Extracellular β-agarase LSL-1 producing neoagarobiose from a newly isolated agar-liquefying soil bacterium, Acinetobacter sp., AG LSL-1

Summary An agar-liquefying Acinetobacter species capable of utilizing agar as sole source of carbon and energy was isolated from soil samples and the culture conditions were standardized for the maximal production of extracellular agarase. The bacterium was capable of liquefying an agar-plate within 3 days of incubation and produced extracellular agarase within a short period of time (16–18 h) when grown in defined mineral salts medium. Bacterium grew in the pH range 4.0–9.0, optimal at pH 7.0; temperature 25–40 °C and optimal at 37 °C. The agarase secreted by the Acinetobacter strain was inducible by agar and not repressed by other simple sugars when supplemented along with agar in the medium. The bacterium did not require NaCl for growth or production of agarase. The bacterium did not utilize other polysaccharides like κ-carrageenan, alginate, cellulose, and CMC. The activity staining of partially purified agarase preparations after native-PAGE and SDS PAGE revealed the presence of a single zone of clearance corresponding to the molecular weight 100 kDa, suggesting that it is a monomer. Neoagarobiose was the end product of agarose hydrolysis by this enzyme. The agarase was an endo-type glycosidase and belongs to Group-III β-agarase family.     

Optimization of Culture Conditions for the Production of Extracellular Agarases from Newly Isolated Pseudomonas Aeruginosa AG LSL-11

Summary An agar-degrading bacterium capable of utilizing agar as sole source of carbon and energy was isolated from sea water by enrichment culture technique. The bacterium was identified as Pseudomonas aeruginosa and the culture conditions were standardized for the maximal production of extracellular agarases. The bacterium grew in the pH range 5.0–11.0, optimal between pH 7.0 and 8.0; temperature between 25 °C and 37 °C, optimal at 30 °C and sodium chloride concentration 0–8% and optimal at 2% respectively. The agarases secreted by Pseudomonas aeruginosa AG LSL-11 were inducible by agar and not by any other simple sugars tested. Maximal agarase production was observed at pH 8.0, and temperature 30 °C. The bacterium had no requirement for NaCl for both growth and production of agarases. The bacterium did not utilize other polysaccharides like ĸ-carrageenan, alginate, cellulose and CMC. The activity staining of partially purified agarase preparation after native-PAGE revealed the presence of three different agarases, agarase LSL-11a, LSL-11b and LSL-11c, whose molecular weights were estimated to be 76, 64 and 46 kDa respectively.     

Production of agarase from a novel <Emphasis Type="Italic">Micrococcus</Emphasis> sp. GNUM-08124 strain isolated from the East Sea of Korea

The agar degrading bacterial strain GNUM-08124 was isolated from Enteromorpha compressa collected in the East Sea of Korea by using a selective artificial sea water (ASW) agar plate containing agar as the sole carbon source. GNUM-08124 grows to produce a circular, smooth, yellow-colored, and raised colony. Its ability to hydrolyze agar was confirmed by staining the ASW agar plate with Lugol’s solution. In liquid culture, the cell density (A₆₀₀) increased exponentially and reached a maximum level on the third day of cultivation. The specific agarase activity also increased in proportion to the cell density and reached maximum agarolytic activity on the third day. The 16S rRNA sequence of GNUM-08124 showed a close relationship to Micrococcus luteus (99.65%) and Micrococcus endophyticus (99.15%), which led us to assign it to the genus Micrococcus. Physiological studies indicated that optimal growth conditions were between 30 and 40°C, pH 4 and 7, using media containing between 5 and 10% NaCl (w/v), respectively. The GNUM-08124 strain was a grampositive, urease-positive, and catalase-positive bacterium. It could not hydrolyze gelatin, cellulose, xylan, or starch, but fermented a broader range of substrates, including Dglucose, D-galactose, D-fructose, D-lactose, D-trehalose, D-mannitol, D-melibiose, D-raffinose, D-xylose, methyl-α-D-glucopyranoside, N-acetyl-glucosamine, and xylitol, than those fermented by M. luteus or M. endophyticus, suggesting GNUM-08124 is a novel agar hydrolyzing microorganism belonging to Genus Micrococcus. Micrococcus sp. GNUM-08124 showed the highest agarase activity when it was cultured in ASW-YP medium supplemented with 0.4% glucose, but demonstrated lower activity in rich media (LB or TSB), in spite of superior cell growth, implying that agarase production is tightly regulated in an agar-dependent manner and repressed in rich conditions.     

