Production and characterization of bioflocculants from bacteria isolated from wastewater treatment plant in South Africa

Bioflocculants produced by six bacteria obtained from activated sludge at a wastewater treatment plant were quantified, purified, and characterized. Effects of pH, temperature, cationic salt content, and specific potential inhibitors on the flocculating activities of the bioflocculants were also determined. Bioflocculants produced by the different bacterial isolates ranged between 6.33 and 27.66 g/L in concentration and were composed of both carbohydrate and protein in varying amounts, as well as a relatively high concentration of uronic acid. The flocculating activity of the broth culture increased during the logarithmic phase of bacterial growth with a maximum ranging from 2.395 to 3.709/OD. Optimum pH for the flocculating activity of the bioflocculants was between 8 and 9, with generally higher flocculating activity observed at 28°C. Of the cations tested, Mg²⁺ and Mn²⁺ improved flocculating activity up to 5.2 fold. The stability of these bacterial bioflocculants under various environmental and nutritional conditions suggests their possible use in the industries and environmental applications. Therefore, this study details important implications in providing a safer alternative flocculation method for wastewater treatment.     

Identification of Petriella setifera LH and characterization of its crude carboxymethyl cellulase for application in denim biostoning

The phylogenetic tree of the partial elongation factor-1 alpha gene fits better than the partial 18S rDNA for generic classification. From the results of the molecular tree and analysis of morphological characters, Petriella setifera LH was identified. It can be induced to produce carboxymethyl cellulase (CMCase). The crude CMCase only shows a 44.1-kDa band by activity staining after SDS-PAGE. It is optimally active at 55°C and pH 6.0, and is stable from pH 5.0–8.0 and at 45°C or below. The crude CMCase, which is not affected by Co²⁺, is strongly activated in the presence of 10 mM Na⁺, K⁺, Ca²⁺, Mg²⁺, EDTA, and Mn²⁺. It is strongly inhibited by 10 mM Fe²⁺, Pb²⁺, Al³⁺, Zn²⁺, Ag⁺, Fe³⁺, and Cu²⁺. When compared with denim treatment by Novoprime A800 (a commercial neutral cellulase), crude CMCase exhibits a similar fabric weight loss and indigo dye removal. These results indicate that crude CMCase has potential application in denim biostoning.     

Bioflocculants’ production in a biomass-degrading bacterium using untreated corn stover as carbon source and use of bioflocculants for microalgae harvest

Background

Bioflocculation has been developed as a cost-effective and environment-friendly method to harvest multiple microalgae. However, the high production cost of bioflocculants makes it difficult to scale up. In the current study, low-cost bioflocculants were produced from untreated corn stover by a biomass-degrading bacterium Pseudomonas sp. GO2.

Results

Pseudomonas sp. GO2 showed excellent production ability of bioflocculants through directly hydrolyzing various biomasses. The untreated corn stover was selected as carbon source for bioflocculants’ production due to its highest flocculating efficiency compared to that when using other biomasses as carbon source. The effects of fermentation parameters on bioflocculants’ production were optimized via response surface methodology. According to the optimal model, an ideal flocculating efficiency of 99.8% was obtained with the fermentation time of 130.46 h, initial pH of 7.46, and biomass content of 0.64%. The relative importance of carboxymethyl cellulase and xylanase accounted for 51.8% in the process of bioflocculants’ production by boosted regression tree analysis, further indicating that the bioflocculants were mainly from the hydrolysates of biomass. Biochemical analysis showed that it contained 59.0% polysaccharides with uronic acid (34.2%), 32.1% protein, and 6.1% nucleic acid in the bioflocculants, which had an average molecular weight as 1.33 × 10⁶ Da. In addition, the bioflocculants showed the highest flocculating efficiency at a concentration of 12.5 mg L^?1 and were stable over broad ranges of pH and temperature. The highest flocculating efficiencies obtained for Chlorella zofingiensis and Neochloris oleoabundans were 77.9 and 88.9%, respectively.

Conclusions

The results indicated that Pseudomonas sp. GO2 can directly utilize various untreated lignocellulolytic biomasses to produce low-cost bioflocculants, which showed the high efficiency to harvest two green microalgae in a low GO2 fermentation broth/algal culture ratio.

     

Characterization of bioflocculants from biologically aerated filter backwashed sludge and its application in dying wastewater treatment

In this study, the feasibility of bioflocculant extraction from backwashing sludge to reduce its production costs was investigated. Results showed that ultrasound and base treatment could significantly enhance bioflocculant extraction efficiency, however, flocculating activity was affected. It was observed that bioflocculants extracted from sludge of pH 11.0 had no flocculating activity. In contrast, bioflocculants extracted from sludge of pH 5.0, named as M-1, had good flocculating activity. To further study the flocculating activity of M-1, factors such as bioflocculant dosage, temperature and pH of the reaction solution were tested. The optimal conditions were 6.0 mg/l bioflocculant dosage and pH 5.0, at a temperature of 10 °C. Under these conditions, the flocculating rate of kaolin clay was 92.67%. The effectiveness of such bioflocculants in the decolorization of synthetically dyed wastewater was then examined. In flocculating methylene blue and fast blue in aqueous solutions, decolorization efficiency levels were 82.9% and 77.8%, respectively.     

Bioflocculant produced by Chlamydomonas reinhardtii

Bioflocculants of Chlamydomonas reinhardtii were investigated under axenic conditions. C. reinhardtii was found to produce significant amounts of bioflocculants. Flocculating activity by C. reinhardtii began in the linear phase of growth and continued until the end of the stationary phase. The highest flocculating efficiency of the culture broth was 97.06%. The purified C. reinhardtii bioflocculant was composed of 42.1% (w/w) proteins, 48.3% carbohydrates, 8.7% lipids, and 0.01% nucleic acid. The optimum condition for bioflocculant production of C. reinhardtii was as follows: under temperature of 15°C to 25°C, pH 6–10 and illumination of 40–60 μmol photons m^?2 s^?1. The bioflocculants produced by C. reinhardtii showed maximum activity in pH ranges from 2 to 10. The flocculating activity was significantly enhanced by the addition of CaCl₂ as a co-flocculant at an optimal concentration of 4.5 mM.     

Characterization of a compound bioflocculant produced by mixed culture of <Emphasis Type="Italic">Rhizobium radiobacter</Emphasis> F2 and <Emphasis Type="Italic">Bacillus sphaeicus</Emphasis> F6

A compound bioflocculant CBF-F26, produced by mixed culture of Rhizobium radiobacter F2 and Bacillus sphaeicus F6, was investigated with regard to its physicochemical and flocculating properties. It was identified as a polysaccharide bioflocculant composed of rhamnose, mannose, glucose, and galactose, respectively, in a 1.3: 2.1: 10.0: 1.0 molar ratio. The average molecular weight was determined as 4.79 × 10⁵ Da by gel-permeation chromatography. Infrared spectrum and X-ray photoelectron spectroscopy revealed the presence of carboxyl, hydroxyl and amino groups in its structure. Thermostability test suggested that CBF-F26 was thermostable and high flocculating activity was maintained. Thermogravimetric property, intrinsic viscosity and surface morphology of CBF-F26 were also studied. CBF-F26 was effective under neutral and weak alkaline conditions (pH 7.0–9.0), and flocculating activities of higher than 90% were obtained in the concentration range of 8–24 mg l⁻¹ at pH 8.0. The flocculation could be stimulated by cations Ca²⁺, Zn²⁺, Fe²⁺, Al³⁺, and Fe³⁺. In addition, the probable flocculation mechanisms were proposed.     

Purification and characterization of an intracellular esterase from a Fusarium species capable of degrading dimethyl terephthalate

Esterase is the key enzyme involved in microbial degradation of phthalate esters (PAEs). In this study, an intracellular esterase was purified from a coastal sediment fungus Fusarium sp. DMT-5-3 capable of utilizing dimethyl terephthalate (DMT) as a substrate. The purified enzyme is a polymeric protein consisting of two identical subunits with a molecular mass of about 84 kDa. The enzyme showed a maximum esterase activity at 50 °C and was stable below 30 °C. The optimal pH was 8.0 and the enzyme was stable between pH 6.0 and 10.0. The esterase activity was inhibited by Cr³⁺, Hg²⁺, Cu²⁺, Zn²⁺, Ni²⁺, and Cd²⁺. Substrate specificity analysis showed that the enzyme was specific to DMT hydrolysis, but had no effect on other isomers of dimethyl phthalate esters (DMPEs) or monomethyl phthalate esters (MMPEs). These findings suggest that the phthalate esterase produced by Fusarium sp. DMT-5-3 is inducible and distinctive esterases involved in hydrolysis of the two carboxylic ester linkages of DMPEs.     

Entamoeba histolytica: Soluble and membrane-associated neutral sphingomyelinase-C and other unidentified esterase activity

Sphingomyelinase (SMase) activity was measured in Entamoeba histolytica particulate and soluble subcellular fractions. The effects on SMase of incubation time, total protein concentration, pH, and several divalent cations were determined. SMase-C and other unidentified esterase activity were detected in soluble and particulate fractions. SMase-C was 94.5-96.0% higher than the unidentified esterase activity. Soluble and insoluble SMase-C specific activities increased with protein dose and incubation time. Soluble and insoluble SMase-C activities were maximum at pH 7.5 and were dependent on Mg²⁺, Mn²⁺, or Co²⁺, and inhibited by Zn²⁺, Hg²⁺, Ca²⁺, and EDTA. SMase-C was active in the pH range of 3-10 and its maximum activity was at pH 7.5. The soluble and insoluble SMases have remarkably similar physicochemical properties, strongly suggesting that E. histolytica has just one isoform of neutral SMase-C that had not been described before and might be essential for E. histolytica metabolism or virulence.     

Influence of Trace Elements Mixture on Bacterial Diversity and Fermentation Characteristics of Liquid Diet Fermented with Probiotics under Air-Tight Condition

Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ are often supplemented to the diet of suckling and early weaning piglets, but little information is available regarding the effects of different Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ mixtures on bacteria growth, diversity and fermentation characteristics of fermented liquid diet for piglets. Pyrosequencing was performed to investigate the effect of Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ mixtures on the diversity, growth and fermentation characteristics of bacteria in the liquid diet fermented with Bacillus subtilis and Enterococcus faecalis under air-tight condition. Results showed that the mixtures of Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ at different concentrations promoted Bacillus growth, increased bacterial diversity and lactic acid production and lowered pH to about 5. The importance of Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ is different for Bacillus growth with the order Zn²⁺> Fe²⁺>Cu²⁺> I⁻ in a 21-d fermentation and Cu²⁺>I⁻>Fe²⁺>Zn²⁺ in a 42-d fermentation. Cu²⁺, Zn²⁺, Fe²⁺ and I⁻ is recommended at a level of 150, 60, 150 and 0.6 mg/kg respectively for the production of fermented liquid diet with Bacillus subtilis. The findings improve our understanding of the influence of trace elements on liquid diet fermentation with probiotics and support the proper use of trace elements in the production of fermented liquid diet for piglets.     

Production of Phanerochaete chrysosporium lignin peroxidase under various culture conditions

Lignin peroxidase production by the white-rot fungus Phanerochaete chrysosporium is markedly influenced by the buffer system employed. In immobilized P. chrysosporium cultures with carbon-limited glucose medium, the use of acetate buffer resulted in higher lignin peroxidase activities than tartrate. With acetate as the buffer in shake-flask cultures a 20% to over 100% improvement in lignin peroxidase production was obtained as compared to tartrate-buffered systems. Of trace elements, Cu²⁺, Mn²⁺ and Zn²⁺ seemed to have the greatest influence on lignin peroxidase production. Furthermore, an increase in the Cu²⁺ and Zn²⁺ concentrations resulted in considerably higher ligninase activities. Although it has been shown previously that high manganese levels repress ligninase production, for maximum ligninase production the presence of some Mn²⁺ appeared to be necessary. The concentration of phosphorus had surprisingly little effect on ligninase production. Highest lignin peroxidase activities were obtained with lower phosphorus concentrations, but reasonably high activities were obtained within the whole studied phosphorus range of 0.12–4.60 g l^–1. Diammonium tartrate alone was a better nitrogen source than a mixture of diammonium tartrate, proteose peptone and yeast extract. The addition of solid manganese (IV) oxide to 3-day-old immobilized biocatalyst cultures increased the maximum ligninase activity obtained by about one-third. Correspondence to: S. Linko     

Influence of Calcium and Manganese on Dechaining of Lactobacillus bulgaricus

The events responsible for the transition of Lactobacillus bulgaricus 1243-F from long filamentous chains to short bacilloid rods were examined in a cation-depleted liquid medium. In the presence of magnesium only, cells grew as long chains of unseparated cells. The addition of 100 μM to 1 mM calcium or manganese to this medium resulted in the dechaining of these cells to short bacilloid rods. Fe²⁺, Zn²⁺, Co²⁺, and Cu²⁺ failed to induce dechaining. Induction of calcium and manganese dechaining functioned under controlled pH maintained at 5.0 and 6.0 but not at pH 7.0. This was consistent with a previous report showing failure in synthesis of dechaining enzymes by L. bulgaricus under pH conditions approaching alkalinity (S. K. Rhee and M. Y. Pack, J. Bacteriol. 144:865-868, 1980). We conclude that under pH conditions which permit synthesis of dechaining enzymes, calcium and manganese are necessary for dechaining activity.     

Further data on the specificity of aequorin luminescence to calcium

The possible triggering of luminescence of the photoprotein aequorin by 11 kinds of metal ions, in concentrations from 10^?3 to 10^?6M, at pH 6.0 and at pH 8.0, were examined under conditions which minimized contamination with extraneous Ca²⁺. Y³⁺ and La³⁺ were found to have activities nearly as great as that of Ca²⁺ at pH 6.0, but gave evidence of quenching effects in the higher concentrations at pH 8.0. Cations indicating no activity, or only negligible activity at either pH 6.0 and 8.0 included Be²⁺, Ba²⁺, Co²⁺, Ni²⁺, and Cu²⁺. Considerable activity was shown by Pb²⁺ and Cd²⁺ at pH 6.0, but very little at pH 8.0.     

Identification, characterization and functional analysis of a GH-18 chitinase from Streptomyces roseolus

A 40 kDa chitinase from Streptomyces roseolus DH was purified to homogeneity from culture medium. The N-terminal sequence was TPPPAKAVKLGYFTNWGVYG, which was highly homologous to the glycoside hydrolase (GH) 18 conserved domain of Streptomyces chitinases and included the two crucial Trp and Tyr sites. The purified enzyme showed maximal activity at 60 °C, pH 6.0 and exhibited good thermal and pH stabilities. The enzyme displayed strict substrate specificity on colloidal or glycol chitin, but not on chitosan derivatives. It was activated by Mg²⁺, Ba²⁺ and Ca²⁺, and inhibited by Cu²⁺, Co²⁺, Mn²⁺, whereas Zn²⁺ and ethylenediamine tetraacetic acid showed little inhibitory effects. Morphological changes observed by scanning electron microscopy revealed the occurrence of regular pores on the surface with the progress of enzymatic chitinolysis. Additionally, this GH-18 chitinase had a marked inhibitory effect on fungal hyphal extensions. In conclusion, this chitinase may have great potential for the enzymatic degradation of chitin.     

兔肌3-磷酸甘油脱氢酶的提纯及性质研究

目的:研究兔肌3-磷酸甘油脱氢酶的分离纯化方法及其酶学性质,为测定血清甘油三酯所用酶联试剂的开发提供试验基础和理论依据。方法:通过硫酸铵分级沉淀、DEAE-Sepharose、Blue-Sepharose和羟磷灰石纯化兔肌3-磷酸甘油脱氢酶,利用凝胶过滤和梯度PAGE(5%～15%)法测定酶分子量,采用常规酶学动力学分析方法,考察pH、温度、底物浓度以及部分金属离子与有机化合物对酶促反应的影响。结果 纯化后的兔肌3-磷酸甘油脱氢酶经PAGE(12%)分析为单一条带;酶分子量为115～122 kDa;酶最适温度45℃,最适pH 9;酸碱稳定范围pH6～9,低于45℃时热稳定性好;最适条件下,以3-磷酸甘油和NAD⁺为底物,测得酶的K_m分别为7.4×10^-3mol/L和1.47×10^-4mol/L;Ba²⁺、Mn²⁺、Fe²⁺、Al³⁺、Cu²⁺、Ni²⁺、Ag⁺、Hg²⁺、NaN₃、EDTA对酶有不同程度的抑制作用,Mg²⁺、Ca²⁺、Co²⁺、Zn²⁺有一定程度的激活作用,其中Co²⁺和Zn²⁺对酶的激活作用能达到200%以上,有机化合物NaF对酶的活性没有影响。     

Absorption of copper, zinc, and manganese by sugarcane leaf tissue

The absorption of Cu²⁺, Zn²⁺, and Mn²⁺ by leaf tissue of 4-month old sugarcane plants (Saccharum officinarum L., var. H53-263) has been investigated. After the “apparent free space” fraction was desorbed, the absorption of Cu²⁺, Mn²⁺, and Zn²⁺ yielded a curve typical of many ion uptake processes when measured as a function of the external concentration. However, only 1 absorption mechanism was evident for each cation. The pH optimum for Cu²⁺ and Zn²⁺ uptake was 5.0 to 6.0, whereas that for Mn²⁺ absorption was 4.5 to 6.0. Absorption was competitively inhibited by H⁺, and this inhibition was reversible when 0.5 mm Ca²⁺ was present. Cu²⁺ and Zn²⁺ were absorbed through the same carrier sites, as concluded from their mutually competitive activities. Mn²⁺ was absorbed through a second, independent mechanism. Uptake of each cation was strongly inhibited by uncouplers of oxidative phosphorylation, by Amytal and Nembutal², by 5 × 10⁻²m succinate, and by ADP and P_i. Absorption of Cu²⁺, Zn²⁺, and Mn²⁺ was concluded to be coupled to oxidative phosphorylation, and specifically to energy-conservation Site I.     

Improved production and properties of β-glucosidase influenced by 2-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-glucose in the culture medium of <Emphasis Type="Italic">Termitomyces clypeatus</Emphasis>

Increased production, secretion, and activity of β-glucosidase in the filamentous fungus Termitomyces clypeatus was achieved in presence of the glycosylation inhibitor 2-deoxy-d-glucose (0.05%, w/v) during submerged fermentation. