EPS Yield Research Articles

Preparation and Antioxidant Activity In Vitro of Fermented Tremella fuciformis Extracellular Polysaccharides

This study was aimed at increasing the capacity of fermented Tremella fuciformis extracellular polysaccharides (TEPS) for possible functional food applications. Thus, strain varieties, fermentation parameters and purification conditions, and the in vitro antioxidant activities of purified EPS fractions were investigated. An EPS high-yield strain Tf526 was selected, and the effects of seven independent fermentation factors (time, temperature, initial pH, inoculum size, shaking speed, carbon, and nitrogen source) on the EPS yield were evaluated. By single factor optimization test, yeast extract and glucose were chosen as nitrogen sources and carbon sources, respectively, and with initial pH of 6.0, inoculum size of 8%, shaking speed of 150 rpm, and culture at 25 °C for 72 h, the optimal yield of TEPS reached 0.76 ± 0.03 mg/mL. Additionally, A-722MP resin showed the most efficient decoloration ratio compared to six other tested resins. Furthermore, optimal decoloration parameters of A-722MP resin were obtained as follows: decoloration time of 2 h, resins dosage of 2 g, and temperature of 30 °C. Decoloration ratio, deproteinization ratio, and polysaccharide retention ratio were 62.14 ± 2.3%, 81.21 ± 2.13%, and 73.42 ± 1.96%, respectively. Furthermore, the crude TEPS was extracted and four polysaccharide fractions were isolated and purified as Tf1-a, Tf1-b, Tf2, and Tf3 by the DEAE-Sepharose FF column and the Sephasryl S100 column. In general, the antioxidant activities of the Lf1-a and Lf1-b were lower compared with Vc at the concentration of 0.1 to 3 mg/mL, but the FRAP assay, DPPH scavenging activity, and hydroxyl radical scavenging activity analysis still revealed that Tf1-a and Tf1-b possess significant antioxidant activities in vitro. At the concentration of 3 mg/mL, the reducing power of Lf1-a and Lf1-b reached 0.86 and 0.70, the maximum DPPH radical were 54.23 ± 1.68% and 61.62 ± 2.73%, and the maximum hydroxyl radicals scavenging rates were 58.76 ± 2.58% and 45.81 ± 1.79%, respectively. Moreover, there were significant correlations (r > 0.8) among the selected concentrations and antioxidant activities of TEPS major fractions Tf1-a and Tf1-b. Therefore, it is expected that Tf1-a and Tf1-b polysaccharide fractions from fermented TEPS may serve as active ingredients in functional foods.

Understanding the N-acylated homoserine lactones(AHLs)-based quorum sensing for the stability of aerobic granular sludge in the aspect of substrate hydrolysis enhancement

Efficient substrate metabolism is the premise for stable operation of aerobic granular sludge and can be regulated by quorum sensing (QS). In this study, starch and acetate were selected to represent complex and simple substrates to provide comparable amount of metabolic energy for granules cultivation. Starch-fed granules were larger in size and contained higher EPS content than acetate-fed granules, though both granules exhibited similar substrate-degradation rates during sequencing batch reactor (SBR) cycle. Three N-acylhomoserine lactones (AHLs), including C8-HSL, 3OHC8-HSL and 3OHC12-HSL, were detected as dominant autoinducers in granules. They accumulated more in starch-fed granules than acetate-fed granules. The batch experiments were implemented to investigate QS regulation for granular stability in terms of substrate hydrolysis and transformation. The addition of three AHLs increased the activity of α-amylase, the main starch hydrolase, 4–6 times, significantly (p < 0.01) higher than the control treatment without AHLs amendment. While activity of dehydrogenase, the main simple substrate degradation enzyme, was increased only 1–2 times. Higher enzyme activity, especially α-amylase, significantly (p < 0.05) promoted the substrate-degradation rate (65 % than control group) and EPS yield in starch-fed system. Overall, QS can facilitate complex substrate uptake via hydrolysis enhancement and EPS secretion, which together promote sludge granulation and stability.

Изменение физико-химических и культуральных свойств бактерий Azospirillum baldaniorum Sp245 под влиянием некоторых синтетических кумаринов

Due to the small number of studies on the role of coumarins in associative symbiotic relationships, some aspects of the influence of synthetic coumarins on the physicochemical and cultural properties of Azospirillum baldaniorum Sp245 were studied for the first time. To reveal the role of hydroxylation in position 7 of the fused aromatic ring – 1-(2-oxo-2H-chromen-3-yl)butan-1,3-dione, comparative studies of the effect of the original and hydroxylated coumarins on the culture of a model strain of azospirilla were carried out. The survival of bacteria after the addition of coumarins was studied by counting CFU on an agar medium. The biofilm formation activity of the culture was assessed using crystal violet. The change in the surface of bacteria under the action of coumarins was studied by the electrical polarizability of bacterial cells on an ELUS electrooptical analyzer (EloSystemGbR, Germany). The yield and monosaccharide composition of extracellular glycopolymers were studied using gas-liquid chromatography.For the first time, an increase in the yield of EPS of bacteria during growth in the presence of 1-(7-hydroxy-2-oxo-2Hchromen-3-yl)butan-1,3-dione by 1.2 and 1.7 times for concentrations of 50 and 100 μM respectively was observed. It has been established for the first time that the hydroxylated substance has a higher antibacterial activity compared to the unsubstituted one. A decrease in the number of viable cells in planktonic culture and inhibition of biofilm growth were revealed. It has been shown by electro-optical analysis that the presence of coumarins in the cultivation medium in all concentrations studied leads to a change in the electrical polarizability of A. baldaniorum Sp245 cells. The use of electrooptical analysis of cell suspensions using monospecific antibodies obtained against lipopolysaccharides of this strain made it possible to reveal the absence of changes in carbohydrate antigenic determinants on the surface of bacterial cells. This is consistent with the data of the analysis of the composition of extracellular polysaccharides by GLC, during which no differences were found in the qualitative composition and ratio of monosaccharides. An increase in the yield of bacterial EPS during growth in the presence of 1-(7-hydroxy-2-oxo-2H-chromen-3-yl) butan-1,3-dione by 1.2 and 1.7 times for concentrations of 50 and 100 μM was shown. The results obtained allow us to consider the changes that have occurred as features of the adaptation of bacteria to the associative conditions of existence.

Homologous Over-Expression of Chain Length Determination Protein EpsC Increases the Molecular Weight of Exopolysaccharide in Streptococcus thermophilus 05-34.

In Streptococcus thermophilus, EpsC is a polysaccharide co-polymerase which is involved in determining the chain length of EPS synthesized by the Wzx/Wzy-dependent pathway. Our previous study found that there was a positive correlation between transcription level of epsC and molecular weight of EPS in S. thermophilus 05-34. To further investigate the effects of EpsC on EPS biosynthesis, this gene was over-expressed in S. thermophilus 05-34 in this study. Reverse transcription qPCR and Western blotting confirmed the successful transcription and translation of epsC in 05-34, respectively. The yield of EPS was not affected by the over-expression of EpsC. Gas chromatography-mass spectrometry (GC-MS) showed that the monosaccharide composition was still composed of galactose and glucose in a molar ratio of 1.0:0.8, whereas high performance gel permeation chromatography (HPGPC) indicated that the molecular weight of EPS was increased from 4.62 × 105 Da to 9.17 × 105 Da by the over-expression of EpsC. In addition, S. thermophilus 05epsC which could produce higher molecular weight EPS improved the viscoelasticity and water-holding capacity of yogurt, but significantly reduced the level of syneresis in yogurt. In summary, these results indicated that homologous over-expression of EpsC in S. thermophilus could increase the molecular weight of EPS and improve the microrheological or physical properties of yogurt.

Assessment of the Impact of Temperature on Biofilm Composition with a Laboratory Heat Exchanger Module.