Isolation of Asticcacaulis sp. SA7, a Novel Agar-Degrading Alphaproteobacterium

An agar-degrading bacterium, strain SA7, was isolated from plant roots cultivated in soil. Analysis of the 16S rDNA sequence showed that strain SA7 is affiliated with the genus Asticcacaulis. Strain SA7 produced extracellular agarase, and grew utilizing agar in the culture medium as sole carbon source. Zymogram analysis showed that strain SA7 extracellularly secreted single agarase protein (about 70 kDa).     

Isolation of Asticcacaulis sp. SA7, a novel agar-degrading alphaproteobacterium

An agar-degrading bacterium, strain SA7, was isolated from plant roots cultivated in soil. Analysis of the 16S rDNA sequence showed that strain SA7 is affiliated with the genus Asticcacaulis. Strain SA7 produced extracellular agarase, and grew utilizing agar in the culture medium as sole carbon source. Zymogram analysis showed that strain SA7 extracellularly secreted single agarase protein (about 70 kDa).     

An organic solvent-tolerant protease from Pseudomonas aeruginosa strain K: Nutritional factors affecting protease production

An organic solvent-tolerant bacterium producing an organic solvent-stable protease was isolated from soil and identified as Pseudomonas aeruginosa strain K. Nutritional requirements for optimized protease production by this strain were investigated. Maximum protease activity was achieved with sorbitol as the sole carbon source, followed by starch and lactose at pH 7.0 and 37 °C. Dextrose, sucrose and glycerol greatly reduced the protease production. The best organic nitrogen source was casamino acid. Tryptone, soytone and yeast extract supported protease production while corn steep liquor and beef extract inhibited the protease activity. Significant protease production was observed with sodium nitrate as a sole nitrogen source however, ammonium nitrate completely inhibit it. More than 62% drop in production occurred in the presence of amino acids. Addition of metal ions such as K⁺, Mg²⁺ and Ca²⁺ maximized the enzyme production.     

Determination of the Chemical Form of Fluorine in Tea Infusions by 19F-NMR

A novel marine ice-nucleating bacterium, KUIN-5, was isolated from a marine algae, Monostroma latissum. Strain KUIN-5 was identified as a Pseudomonas sp. from its characteristics and taxonomies; the optimum temperature and pH for its growth were 25°C and 6.0, respectively. When strain KUIN-5 was aerobically cultured in Carlucci-Pramer medium (pH 6.0) for 50 h at 25°C, the highest ice-nucleating activity of the cells among the media for marine bacteria was obtained, and the ice-nucleating temperature, T₅₀, was indicated to be ? 3.2°C. Also, the optimum concentration of NaCl for the growth in this medium, which was prepared with distilled water instead of seawater, was 2.0% (w/v) and then the ice-nucleating activity was inversely proportional to the NaCl concentration. Moreover, when strain KUIN-5 was cultured in Davis medium under optimum conditions, it produced insoluble polysaccharide (IPS) in the culture. The maximum amount of IPS production by strain KUIN-5 was 84.5 mg/ml of medium under optimum conditions. Therefore, this IPS was isolated and could be identified as cellulose, based on TLC or HPLC of the acid hydrolysate, and GC-MS of the acetylated polyalcohol prepared by periodate oxidation and Smith degradation of this polysaccharide. This is the first report of cellulose production by a marine ice-nucleating bacterium.     

Isolation and Characterization of Agar-degrading Paenibacillus spp. Associated with the Rhizosphere of Spinach

Agar-degrading bacteria in spinach plant roots cultivated in five soils were screened, and four strains of Paenibacillus sp. were isolated from roots cultivated in three soils. The agar-degrading bacteria accounted for 1.3% to 2.5% of the total bacteria on the roots. In contrast, no agar-degrading colony was detected in any soil (non-rhizosphere soil samples) by the plate dilution method, and thus these agar-degrading bacteria may specifically inhabit plant roots. All isolates produced extracellular agarase, and could grow using agar in the culture medium as the sole carbon source. Zymogram analyses of agarase showed that all four isolates extracellularly secreted multiple agarases (75-160 kDa). In addition, the isolates degraded not only agar but also various plant polysaccharides, i.e., cellulose, pectin, starch, and xylan.     

1株产琼脂酶细菌的分离鉴定及其胞外酶活性测定

从四川省成都市青城山采集土壤,以琼脂作为唯一碳源,筛选到产琼脂酶细菌CMCK136;通过形态观察、生化鉴定、16S r DNA测序及序列分析鉴定其种属;随后测定了菌株CMCK136的胞外酶活性。菌株CMCK136被鉴定为芽胞杆菌属细菌,命名为Bacillus sp.CMCK136。菌株CMCK136的胞外琼脂酶的最适酸碱度为p H 7.0,最适温度为35℃。菌株CMCK136是产琼脂酶细菌家族的新成员,该菌株的发现进一步提示芽胞杆菌属很可能蕴含有尚待开发的琼脂酶资源。     

Isolation and characterization of agar-degrading Paenibacillus spp. associated with the rhizosphere of spinach

Agar-degrading bacteria in spinach plant roots cultivated in five soils were screened, and four strains of Paenibacillus sp. were isolated from roots cultivated in three soils. The agar-degrading bacteria accounted for 1.3% to 2.5% of the total bacteria on the roots. In contrast, no agar-degrading colony was detected in any soil (non-rhizosphere soil samples) by the plate dilution method, and thus these agar-degrading bacteria may specifically inhabit plant roots. All isolates produced extracellular agarase, and could grow using agar in the culture medium as the sole carbon source. Zymogram analyses of agarase showed that all four isolates extracellularly secreted multiple agarases (75-160 kDa). In addition, the isolates degraded not only agar but also various plant polysaccharides, i.e., cellulose, pectin, starch, and xylan.     

Biodegradation of Crude Oil by an Arctic Psychrotrophic Bacterium <Emphasis Type="Italic">Pseudoalteromomas</Emphasis> sp. P29

A psychrotrophic petroleum-degrading bacterium Pseudoalteromonas sp. P29 was isolated from marine sediment, which was collected during 2nd Chinese Arctic Scientific Expedition. The phenotypic character and biodegradation efficiency on mixed oil or vacuum oil were tested at low temperature. The strain Pseudoalteromonas sp. P29 grew in a range of temperature from 5 to 35°C and the optimum temperature was 25°C. Gas chromatography analysis indicated that the strain might preferentially metabolize shorter-chain alkanes. The biodegradation efficiency were nearly 90 and 80%, respectively, after incubation at 5°C for 28 days in the mineral medium supplement with mixed oil or vacuum oil as the sole carbon and energy source. The results showed a possible exploitation of the strain in future biotechnological processes especially in cold contaminated environments.     

A serum-free medium for colony growth and hyaluronic acid production by Streptococcus zooepidemicus NJUST01

A hyaluronic acid (HA)-producing strain, Streptococcus zooepidemicus NJUST01, can grow in a serum-free agar medium, with starch as exclusive carbon source, but not glucose, sucrose, dextrine, xylose, or lactose. In this starch medium, the strain NJUST01 reproduced successively at 37°C for 60 generations, with no obvious variation on morphology and physiology, but colonies of the strain after 60th generation could not produce a clear hemolytic zone on sheep blood agar plates. Hyaluronic acid production by the strain NJUST01 was analyzed relative to the starch medium. Employing a multifactor cross experiment, an optimum medium revealed for hyaluronic acid production was composed of 5% starch, 0.3% glucose, 0.5% peptone, 0.15% MgSO₄, and 2.0% K₂HPO₄. The amount of HA 6.7 g/l was obtained in batch fermentation on a rotary shaker at 37°C, 220 rpm for 36 h.     

Optimization of medium composition and culture conditions for agarase production by Agarivorans albus YKW-34

Effect of medium composition and culture conditions on agarase production by Agarivorans albus YKW-34 was investigated in shake flasks. The most suitable carbon source, nitrogen source, and culture temperature were agar, yeast extract, and 25 °C, respectively, for agarase production by one-factor-at-a-time design. The nutritional components of the medium and culture conditions were analyzed by Plackett–Burman design. Among the nine factors studied, agar, yeast extract, and initial pH had significant effects on agarase production (p < 0.05). The optimum levels of these key variables were further determined using a central composite design. The highest agarase production was obtained in the medium consisting of 0.23% agar and 0.27% yeast extract at initial pH 7.81. The whole optimization strategy enhanced the agarase production from 0.23 U/ml to 0.87 U/ml. The economic medium composition and culture condition as well as the dominant occupation of agarase with high activity in culture fluid enlighten the potential application of A. albus YKW-34 for the production of agarase.     

