Optimization of exopolysaccharide production and diesel oil emulsifying properties in root nodulating bacteria

Bioremediation, a strategy mediated by microorganisms, is a promising way used in the degradation or removal of organic contaminants from soil or aquatic system. Exopolysaccharide (EPS) which was produced by a variety of Gram-negative bacteria has been demonstrated to be a potential bioemulsifier used in the degradation of hydrocarbons. In the present study, attempts were made to optimize the production of EPS from our newly isolates by adjusting the culture conditions and medium components. Besides, the performance of diesel oil emulsification using partially purified EPS derived from different conditions was also demonstrated. Out of 40 root nodulating bacteria the better emulsifying abilities were recorded from three strains namely Rhizobium miluonense CC-B-L1, Burkholderia seminalis CC-IDD2w and Ensifer adhaerens CC-GSB4, as can be seen from their emulsification index (E₂₄) 66, 64 and 60%, respectively. These three strains produced 212, 203 and 198 mg l⁻¹ of EPS, respectively, in yeast extract mannitol (YEM) medium. After modifying culture conditions, better performances can be achieved from these three strains, with increases of 21.7, 21.4, 16.7% in the EPS production and 12.1, 10.9, 8.3% in E₂₄, respectively. When considered for strain CC-B-L1 and CC-IDD2w, the addition of 1.5% (v/v) of mannitol and 0.1% (v/v) of asparagine in YEM enhanced 42.9 and 34.7% in EPS production along with 28.8 and 37.5% higher in E₂₄. The supplement of 2.0% (v/v) glucose and 0.2% (v/v) asparagine in YEM increased 65.2% of EPS and 38.3% of E₂₄ in strain CC-GSB4. This is the first report demonstrating the optimization of diesel emulsification by EPS from root nodulating isolates, and these microbial agents might be used in the remediation of hydrocarbon contaminated soils in a near future.     

Efficiency of the EPS emulsifier produced by <Emphasis Type="Italic">Ochrobactrum anthropi</Emphasis> in different hydrocarbon bioremediation assays

Ochrobactrum anthropi strain AD2 was isolated from the waste water treatment plant of an oil refinery and was identified by analysis of the sequence of the gene encoding 16S rDNA. This bacterium produced exopolysaccharides in glucose nutrient broth media supplemented with various hydrocarbons (n-octane, mineral light and heavy oils and crude oils). The exopolysaccharide AD2 (EPS emulsifier) synthesized showed a wide range of emulsifying activity but none of them had surfactant activity. Yield production varied from 0.47 to 0.94 g of EPS l⁻¹ depending on the hydrocarbon added. In the same way, chemical composition and emulsification activity of EPS emulsifier varied with the culture conditions. Efficiency of the EPS emulsifier as biostimulating agent was assayed in soil microcosms and experimental biopiles. The AD2 biopolymer was added alone or combined with commercial products frequently used in oil bioremediation such as inorganic NPK fertilizer and oleophilic fertilizer (S200 C). Also, its efficiency was tested in mixture with activated sludge from an oil refinery. In soil microcosms supplemented with S200 C + EPS emulsifier as combined treatment, indigenous microbial populations as well as hydrocarbon degradation was enhanced when compared with microcosms treated with NPK fertilizer or EPS emulsifier alone. In the same way EPS emulsifier stimulated the bioremediation effect of S200 C product, increasing the number of bacteria and decreasing the amount of hydrocarbon remained. Finally, similar effects were obtained in biopile assays amended with EPS emulsifier plus activated sludge. Our results suggest that the bioemulsifier EPS emulsifier has interesting properties for its application in environment polluted with oil hydrocarbon compounds and may be useful for bioremediation purposes.     

Extracellular polysaccharides produced by cooling water tower biofilm bacteria and their possible degradation

The extracellular polymers (EPS) of biofilm bacteria that can cause heat and mass transfer problems in cooling water towers in the petrochemical industry were investigated. In addition, these microorganisms were screened for their ability to grow and degrade their own EPS and the EPS of other species. Twelve bacteria producing the most EPS were isolated from cooling water towers and characterized biochemically by classic and commercial systems. These were species of Pseudomonas, Burkholderia, Aeromonas, Pasteurella, Pantoea, Alcaligenes and Sphingomonas. EPS of these species were obtained by propan-2-ol precipitation and centrifugation from bacterial cultures in media enriched with glucose, sucrose or galactose. EPS yields were of 1.68-4.95 g l(-1). These EPS materials were characterized for total sugar and protein contents. Their total sugar content ranged from 24 to 56% (g sugar g(-1) EPS), and their total protein content ranged from 10 to 28% (g protein g(-1) EPS). The monosaccharide compositions of EPS were determined by HPLC. Generally, these compositions were enriched in galactose and glucose, with lesser amounts of mannose, rhamnose, fructose and arabinose. All bacteria were investigated in terms of EPS degradation. Eight of the bacteria were able to utilize EPS from Burkholderia cepacia, seven of the bacteria were able to utilize EPS from Pseudomonas sp. and Sphingomonas paucimobilis. The greatest viscosity reduction of B. cepacia was obtained with Pseudomonas sp. The results show that the bacteria in this study are able to degrade EPS from biofilms in cooling towers.     

Extracellular polysaccharides of Rhodococcus rhodochrous S-2 stimulate the degradation of aromatic components in crude oil by indigenous marine bacteria

Rhodococcus rhodochrous S-2 produces extracellular polysaccharides (S-2 EPS) containing D-glucose, D-galactose, D-mannose, D-glucuronic acid, and lipids, which is important to the tolerance of this strain to an aromatic fraction of (AF) Arabian light crude oil (N. Iwabuchi, N. Sunairi, H. Anzai, M. Nakajima, and S. Harayama, Appl. Environ. Microbiol. 66:5073-5077, 2000). In the present study, we examined the effects of S-2 EPS on the growth of indigenous marine bacteria on AF. Indigenous bacteria did not grow significantly in seawater containing AF even when nitrogen, phosphorus, and iron nutrients were supplemented. The addition of S-2 EPS to seawater containing nutrients and AF resulted in the emulsification of AF, promotion of the growth of indigenous bacteria, and enhancement of the degradation of AF by the bacteria. PCR-denaturing gradient gel electrophoresis analyses show that addition of S-2 EPS to the seawater containing nutrients and AF changed the composition of the bacterial populations in the seawater and that bacteria closely related to the genus Cycloclasticus became the major population. These results suggest that Cycloclasticus was responsible for the degradation of hydrocarbons in AF. The effects of 15 synthetic surfactants on the degradation of AF by indigenous marine bacteria were also examined, but enhancement of the degradation of AF was not significant. S-2 EPS was hence the most effective of the surfactants tested in promoting the biodegradation of AF and may thus be an attractive agent to use in the bioremediation of oil-contaminated marine environments.     

Physiological adaptations involved in alkane assimilation at a low temperature by Rhodococcus sp. strain Q15.

We examined physiological adaptations which allow the psychrotroph Rhodococcus sp. strain Q15 to assimilate alkanes at a low temperature (alkanes are contaminants which are generally insoluble and/or solid at low temperatures). During growth at 5 degrees C on hexadecane or diesel fuel, strain Q15 produced a cell surface-associated biosurfactant(s) and, compared to glucose-acetate-grown cells, exhibited increased cell surface hydrophobicity. A transmission electron microscopy examination of strain Q15 grown at 5 degrees C revealed the presence of intracellular electron-transparent inclusions and flocs of cells connected by an extracellular polymeric substance (EPS) when cells were grown on a hydrocarbon and morphological differences between the EPS of glucose-acetate-grown and diesel fuel-grown cells. A lectin binding analysis performed by using confocal scanning laser microscopy (CSLM) showed that the EPS contained a complex mixture of glycoconjugates, depending on both the growth temperature and the carbon source. Two glycoconjugates [beta-D-Gal-(1-3)-D-GlcNAc and alpha-L-fucose] were detected only on the surfaces of cells grown on diesel fuel at 5 degrees C. Using scanning electron microscopy, we observed strain Q15 cells on the surfaces of octacosane crystals, and using CSLM, we observed strain Q15 cells covering the surfaces of diesel fuel microdroplets; these findings indicate that this organism assimilates both solid and liquid alkane substrates at a low temperature by adhering to the alkane phase. Membrane fatty acid analysis demonstrated that strain Q15 adapted to growth at a low temperature by decreasing the degree of saturation of membrane lipid fatty acids, but it did so to a lesser extent when it was grown on hydrocarbons at 5 degrees C; these findings suggest that strain Q15 modulates membrane fluidity in response to the counteracting influences of low temperature and hydrocarbon toxicity.     

Changes in Intestinal Microbiota Due to the Expanded Polystyrene Diet of Mealworms (Tenebrio molitor)

Expanded polystyrene (EPS), which is difficult to decompose, is usually buried or incinerated, causing the natural environment to be contaminated with microplastics and environmental hormones. Digestion of EPS by mealworms has been identified as a possible biological solution to the problem of pollution, but the complete degradation mechanism of EPS is not yet known. Intestinal microorganisms play a significant role in the degradation of EPS by mealworms, and relatively few other EPS degradation microorganisms are currently known. This study observed significant differences in the intestinal microbiota of mealworms according to the dietary results of metagenomics analysis and biodiversity indices. We have proposed two new candidates of EPS-degrading bacteria, Cronobacter sakazakii and Lactococcus garvieae, which increased significantly in the EPS feeding group population. The population change and the new two bacteria will help us understand the biological mechanism of EPS degradation and develop practical EPS degradation methods.     

Exopolysaccharide biosynthesis is important for <Emphasis Type="Italic">Mesorhizobium tianshanense</Emphasis>: plant host interaction

Mesorhizobium tianshanense is a nitrogen-fixing bacterium that can establish symbiotic associations with Glycyrrhiza uralensis in the form of root nodules. Nodule formation in rhizobia often requires various secreted carbohydrates. To investigate exopolysaccharide (EPS) production and function in M. tianshanense, we performed a genome-wide screen using transposon mutagenesis to identify genes involved in EPS production. We identified seven mutants that produced significantly lower amounts of EPS as well as a two-component sensor kinase/response regulator system that is involved in the activation of EPS synthesis. EPS mutants formed significantly less biofilm and displayed severely reduced nodulation capacity than wild type bacteria, suggesting that EPS synthesis can play important roles in the symbiosis process. Peng Wang, Zengtao Zhong and Jing Zhou have contributed equally to this work.     

灵芝胞外多糖分批发酵动力学模型

利用分批发酵研究了灵芝（Ganoderma lucidum）胞外多糖的合成特性,结果表明Ganoderma lucidum多糖合成和菌体生长呈部分生长关联型。菌体干重、胞外多糖分别达到15.56g·L^-1<、3.02g·L^-1<,胞外多糖对细胞干重得率系数(Y_p/x）为0.19。根据分批发酵试验结果采用Logistic方程、Luedeking-Piret方程和类似Luedeking-Piret方程,得到了描述灵芝生长、胞外多糖以及葡萄糖底物消耗分批发酵动力学模型。同时在初始葡萄糖变化较大范围内,试验数据与模型预测值进行了比较拟合,平均相对误差小于5％,表现出很好的适用性。表明该动力学模型对指导灵芝胞外多糖的发酵生产具有实际意义。     

Verification of Degradation of <Emphasis Type="Italic">n</Emphasis>-Alkanes in Diesel Oil by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Strain WatG in Soil Microcosms

Degradation of n-alkanes in diesel oil by Pseudomonas aeruginosa strain WatG (WatG) was verified in soil microcosms. The total petroleum hydrocarbon (TPH) degradation level in two bioaugmentation samples was 51% and 46% for 1 week in unsterilized and sterilized soil microcosms, respectively. The TPH degradation in the biostimulation was of control level (15%). The TPH degradation in aeration-limited samples was clearly reduced when compared with that in aeration-unlimited ones under both sterilized and unsterilized conditions. Addition of WatG into soil microcosms was accompanied by dirhamnolipid production only in the presence of diesel oil. These findings suggest that degradation of n-alkanes in diesel oil in soil microcosms would be facilitated by bioaugmentation of WatG, with production of dirhamnolipid, and also by participation of biostimulated indigenous soil bacteria.     

嗜热链球菌MGB80-7所产胞外多糖的表型结构及其抗氧化活性

【背景】胞外多糖（exopolysaccharide,EPS）是乳酸菌生长代谢过程中所产生的一种次级代谢产物,除了可以改善产品质构和品质外,其生理功能也是近年来研究人员追捧的热点。【目的】探究乳酸菌EPS的表征特性和分子结构,揭示其与EPS益生特性之间的联系。【方法】以产EPS的嗜热链球菌（Streptococcus thermophilus,S. thermophilus） MGB80-7为研究对象,利用苯酚-硫酸法测定菌株EPS产量。采用离子交换柱层析和凝胶分子筛层析对该菌株所产EPS进行分离纯化,结合凝胶色谱、红外光谱及高效液相色谱对EPS表型结构进行剖析。此外,为确定EPS表型特征对其抗氧化活性的影响,测定了EPS对超氧阴离子、羟自由基及DPPH自由基等的清除能力。【结果】S. thermophilus MGB80-7在M17培养基中EPS产量较高,为（268.25±5.36） mg/mL,分离纯化后共得到2种多糖组分,其中中性多糖（WPS-807）分子量为1.028×10⁵ Da,主要由葡萄糖、半乳糖和甘露糖组成,并含有少量的鼠李糖和阿拉伯糖,酸性多糖（...     

Spent growth medium of Pantoea agglomerans primes wheat suspension cells for augmented accumulation of hydrogen peroxide and enhanced peroxidase activity upon elicitation

Induced disease resistance in plants is based on multiple mechanisms, including cell “priming”, i.e. an enhancement of the capacity to mobilize cellular defense responses upon pathogen attack. Potent inducers of priming are, for example, salicylic acid, synthetic compounds such as a benzothiadiazole, and certain rhizosphere bacteria. While priming is well characterized for a number of dicot plants, only few cases of priming are documented in monocots. Here, we report that the spent growth medium of the Gram negative bacterium Pantoea agglomerans is capable of priming wheat cells (Triticum aestivum L. cv Prelude-Sr5) for elicitor-induced defense responses. Pre-incubation of suspension-cultured wheat cells with growth medium of P. agglomerans led to a strong enhancement of an oxidative burst that has been induced by chitin or chitosan and to an increase in extracellular peroxidase activity. Moreover, exopolysaccharides (EPS) were isolated from the spent growth medium and demonstrated to be sufficient for the induction of H₂O₂ priming. The EPS-induced priming was shown to be time- and concentration-dependent. We conclude that EPS are the or one of several priming-active component(s) in the spent growth medium of P. agglomerans. The present work is the first report of priming in a monocot plant by a specific component of bacterial origin. A comparison with known chemical inducers of resistance revealed that a benzothiadiazole was able to enhance the oxidative burst similar to the spent growth medium or the EPS of P. agglomerans, while salicylic acid was not.     

Culture and exopolysaccharide production from sugarcane molasses by Gordonia polyisoprenivorans CCT 7137, isolated from contaminated groundwater in Brazil

Gordonia polyisoprenivorans CCT 7137 was isolated from groundwater contaminated with leachate in an old controlled landfill (São Paulo, Brazil), and cultured in GYM medium at different concentrations of sugarcane molasses (2%, 6%, and 10%). The strain growth was analyzed by monitoring the viable cell counts (c.f.u. mL^?1) and optical density and EPS production was evaluated at the end of the exponential phase and 24 h after it. The analysis of the viable cell counts showed that the medium that most favored bacterial growth was not the one that favored EPS production. The control medium (GYM) was the one that most favored the strain growth, at the maximum specific growth rate of 0.232 h^?1. Differences in bacterial growth when cultured at three different concentrations of molasses were not observed. Production of EPS, in all culture media used, began during the exponential phase and continued during the growth stationary phase. The highest total EPS production, after 24 h of stationary phase, was observed in 6% molasses medium (172.86 g L^?1) and 10% (139.47 g L^?1) and the specific total EPS production was higher in 10% molasses medium (39.03 × 10^?11 g c.f.u.^?1). After the exponential phase, in 2%, 6%, and 10% molasses media, a higher percentage of free exopolysaccharides (EPS) was observed, representing 88.4%, 62.4%, and 64.2% of the total, respectively. A different result was observed in pattern medium, which presented EPS made up of higher percentage of capsular EPS (66.4% of the total). This work is the first study on EPS production by G. polyisoprenivorans strain in GYM medium and in medium utilizing sugarcane molasses as the sole nutrient source and suggests its potential use for EPS production by G. polyisoprenivorans CCT 7137 aiming at application in biotechnological processes.     

