention in the scientific community due to its desirable properties. However, challenges, such as high viscosity, residual bacterial cells, carotenoids, and protein complexation, hinder the widespread application of welan gum. In this study, we established a method for the extraction and purification of welan gum using a synergistic approach with lysozyme and alkaline protease. Lysozyme hydrolysis conditions were optimized by applying response surface methodology, and the best results for bacterial cell removal were achieved at 11 000 U/g, 44 °C, and pH 9 after 3 h of treatment. Subsequently, we evaluated protein hydrolysis through computer simulation and identified alkaline protease as the most suitable enzyme. Through experimental investigations, we found that the optimal conditions for alkaline protease hydrolysis were 7500 U/g, 50 °C, pH 10, and 600 rpm. These conditions resulted in a sugar recovery rate of 76.1%, carotenoid removal rate of 89.5%, bacterial removal rate of 95.2%, and protein removal rate of 87.3% after 3 h of hydrolysis. The purified welan gum exhibited high transparency and purity. Structural characterization and antioxidant activity evaluation revealed that enzymatically purified welan gum has potential application prospects. Our study provides valuable insights into the optimal method for the enzymatic extraction and purification of welan gum. Such a method is conducive to the development of the multiple potential applications of welan gum. KEY POINTS: • A novel process for the synergistic purification of welan gum using lysozyme and alkaline protease was established. • In silico virtual digestion was employed to select the purification enzyme. • Welan gum with high transparency and purity was obtained.

Fed-batch fermentation strategy for efficient welan gum production by Sphingomonas sp. FM01.

BACKGROUND: As a novel type of extracellular polysaccharide produced by Sphingomonas sp., welan gum has been widely applied in various fields because of its excellent properties. The study has improved the fermentation process. RESULTS: The initial sucrose concentration, temperature and stirring speed were set to 20 g L-1, 33 °C and 400 rpm, respectively, and 13.3 g L-1 sucrose was added at 24, 40 and 56 h. The temperature and stirring speed were then set at 28 °C and 600 rpm from 24 to 48 h and 28 °C and 600 rpm from 48 to 72 h, respectively. As a result, welan gum production, dry cell weight, sucrose conversion rate and viscosity were correspondingly increased to 38.60 g L-1, 5.47 g L-1, 0.64 g g-1 and 3779 mPa·s, respectively. In addition, the mechanism by which fermentation strategy promotes welan gum synthesis was investigated by transcriptome analysis. CONCLUSION: Improving respiration and ATP supply, reducing unnecessary protein synthesis, and alleviating competition between cell growth and welan gum synthesis contribute to promoting the fermentation performance of Sphingomonas sp., thus providing a practical strategy for efficient welan gum production. © 2024 Society of Chemical Industry.

Efficient biosynthesis of polysaccharide welan gum in heat shock protein-overproducing Sphingomonas sp. via temperature-dependent strategy.

Cell growth and product formation are two critical processes in polysaccharide welan biosynthesis, but the conflict between them is often encountered. In this study, a temperature-dependent strategy was designed for two-stage welan production through overexpressing heat shock proteins in Sphingomonas sp. The first stage was cell growth phase with higher TCA cycle activity at 42 °C; the second stage was welan formation phase with higher precursor synthesis pathway activity at 37 °C. The highest welan concentration 37.5 g/L was achieved after two-stage process. Ultimately, this strategy accumulated welan yield of 79.2 g/100 g glucose and productivity of 0.62 g/L/h at 60 h, which were the best reported results so far. The duration of fermentation was shortened. Besides, rheological behavior of welan gum solutions remained stable at wide range of temperature, pH, and NaCl. These results indicated that this approach efficiently improved welan synthesis.

Different nitrogen sources change the transcriptome of welan gum-producing strain Sphingomonas sp. ATCC 31555.

To reveal effects of different nitrogen sources on the expressions and functions of genes in Sphingomonas sp. ATCC 31555, it was cultivated in medium containing inorganic nitrogen (IN), organic nitrogen (ON), or inorganic-organic combined nitrogen (CN). Welan gum production and bacterial biomass were determined, and RNA sequencing (RNA-seq) was performed. Differentially expressed genes (DEGs) between the different ATCC 31555 groups were identified, and their functions were analyzed. Welan gum production and bacterial biomass were significantly higher in the ON and CN groups compared with those in the IN group. RNA-seq produced 660 unigenes, among which 488, 731, and 844 DEGs were identified between the IN vs. ON, IN vs. CN, and ON vs. CN groups, respectively. All the DEGs were related significantly to metabolic process and signal transduction. DEGs between the IN vs. CN and ON vs. CN groups were potentially associated with bacterial chemotaxis. Real-time PCR confirmed the expressions of selected DEGs. Organic nitrogen led to higher bacterial biomass and welan gum production than inorganic nitrogen, which might reflect differences in gene expression associated with metabolic process, signal transduction, and bacterial chemotaxis induced by different nitrogen sources.

Improved production of carotenoid-free welan gum in a genetic-engineered Alcaligenes sp. ATCC31555.

OBJECTIVE: To improve the production of welan gum and obtain a carotenoid-free strain while reducing the fermentation and post-treatment costs. RESULTS: The vitreoscilla globin (vgb) gene combined with the ß-galactosidase (lacZ) promoter was inserted into the phytoene synthase (crtB) gene region of the chromosome in Alcaligenes sp. ATCC31555. When the recombinant strain was grown in a 5 l fermentor, welan gum was produced at 24 ± 0.4 g l(-1) compared to 21 g ± 0.4 g l(-1) in the wild type. Furthermore, the carotenoid-free welan gum produced using Alcaligenes sp. ATCC31555 VHb strain was less expensive with improved properties. CONCLUSIONS: Alcaligenes sp. ATCC31555 VHb strain was a better neutral welan-producing strain with a higher production than the wild-type strain.

NMR analysis of the side-group substituents in welan gum in comparison to gellan gum.

The physicochemical properties and applications of polysaccharides are highly dependent on their chemical structures, including the monosaccharide composition, degree of substitution, and position of substituent groups in the backbone. The occurrence of side groups or side chains in the chain backbone of polysaccharides is often an essential factor influencing their conformational and physicochemical properties. Welan gum produced by the fermentation of Sphingomonas sp. ATCC 31555 microorganisms has been widely used in food, construction, and oil drilling fields. While understanding the physicochemical properties of welan gum solution has been highly developed, there is still little information about the determination strategy of the glycosyl side groups in welan gum. In this study, the NMR method was established to quantitatively determine the substituent groups in the chain backbone of welan gum. The delicate chemical structures of welan gum obtained at different fermentation conditions were clarified. The composition and content of side substituents were also identified by high-performance liquid chromatography to confirm the accuracy of NMR analysis. The quantitative determination of substituent groups in gellan gum based on NMR analysis was also elaborated for comparison. This work provides insights for profoundly understanding the structure-function relationship of welan gum.

Modification of Welan gum with poly(2-oxazoline) to obtain thermoviscosifying polymer for enhanced oil recovery.

Thermoviscosifying polymers refer to a category of smart materials that exhibit a responsive behavior to environmental stimuli, specifically demonstrating a natural rise in viscosity of solutions as the temperature increases. The temperature-dependent behavior exhibited by thermally viscous polymers renders them potentially advantageous in the context of Enhanced Oil Recovery (EOR). There exists a dearth of research pertaining to the application of thermoviscosifying polymer for better recovery in reservoirs characterized by high temperatures and high salt content. In order to tackle the mentioned concerns, this study examined the utilization of welan gum modified with poly(2-oxazoline) as thermally responsive chain segments to enhance viscosity. The objective was to evaluate the ability to enhance viscosity under thermal conditions and to assess their effectiveness in displacement of reservoir oil in high temperature and high salt environments. This study aimed to establish a theoretical framework for understanding the correlation between the molecular structure and performance of novel thermally viscous polymers. Additionally, it sought to offer practical insights into designing the molecular structure of thermally viscous polymers suitable for polymer flooding in high temperature and high salt environments. Furthermore, the study proposed the application of these new thermoviscosifying polymers for EOR.

Glycerol-driven adaptive evolution for the production of low-molecular-weight Welan gum: Characterization and activity evaluation.

Through adaptive laboratory evolution (ALE) of Sphingomonas sp. ATCC 31555, fermentation for production of low-molecular-weight welan gum (LMW-WG) was performed using glycerol as sole carbon source. During ALE, GPC-MALS analysis revealed a gradual decrease in WG molecular weight with the increase of adaptation cycles, accompanied by changes in solution conformation. LMW-WG was purified and structurally analyzed using GPC-MALS, monosaccharide composition analysis, infrared spectroscopy, NMR analysis, atomic force microscopy, and scanning electron microscopy. Subsequently, LMW-WG obtains hydration, transparency, antioxidant activity, and rheological properties. Finally, an in vitro simulation colon reactor was used to evaluate potential prebiotic properties of LMW-WG as dietary fiber. Compared with WG produced using sucrose as substrate, LMW-WG exhibited a fourfold reduction in molecular weight while maintaining moderate viscosity. Structurally, L-Rha nearly completely replaced L-Man. Furthermore, LMW-WG demonstrated excellent hydration, antioxidant activity, and high transparency. It also exhibited resistance to saliva and gastrointestinal digestion, showcasing a favorable colonization effect on Bifidobacterium, making it a promising symbiotic agent.

Adsorption of Welan Gum on Montmorillonite and Its Influencing Factors.

Welan gum is one of the most promising polymers used in polymer flooding for enhancing oil recovery, due to its excellent temperature resistance and salt-tolerance performance. However, welan gum, as a polymer with higher molecular weight, can be adsorbed and detained in the pore throat of the reservoir, which is characterized by a smaller size. Montmorillonite, a kind of clay mineral with high content in reservoir rocks, has strong adsorption capacity. Therefore, the adsorption behavior of welan gum on montmorillonite, as well as its influencing factors, are studied in this paper. The results show that the adsorption capacity is 2.07 mg/g. The adsorption capacity decreased with the increase in temperature. Both acidic and alkaline conditions reduced the adsorption capacity. The existence of inorganic salt affected the adsorption capacity. In addition, the higher the cation value, the lower the adsorption capacity. The characterization tests showed that the adsorption of welan gum on montmorillonite was characterized by physical adsorption and surface adsorption, indicating that there were no changes in the internal structure of montmorillonite. This study provides feasible methods to reduce the amount of welan gum adsorbed on montmorillonite, which is of great significance for reducing the permeability damage caused by welan gum adsorption and promoting the application of welan gum in polymer flooding for enhancing oil recovery.

Effect of the Welan Gum Concentration on the Rheological and Structural Behaviour of Biocomposite Hydrogels with Sepiolite as Filler.

A very positive and effective approach to tuning the mechanical properties of polymers has been the development of composites. This paper deals with novel biocomposite hydrogels composed by two biocompatible materials: welan gum as biopolymer matrix and sepiolite as filler. Welan gum content was studied as a tuning parameter to control the rheological properties of the developed biocomposites. The rheological and microstructural behaviour of the composites was investigated by mean of steady-state flow curves, creep-recovery tests, small amplitude oscillatory shear tests, and electron microscopy. An increase in welan gum content provoked the progressive disappearance of the shear-thinningzero-shear-thinning behaviour with a yield point which was clearly defined, characteristic of sepiolite gels, leading to a conventional shear-thinning behaviour, typical of polymeric systems. Also, a higher content of biopolymer in the mixtures led to a more elastic and compact structure characterized by higher values of both G' and G". The fundamental novelty was based on taking the flowability provided by the biopolymer as the main objective and reinforcing the viscosity yielded by welan gum with sepiolite, which contributed to increasing the biocomposite consistency. Thus, rheological properties can be adjusted, taking into account the balance of the components to adapt them to the requirements of each application.

Sustainable production and characterization of medium-molecular weight welan gum produced by a Sphingomonas sp. RW.

Welan gum is widely used in food, concrete, and oil recovery for its excellent stability and unique rheological properties. This study describes an engineered Sphingomonas sp. RW strain deficient in the lyase gene (welR) and outlines its application in an industrial by-product fermentation strategy to produce welan gum. The yield of the engineered strain was enhanced to 25.11 ± 0.05 g/L, whereas the broth viscosity (2110 mPas) increased by 281.81%. The welan gum produced by Sphingomonas sp. RW was named medium-molecular weight welan gum (MMWG, ~350 kDa). FT-IR and monosaccharide composition analysis revealed that the MMWG was composed of l-mannose, l-rhamnose, glucuronic acid, and glucose (with mole ratios of 1:4.46:1.26:2.82). Significantly, MMWG had favorable hydroxyl radical scavenging activity and emulsifying capacity. These results provide a strategy for cost-effective welan gum production and enrich the properties of welan gum with different molecular weight.

Effect of an inorganic nitrogen source (NH4)2SO4 on the production of welan gum from Sphingomonas sp. mutant obtained through UV-ARTP compound mutagenesis.

As one of the most expensive extracellular polysaccharides, welan gum is widely used in biomedicine, food products, and petroleum because of its unique structure and excellent rheological properties. To reduce the cost of welan gum fermentation, together with (NH4)2SO4, which served as the sole nitrogen source, a high-welan-gum-producing mutant, B-8, screened through UV-ARTP compound mutagenesis was used. Under optimum conditions (C:N ratio 25:1, sucrose 50 g/L, (NH4)2SO4 4 g/L, and adding 8 mM NaCl at 32 h fermentation), the yield of welan gum and sucrose conversion were 18.86 g/L and 0.38 g/g, respectively, which were 98.95% and 137.50% higher than those achieved with the parent strain FM01, respectively. After the same treatment process, IN-welan (obtained with (NH4)2SO4) consumed less 95% ethanol, had higher molecular weight, and exhibited better rheological properties than ON-welan (obtained with beef extract). Transcriptome analysis revealed that (NH4)2SO4 could affect the synthetic pathway and monosaccharide content of welan gum by increasing bacterial chemotaxis and the availability of key intermediates. The fermentation performance of Sphingomonas sp. mutants could further be improved by providing several target genes to the mutants through metabolic engineering.

A strategy for the synthesis of low-molecular-weight welan gum by eliminating capsule form of Sphingomonas strains.

Welan gum is widely used in food, concrete additives, and oil recovery. Here we changed the capsule form of Sphingomonas strains by knocked out the sortase gene (srtW). The obtained welan gum was mainly composed of mannose, glucose, rhamnose, and glucuronic acid at a molar ratio of 4.0:5.8:1.6:1, respectively. Meanwhile, the molecular weight of welan gum decreased sharply (about 68 kDa). Moreover, the low molecular weight (LMW) welan gum was characterized by FT-IR and NMR spectroscopy. The rheological results revealed that the LMW welan gum solution is a pseudoplastic fluid with a lower apparent viscosity. Furthermore, the oscillation test illustrated stable dynamic viscoelasticity within the temperature range of 5-68 °C and frequency range of 0.01-15 rad/s. To the best of our knowledge, this is the first report of LMW welan gum production and characterization. These results provide references for LMW welan gum applications, and likely applicable for other biopolymers production.

Effect of NaCl addition on the production of welan gum with the UV mutant of Sphingomonas sp.

Because of its excellent stability, non-toxicity, biodegradability and unique rheology, welan gum can be widely used in various fields, such as petroleum, biomedicine and food products. In this study, a high-yield mutant strain FM01-S09 was screened through two rounds of UV mutagenesis. Remarkably, the production of welan gum could be further increased by adding 4 mM NaCl at 32 h fermentation, reaching 30.12 ± 0.25 g/L (28.66% higher than no adding), and the NaCl-WG solution had stronger structural, impact resistance, and temperature resistance than H2O2-WG and WG solutions. Furthermore, the mechanism by which NaCl promotes welan gum synthesis was also investigated. It was found that cell membrane characteristics, intracellular microenvironment makeup, and key enzyme gene expression levels were significantly altered in different fermentation stages. Therefore, the addition of NaCl could effectively promote the growth and fermentation performance of Sphingomonas sp., providing a novel strategy for cost-effective welan gum production.

Characterization and Function of a Novel Welan Gum Lyase From Marine Sphingomonas sp. WG.

Welan gum, a kind of microbial exopolysaccharides, produced by the genus Sphingomonas, have great potential for application in many fields, such as the food industry, cement production, and enhanced oil recovery. But there are still challenges to reduce the cost, enhance the production and the quality. Herein, the bioinformatics analysis of WelR gene was preformed, and the characterization and function of WelR, welan gum lyase, from Sphingomonas sp. WG were investigated for the first time. The results indicated that 382nd (Asn), 383rd (Met), 494th (Asn), and 568th (Glu) were the key amino acid residues, and C-terminal amino acids were essential to keeping the stability of WelR. The optimal temperature and pH of the enzymatic activity were found to be 25°C and 7.4, respectively. And WelR was good low temperature resistance and alkali resistant. K+, Mg2+, Ca2+, Mn2+, and EDTA increased WelR activities, in contrast to Zn2+. Coupled with the change in glucose concentration and growth profile, the qRT-PCR results indicated that WelR may degrade welan gum existing in the culture to maintain bacterial metabolism when glucose was depleted. This work will lay a theoretical foundation to establish new strategies for the regulation of welan gum biosynthesis.

Production of welan gum from cane molasses by Sphingomonas sp. FM01.

As an extracellular exopolysaccharide, welan gum could serve as a suspending agent, stabilizer, emulsifier and thickener with characteristics that include bio-adhesion, non-toxicity, biocompatibility and biodegradability. In this study, Sphingomonas sp. FM01 was applied to effectively produce welan gum using cane molasses as the carbon source. After pretreatment with phosphoric acid and polyacrylamide, 60 g/L cane molasses combined with 6.0 g/L beef extract, 3 g/L KH2PO4, 0.5 g/L MgSO4, 15 mmol/L H2O2 and 4 mg/L niacin significantly improved the fermentation performance of Sphingomonas sp. FM01, increasing the production of welan gum to 37.65 g/L. Investigation of the rheological behavior of the welan gum obtained from the molasses-welan gum mixture showed it had an acceptable molecular weight and similar rheological properties and better viscosity stability compared to that obtained from sucrose, indicating that cane molasses could be explored as a suitable and inexpensive substrate for cost-effective welan gum production.

Welan gum promoted the growth of rice seedlings by enhancing carbon and nitrogen assimilation.

Based on the characteristics of natural polysaccharides in film-forming, chelating, and environmental friendly, a natural polysaccharide fertilizer agent was selected to increase the utilization of nitrogen fertilizer and increase plant growth. Five polysaccharides: xanthan gum, guar gum, fenugreek gum, welan gum and chitosan were screened for plant growth promoting effect. The results showed that welan gum had the most significant effect on promoting the growth of rice seedlings, and the concentrations of 0.1 mg mL-1 and 0.15 mg mL-1 showed the best growth effects. The effects of welan gum on nitrogen utilization in rice seedlings were investigated. Results showed welan gum increased the contents of ammonium, nitrate, free amino acids, and proteins in rice seedlings. There were four key enzymes of nitrogen metabolism which are nitrate reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase significantly enhanced by welan gum though up-regulating the transcriptional levels of these enzymes. Therefore, nitrogen uptake and nitrogen metabolism in rice seedlings were promoted to increase the biomass of rice seedlings. Based on the research, results showed that welan gum could constitute a promising fertilizer in the future.

Construction of a Robust Sphingomonas sp. Strain for Welan Gum Production via the Expression of Global Transcriptional Regulator IrrE.

Welan gum is a widely used microbial polysaccharide produced by Sphingomonas sp. However, an important factor hindering the expansion of its production is the maladaptation of strain to fermentation conditions. In this work, the global transcriptional regulator gene irre was selected as a stress-resistant element. And it was integrated into the site of the genomic carotene synthesis key enzyme gene crtB to construct a robust carotenoid-free welan gum producing strain. Fermentation with the recombinant strain effectively reduced the ethanol consumption and pigment content in the product. The tolerance temperature increased by 10°C without the need for controlling the pH. Under this fermentation condition, welan gum concentration could still reach 20.26 ± 0.25 g/L, which was 187.38% higher than that of the wild-type strain (7.05 ± 0.15 g/L). Transcriptome analysis showed that with the control of IrrE, more than 1000 genes that are involved in multiple pathways, including two-component system, bacterial chemotaxis, flagellar assembly, and cell cycle, exhibited changes at the transcriptional level and jointly allowed the strain to protect against environmental stresses.

Effect of different nitrogen sources on the viscosity and rheological properties of welan gum produced by Sphingomonas sp. ATCC 31555.

The current study aimed to determine the rheological properties of welan gum produced by Sphingomonas sp. ATCC 31555 using different nitrogen sources. The viscosity and rheological properties of welan gum produced by Sphingomonas sp. ATCC 31555 from different ratios of inorganic, organic, and mixture of organic and inorganic nitrogen sources were measured under different conditions of pH, rotational speeds, and temperatures and also in the presence of various metal ions. Increasing the proportion of sodium nitrate (NaNO3 ) as the nitrogen source enhanced the viscosity and the ability of welan gum solution to resist high temperatures and salinity. The viscosity of welan gum solution derived from all three nitrogen sources gradually declined when the rotational speed was increased and stabilized at rotational speeds >30 rpm. The elastic modulus and viscous modulus were highest for a welan gum solution derived from 4.0 g/L NaNO3 . The viscosity, temperature, and salt tolerance were superior when a welan gum solution was produced from NaNO3 in comparison with that produced from organic nitrogen source.