Production and characterization of a novel extracellular metalloproteinase by a newly isolated moderate halophile, <Emphasis Type="Italic">Halobacillus</Emphasis> sp. LY6

A moderately halophilic bacterium LY6 with high proteolytic activity was isolated. Biochemical and physiological characterization, along with 16S rDNA sequence analysis placed the isolate in the genus Halobacillus. The salinity of the culture medium strongly influenced the proteinase production of LY6. Maximum enzyme production was observed in the medium containing 5% Na₂SO₄ or 10% NaCl. Proteinase production was synchronized with bacterial growth and reached a maximum level during the mid-stationary phase. Enzyme purification was carried out by a simple approach including a combination of ammonium sulfate precipitation and Sephacryl S-100 gel filtration chromatography. SDS-PAGE and gelatin zymography analysis revealed it was a monomer with high molecular weight of 69 kDa. Optimal proteinase activity was obtained at pH 10.0, 40°C, and 10% NaCl. It was high active over broad temperature (30–80°C), pH (6.0–12.0), and NaCl concentration (0–25%) ranges, indicating its thermostable, alkali-stable, and halotolerant nature. Moreover, the enzyme activity was markedly enhanced by Ca²⁺ and Cu²⁺, but strongly inhibited by EDTA, PAO, and DEPC, indicating that it probably was a metalloproteinase with cysteine and histidine residues located in its active site.     

1,3‐Propanediol production from glycerol by a newly isolated Trichococcus strain

A coccal bacterium (strain ES5) was isolated from methanogenic bioreactor sludge with glycerol as the sole energy and carbon source. Strain ES5 fermented glycerol to 1,3‐propanediol as main product, and lactate, acetate and formate as minor products. The strain was phylogenetically closely related to Trichococcus flocculiformis; the rRNA gene sequence similarity was 99%. However, strain ES5 does not show the typical growth in chains of T. flocculiformis. Moreover, T. flocculiformis does not ferment glycerol. Strain ES5 used a variety of sugars for growth. With these substrates, lactate, acetate and formate were the main products, while 1,3‐propanediol was not formed. The optimum growth temperature of strain ES5 ranges from 30–37°C, but like several other Trichoccoccus strains, strain ES5 is able to grow at low temperature (< 10°C). Therefore, strain ES5 may be an appropriate catalyst for the biotechnological production of 1,3‐propanediol from glycerol at low ambient temperature.     

A moderately thermophilic and mercaptan-degrading Bacillus licheniformis strain CAN55 isolated from gas-washing wastewaters of the phosphate industry,Tunisia

A moderately thermophilic bacterium, designated strain CAN55, was isolated from the gas-washing wastewaters of the phosphate industry, located in Sfax, Tunisia, after enrichment on 1-decanethiol, a mercaptan used as the sole carbon and energy source. Strain CAN55 belonged to the Bacillus genus and was closely related to Bacillus licheniformis, with 16S rRNA gene sequence similarity of 99.2%. It was able to degrade 83% of 1-decanethiol (3 mM), and 39% of decyl disulfide, the product of spontaneous autoxidation, after 14 days of incubation at 55 °C, and without yeast extract added. Strain CAN55 was also capable of degrading 70% of 1-dodecanethiol (3 mM) after 14 days of incubation at 55 °C. During the growth on decanethiol and dodecanethiol, the decrease of surface tensions of cell-free culture supernatants from 55.1 to 40 mN/m and from 56.1 to 41 mN/m, respectively, after 26 h of incubation at 55 °C, was an agreement with the production of biosurfactant(s). In addition, strain CAN55 degraded 1-heptanethiol and cyclohexylmercaptan thus suggesting that this isolate may be useful for the bioremediation of mercaptan contaminated sites.     

Plant growth-promoting activity in newly isolated <Emphasis Type="Italic">Bacillus thioparus</Emphasis> (NII-0902) from Western <Emphasis Type="Italic">ghat</Emphasis> forest,India

A Gram-positive rod-shaped bacterium isolated on nutrient agar plates incubated at 28 ± 2°C. The identity of the bacterium was confirmed by sequencing of the 16S rRNA gene and it reveals that it shares highest similarity with Bacillus thioparus CECT 7196^T (99.08%). It was capable of growing at temperatures ranging from 4 to 40°C, but optimum growth was observed at 28 ± 2°C. Strain NII-0902 is endowed with multiple plant growth promotion attributes such as phosphate solubilization, Indole acetic acid (IAA), siderophore and HCN production, which were expressed differentially at sub-optimal temperatures (5–40°C). It was able to solubilize phosphate (17.7 μg ml⁻¹), and produce IAA (139.7 μg ml⁻¹) at 28 ± 2°C. Qualitative detection of siderophore production and HCN were also observed. At 5°C it was found to express all the plant growth promotion attributes except HCN production. The ability to colonize roots is a sine qua non condition for a rhizobacteria to be considered a true plant growth-promoting rhizobacteria (PGPR). Bacillus sp. NII-0902 has a potential ability to colonize roots visualized by transparency, bacterial growth (turbid, milky and narrow zone) along and around roots and truly supported by scanning electron micrograph. Hence, it is proposed that, Bacillus thioparus sp. NII-0902 could be deployed as an inoculant to attain the desired results of bacterization.     

Zobellella denitrificans strain MW1, a newly isolated bacterium suitable for poly(3-hydroxybutyrate) production from glycerol

Aims: To search for new bacteria for efficient production of polyhydroxyalkanoates (PHAs) from glycerol. Methods and Results: Samples were taken from different environments in Germany and Egypt, and bacteria capable of growing in mineral salts medium with glycerol as sole carbon source were enriched. From a wastewater sediment sample in Egypt, a Gram‐negative bacterium (strain MW1) was isolated that exhibited good growth and that accumulated considerable amounts of polyhydroxybutyrate (PHB) from glycerol and also from other carbon sources. The 16S rRNA gene sequence of this isolate exhibited 98·5% and 96·2% similarity to Zobellella denitrificans strain ZD1 and to Zobellella taiwanensis strain ZT1 respectively. The isolate was therefore affiliated as strain MW1 of Z. denitrificans. Strain MW1 grows optimally on glycerol at 41°C and pH 7·3 and accumulated PHB up to 80·4% (w/w) of cell dry weight. PHB accumulation was growth‐associated. Although it was not an absolute requirement, 20 g l^?1 sodium chloride enhanced both growth (5 g cell dry weight per litre) and PHB content (87%, w/w). Zobellella denitrificans strain MW1 is also capable to accumulate the poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) copolymer if sodium propionate was used as cosubstrate in addition to glycerol. Conclusions: A new PHB‐accumulating strain was isolated and identified. This strain is able to utilize glycerol for growth and PHB accumulation to high content especially in the presence of NaCl that will enable the utilization of waste glycerol from biodiesel industry. Significance and Impact of the Study: This study is the first report on accumulation of PHA in a member of the new genus Zobellella. Furthermore, utilization of glycerol as the sole carbon source for fast growth and PHB biosynthesis, growth in the presence of NaCl and high PHB contents of the cells will make this newly isolated bacterium a potent candidate for industrial production of PHB from crude glycerol occurring as byproduct during biodiesel production.     

Characterization of an organic solvent‐tolerant thermostable glucoamylase from a halophilic isolate,Halolactibacillus sp. SK71 and its application in raw starch hydrolysis for bioethanol production

A halophilic bacterium Halolactibacillus sp. SK71 producing extracellular glucoamylase was isolated from saline soil of Yuncheng Salt Lake, China. Enzyme production was strongly influenced by the salinity of growth medium with maximum in the presence of 5% NaCl. The glucoamylase was purified to homogeneity with a molecular mass of 78.5 kDa. It showed broad substrate specificity and raw starch hydrolyzing activity. Analysis of hydrolysis products from soluble starch by thin‐layer chromatography revealed that glucose was the sole end‐product, indicating the enzyme was a true glucoamylase. Optimal enzyme activity was found to be at 70°C, pH 8.0, and 7.5% NaCl. In addition, it was highly active and stable over broad ranges of temperature (0–100°C), pH (7.0–12.0), and NaCl concentration (0–20%), showing excellent thermostable, alkali stable, and halotolerant properties. Furthermore, it displayed high stability in the presence of hydrophobic organic solvents. The purified glucoamylase was applied for raw corn starch hydrolysis and subsequent bioethanol production using Saccharomyces cerevisiae. The yield in terms of grams of ethanol produced per gram of sugar consumed was 0.365 g/g, with 71.6% of theoretical yield from raw corn starch. This study demonstrated the feasibility of using enzymes from halophiles for further application in bioenergy production. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1262–1268, 2014     

氰戊菊酯降解菌FDB的分离鉴定及其生长特性

从长期受农药污染的农田土壤中分离筛选到一株降解氰戊菊酯杀虫剂的细菌菌株FDB。经形态和生理生化特征鉴定以及对16SrDNA序列进行同源性比较,将该菌株鉴定为铜绿假单胞菌Pseudomonas aeruginosa。FDB能以氰戊菊酯杀虫剂为唯一碳源生长,在30°C培养5d对100mg/L氰戊菊酯异构体的降解率分别达到69.06%(SR+RS)和64.32%(SS+RR)。FDB的最适生长条件为:温度35°C,初始pH值7.0,250mL摇瓶装液量75mL。采用超声波方法破碎菌体细胞,得到粗酶液。胞内和胞外粗酶液对氰戊菊酯异构体的降解试验表明,FDB的氰戊菊酯降解酶属于胞内蛋白组分。