Modeling the Impact of Interspecies Competition on Performance of a Microbial Fuel Cell

Previous models of biofilms growing in a microbial fuel cell (MFC) have primarily focused on modeling a single growth mechanism: growth via a conductive biofilm matrix, or growth utilizing diffusible electron shuttles or mediators. In this work, we implement both flavors of models in order to explore the competition for space and nutrients in a MFC biofilm populated by both species types. We find that the optimal growth conditions are for bacteria that utilize conductive EPS provided a minimal energy used to create the EPS matrix. Mediator-utilizing bacteria do have favorable niche regions, most notably close to the anode and where exposed to the bulk inflow, where oxidized mediator is readily available.     

Fermentation performance of an exopolysaccharide-producing strain of<Emphasis Type="Italic"> Lactobacillus delbrueckii</Emphasis> subsp.<Emphasis Type="Italic"> bulgaricus</Emphasis>

The formation of exopolysaccharide (EPS) and extracellular metabolites was studied in a strain of Lactobacillus delbrueckii subsp. bulgaricus (NCFB 2483), grown under batch culture conditions in a semi-defined medium incorporating lactose and casein hydrolysate. Performance parameters were derived from the fermentation data, and kinetic models were applied in order to describe the production of EPS, extracellular metabolites, and biomass produced. Lactose was split intracellularly, with the resultant galactose being exported from the cell, and the glucose being metabolised further to EPS and lactic acid. Production of EPS, lactate, and galactose was closely growth-associated and followed a pattern of primary kinetics. A marginally lower galactose level relative to the modelled levels throughout most of the time course of the fermentation suggests that not all galactose is exported from the cell, and that a low level of flux to other metabolites, such as EPS, might exist.     

Isolation and characterization of <Emphasis Type="Italic">Pseudoalteromonas ruthenica</Emphasis> (SBT033), an EPS-producing biofilm bacterium from the seawater intake point of a tropical power station

Biofilm formation in bacteria is closely linked with production of exopolysaccharides (EPS). This study examined the quantitative variations in EPS production and biofilm-forming ability among bacteria isolated from the seawater intake point of a power station located on the east coast of India. Of the 233 isolates obtained from the intake site, 71 bacterial isolates displayed different colony morphological characteristics. Thirteen isolates that produced wide and thick mucoid colonies were further tested for their ability to attach and form biofilms by microtitre plate assay and confocal microscopy. EPS production among the selected bacterial isolates ranged from 826 to 1838 μg ml⁻¹. Strain SBT033, which produced the maximum amount of EPS also displayed the maximum biofilm-forming ability among the 13 isolates. This strain was selected for further characterization using biochemical and molecular methods. The pale orange-pigmented isolate was a Gram negative, aerobic, short rod-shaped and grew well only in the presence of 2% NaCl. On the basis of phenotypic characteristics the isolate SBT033 is shown to belong to the genus Pseudoalteromonas. Analysis of 16S rRNA of the isolate revealed 99% homology with Pseudoalteromonas ruthenica.     

Arabinose content of extracellular polysaccharide plays a role in cell aggregation of Azospirillum brasilense

Extracellular polysaccharides play an important role in aggregation and surface colonization of plant-associated bacteria. In this work, we report the time course production and monomer composition of the exopolysaccharide (EPS) produced by wild type strain and several mutants of the plant growth promoting rhizobacterium (PGPR) Azospirillum brasilense. In a fructose synthetic medium, wild type strain Sp7 produced a glucose-rich EPS during exponential phase growth and an arabinose-rich EPS during stationary and death phase growth. D-glucose or L-arabinose did not support cell growth as sole carbon sources. However, glucose and arabinose-rich EPSs, when used as carbon source, supported bacterial growth. Cell aggregation of Sp7 correlated with the synthesis of arabinose-rich EPS. exoB (UDP-glucose 4'-epimerase), exoC (phosphomannomutase) and phbC (poly-beta-hydroxyburyrate synthase) mutant strains, under tested conditions, produced arabinose-rich EPS and exhibited highly cell aggregation capability. A mutant defective in LPS production (dTDP 4-rhamnose reductase; rmlD) produced glucose-rich EPS and did not aggregate. These results support that arabinose content of EPS plays an important role in cell aggregation. Cell aggregation appears to be a time course phenomenon that takes place during reduced metabolic cell activity. Thus, aggregation could constitute a protected model of growth that allows survival in a hostile environment. The occurrence of exoC and rmlD was detected in several species of Azospirillum.     

Composition and immunochemical characteristics of exopolysaccharides from the rhizobacterium <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> 1465

Exopolysaccharides (EPS) synthesized by Paenibacillus polymyxa 1465 in the course of batch cultivation were proven to contain neutral and acidic fractions. EPS are heterogeneous polysaccharides, represented by a complex of macromolecules with molecular mass of 7 × 10⁴ to 2 × 10⁶ Da. The acidic component was shown to be predominant in EPS preparations isolated from bacteria cultivated on glucose, which corresponds to a higher viscosity of EPS water solutions. The exoglycans were shown to contain glucose, mannose, galactose, and uronic acids. Polyclonal rabbit antibodies against the isolated P. polymyxa 1465 EPS preparations were used in a comparative immunodiffusion analysis of a number of P. polymyxa strains.     

Microbial structure and community of RBC biofilm removing nitrate and phosphorus from domestic wastewater

Using a rotating biological contactor modified with a sequencing bath reactor system (SBRBC) designed and operated to remove phosphate and nitrogen [58], the microbial community structure of the biofilm from the SBRBC system was characterized based on the extracellular polymeric substance (EPS) constituents, electron microscopy, and molecular techniques. Protein and carbohydrate were identified as the major EPS constituents at three different biofilm thicknesses, where the amount of EPS and bacterial cell number were highest in the initial thickness of 0-100 mum. However, the percent of carbohydrate in the total amount of EPS decreased by about 11.23%, whereas the percent of protein increased by about 11.15% as the biofilm grew. Thus, an abundant quantity of EPS and cell mass, as well as a specific quality of EPS were apparently needed to attach to the substratum in the first step of the biofilm growth. A FISH analysis revealed that the dominant phylogenetic group was beta- and gamma-Proteobacteria, where a significant subclass of Proteobacteria for removing phosphate and/or nitrate was found within a biofilm thickness of 0-250 mum. In addition, 16S rDNA clone libraries revealed that Klebsiella sp. and Citrobacter sp. were most dominant within the initial biofilm thickness of 0-250 mum, whereas sulfur-oxidizing bacteria, such as Beggiatoa sp. and Thiothrix sp., were detected in a biofilm thickness over 250 mum. The results of the bacterial community structure analysis using molecular techniques agreed with the results of the morphological structure based on scanning electron microscopy. Therefore, the overall results indicated that coliform bacteria participated in the nitrate and phosphorus removal when using the SBRBC system. Moreover, the structure of the biofilm was also found to be related to the EPS constituents, as well as the nitrogen and phosphate removal efficiency. Consequently, since this is the first identification of the bacterial community and structure of the biofilm from an RBC simultaneously removing nitrogen and phosphate from domestic wastewater, and it is hoped that the present results may provide a foundation for understanding nitrate and phosphate.     

Production and characterization of the exopolysaccharide produced by <Emphasis Type="Italic">Paenibacillus jamilae</Emphasis> grown on olive mill-waste waters

Paenibacillus jamilae, a strain isolated from compost prepared with olive-mill wastewaters, produced an extracellular polysaccharide (EPS) when it was grown in a culture containing olive-mill waste waters (OMWW) as sole carbon and energy sources. Maximal EPS production in 100 mL batch-culture experiments (5.1 g L⁻¹) was reached with a concentration of 80% of OMWW as fermentation substrate (v/v). Although an inhibitory effect was observed on growth and EPS production when OMWW concentration was increased, an appreciable amount of EPS (2.7 g L⁻¹) was produced with undiluted OMWW. Sepharose CL-2B chromatography showed that the EPS presented two fractions, EPS I (>2000 kDa) and EPS II (500 kDa). Both fractions were characterized by GC-MS as two different acidic heteropolysaccharides containing glucose, galactose and mannose as the major components. The performed study made evident the possibility of using OMWW as substrate for the production of EPS by P. jamilae with a satisfactory yield.