Temperature change over the length of heat exchangers might be an important factor affecting biofouling. This research aimed at assessing the impact of temperature on biofilm accumulation and composition with respect to bacterial community and extracellular polymeric substances. Two identical laboratory-scale plate heat exchanger modules were developed and tested. Tap water supplemented with nutrients was fed to the two modules to enhance biofilm formation. One “reference” module was kept at 20.0 ± 1.4 °C and one “heated” module was operated with a counter-flow hot water stream resulting in a bulk water gradient from 20 to 27 °C. Biofilms were grown during 40 days, sampled, and characterized using 16S rRNA gene amplicon sequencing, EPS extraction, FTIR, protein and polysaccharide quantifications. The experiments were performed in consecutive triplicate. Monitoring of heat transfer resistance in the heated module displayed a replicable biofilm growth profile. The module was shown suitable to study the impact of temperature on biofouling formation. Biofilm analyses revealed: (i) comparable amounts of biofilms and EPS yield in the reference and heated modules, (ii) a significantly different protein to polysaccharide ratio in the EPS of the reference (5.4 ± 1.0%) and heated modules (7.8 ± 2.1%), caused by a relatively lower extracellular sugar production at elevated temperatures, and (iii) a strong shift in bacterial community composition with increasing temperature. The outcomes of the study, therefore, suggest that heat induces a change in biofilm bacterial community members and EPS composition, which should be taken into consideration when investigating heat exchanger biofouling and cleaning strategies. Research potential and optimization of the heat exchanger modules are discussed.

Performance and working mechanism of a novel anaerobic self-flotation reactor for treating wastewater with high suspended solids.

The new design of internal flotation components and the use of biogas were employed to develop a novel anaerobic self-flotation (ASF) reactor. Compared with the upflow anaerobic sludge blanket (UASB) reactor, the removal efficiencies of total chemical oxygen demand (COD) and suspended solids (SS) of the ASF reactor were higher than 90% under high SS concentration and high volumetric organic loading rate (OLR). The biogas flotation, sludge bed retention, and effluent washout accounted for 60%, 30%, and 10% of SS mass, respectively, proving that the biogas flotation was the main mechanism of SS removal in the ASF reactor. Extracellular polymer substance, mainly consisting of polysaccharide (PS) and protein (PN), was found to promote the SS removal by biogas flotation via the scum formation at the ratio of 294.12 g-VS/g-PS and 103.09 g-VS/g-PN. The EPS yield was determined as 2.3 ± 0.6 g-PS/g-COD and 11.5 ± 2.6 g-PN/g-COD at the OLR of 60 kg/(m3 day). The biogas production was revealed to enhance the SS removal by providing flotation driving force and by decreasing the scum density. A model was established to describe the quantitative relationship between flotation scum and OLR. This work would shed light on the high SS wastewater treatment challenge of high-rate anaerobic processes by using biogas flotation.

PRODUCTION AND CHARACTERIZATION OF EXOPOLYSACCHARIDE FROM MARINE MODERATELY HALOPHILIC BACTERIUM HALOMONAS SMYRNENSIS SVD III

Objective: To study 1) Optimization of nutritional and environmental parameters to enhance the yield of EPS by Halomonas smyrnensis SVD III isolated from seawater, West Coast of Maharashtra, India and 2) Purification and characterization of the EPS produced.Methods: The isolate was grown in Sehgal and Gibbons (SG) medium broth supplemented with 3% glucose, at 37 °C, 120 rpm for 7 d. Optimization of different parameters was carried out with one factor at a time approach. EPS was isolated from cell-free supernatant of the culture broth by centrifugation and precipitation using chilled ethanol, after removal of proteins by trichloroacetic acid (TCA) treatment. Characterization of the purified EPS was carried out with respect to fourier-transform infrared (FTIR) spectrum, 1H nuclear magnetic resonance (NMR) spectrum and mass spectrometry (MS) analysis.Results: Two-fold increase in the yield of EPS (23 g/l) by the selected isolate was obtained by using culture conditions as 10% inoculum size having cell density of 107 cells/ml, pH 6, incubation temperature 45 °C, 3% carbohydrate, 0.5% yeast extract as nitrogen source, 20% salt concentration and 7 d of incubation period. Characterization of the purified EPS suggested the presence of dominated glycosidic linkages and heptasaccharide nature of the molecule. As the present strain is halophilic, 20% NaCl was found to be optimum.Conclusion: Optimization studies resulted in two-fold increase in the yield of EPS which is of heptasaccharide nature.

New advances in exopolysaccharides production of Streptococcus thermophilus.

Streptococcus thermophilus is the most important thermophilic dairy starter, and is widely used in the dairy industry. Streptococcus thermophilus exopolysaccharides received wide attention over recent decades, because they can improve the properties of the dairy product and confer beneficial health effects. The understanding of the regulatory and biosynthetic mechanisms of EPS will improve the EPS biosynthesis, increase the productivity of EPSs, and develop EPSs with desirable properties. The structure of EPSs is the focus of this study. Revealing the structure-function relationship can lead to increase the knowledge base and from there to increased research of EPS. The EPS yield is a key limiting factor in the research and utilization of EPS. In the present review, biosynthetic pathways and genetics of S. thermophilus EPSs were described and reviewed. At the same time, functional properties and applications of EPS, and strategies for enhancement of EPS production are discussed.

0502 Effect of incubation temperature on yield and molar mass of EPS during fermentation of milk by Streptococcus thermophilus DGCC 7785 and the impact on the rheological properties of acid milk gels

0502 Effect of incubation temperature on yield and molar mass of EPS during fermentation of milk by Streptococcus thermophilus DGCC 7785 and the impact on the rheological properties of acid milk gels

DIVERSITY ASSESSMENT AND EPS PRODUCTION POTENTIAL OF CULTIVABLE BACTERIA FROM THE SAMPLES OF COASTAL SITE OF ALANG

In this study diversity of cultivable bacteria and their exopolysaccharide production potential were investigated from the water and sediment samples of coastal site of ALang. The studied ten samples represented diversity in pH, conductivity, salinity and TDS, in the range of 7.25 to 8.92, 9.4 to 76.8 mS, 3570 to 35100 mg/L and 15120 to 53100 mg/L respectively. Total 141 bacteria were isolated from the collected ten samples, from which 105 were gram positive and 36 were gram negative in nature. Amongst the isolates 33% were able to produce variety of pigments. The diversity indices, including Shannon-Wiener index (H’), Richness, and Evenness Indices based on the metabolic characteristics of the organisms were calculated. The isolates were characterized morphologically and biochemically. Identification of 41 isolates was confirmed by 16S rRNA gene sequencing. They represented 22 genera and various species of these genera. The bacterial isolates were able to grow in the range of 3-25% NaCl concentrations. When 141 cultures were grown in liquid medium, the viscosity of the medium ranged between 9.20x10-6 to 2.09x10-4 m.pa.s-1. The EPS yield in terms of dry weight also ranged from 0.76 to 10.7g/L.

인삼 근계로부터 다당 생성세균의 분리 및 특성

인삼근계(근권, 근면, 근내부) 내 EPS 생성세균의 밀도를 측정한 결과, 근권토양 내에는 <TEX>$2.4{\times}10^6$</TEX> CFU/g, 근면에는 <TEX>$9.1{\times}10^6$</TEX> CFU/g, 그리고 근내부에는 <TEX>$2.0{\times}10^4$</TEX> CFU/g로 확인되어 다수의 EPS 생성세균이 분포하고 있음이 확인되었다. 인삼 근계로부터 EPS 생성 우수 균주 24균주를 순수분리하고 계통학적 특성을 확인한 결과, 근권(RS)으로부터 분리된 EPS 생성세균은 Arthrobacter 속 6균주, 그리고 Rhizobium 속 1균주로 나타났다. 근면(RP)으로 부터 분리된 EPS 생성세균은 Arthrobacter 속 6균주, Rhodococcus 속 1균주, Pseudomonas 속 1균주로 나타났다. 근내부(IR)에서 분리된 EPS 생성세균은 Rhizobium 속 6균주, Bacillus 속 1균주 그리고 Rhodococcus 속 1균주, Pseudomonas 속 1균주로 나타났다. 근권과 근면에서 분리된 EPS 세균 중 Arthrobacter 속에 속하는 균주는 가장 특징적인 세균으로 밝혀졌으며, Rhizobium 속은 근내부에서 분리된 가장 특징적인 EPS 생성세균으로 나타났다. EPS 생성 우수균주 Rhizobium sp. 1NP2 (KACC 17637)는 10 g/L 그리고 Arthrobacter sp. 5MP1 (KACC 17636)는 4.9 g/L의 다당을 생성하였으며, 당단백질의 구성당 성분을 확인한 결과, galactose, glucose, mannose를 구성하고 있었으며, glucosamine의 아미노당이 나타났다. 특히, glucose는 72.7-84.9%로 주요 구성당임이 확인되었다. EPS producing bacteria were enumerated in ginseng root system (rhizosphere soil, rhizoplane, inside of root). EPS producing bacterial density of rhizosphere soil, rhizoplane and inside of root were distributed <TEX>$9.0{\times}10^6$</TEX> CFU/g, <TEX>$7.0{\times}10^6$</TEX> CFU/g, and <TEX>$1.4{\times}10^3$</TEX> CFU/g, respectively. Phylogenetic analysis of the 24 EPS producing isolates based on the 16S rRNA gene sequences, EPS producing isolates from rhizosphere soil (RS) belong to genus Arthrobacter (6 strains) and Rhizobium (1 strain). EPS producing bacteria from rhizoplane (RP) were Arthrobacter (6 strains), Rhodococcus (1 strain) and Pseudomonas (1 strain). EPS producing bacteria from inside of root (IR) were categorized into Rhzobium (6 strains), Bacillus (1 strain), Rhodococcus (1 strain), and Pseudomonas (1 strain). Phylogenetic analysis indicated that Arthrobacter may be a member of representative EPS producing bacteria from ginseng rhizosphere soil and rhizoplane, and Rhizobium is typical EPS producing isolates from inside of ginseng root. The yield of EPS was 10.0 and 4.9 g/L by Rhizobium sp. 1NP2 (KACC 17637) and Arthrobacter sp. 5MP1 (KACC 17636). The purified EPS were analyzed by Bio-LC and glucose, galactose, mannose and glucosamine were detected. The major EPS sugar of these strains was glucose (72.7-84.9%).

The use of mushroom hydrolysate from waste bag-log as the nitrogen source to mycelium biomass and exopolysaccharide production in Pleurotus eryngii cultivation

In this study, Pleurotus eryngii head part waste in a bag-log was collected and processed to yield mushroom hydrolysate powder (MHP). To test its stimulatory effect, a P. eryngii submerged culture was conducted. The prepared MHP (1.6%) and glucose (2%) were found to be the best sources for both mycelial biomass and EPS productions. A specific growth rate of 0.68day−1, and a specific EPS yield of 9.55g/g-dry-cell-weight were obtained under these conditions. It was also found that MHP gave higher mycelial biomass growth rate (2.05g/L/day), and EPS yield (92mg/L) than those of yeast extract. In 5-L fermentation with MHP as nitrogen source, high mycelial biomass growth rate (2.44g/L/day) and EPS yield (312mg/L) were obtained. In the solid state culture, P. eryngii hyphae growth rate in MHP agar (6.43±0.21mm/day) was better than that in PDA (4.57±0.23mm/day). By using PCA and cluster analysis, the major amino acids of MHP displayed a close similarity to those of P. eryngii mycelial hydrolysate powder (PEMHP).

Growth dependence of conjugation explains limited plasmid invasion in biofilms: an individual‐based modelling study

Plasmid invasion in biofilms is often surprisingly limited in spite of the close contact of cells in a biofilm. We hypothesized that this poor plasmid spread into deeper biofilm layers is caused by a dependence of conjugation on the growth rate (relative to the maximum growth rate) of the donor. By extending an individual-based model of microbial growth and interactions to include the dynamics of plasmid carriage and transfer by individual cells, we were able to conduct in silico tests of this and other hypotheses on the dynamics of conjugal plasmid transfer in biofilms. For a generic model plasmid, we find that invasion of a resident biofilm is indeed limited when plasmid transfer depends on growth, but not so in the absence of growth dependence. Using sensitivity analysis we also find that parameters related to timing (i.e. a lag before the transconjugant can transfer, transfer proficiency and scan speed) and spatial reach (EPS yield, conjugal pilus length) are more important for successful plasmid invasion than the recipients' growth rate or the probability of segregational loss. While this study identifies one factor that can limit plasmid invasion in biofilms, the new individual-based framework introduced in this work is a powerful tool that enables one to test additional hypotheses on the spread and role of plasmids in microbial biofilms.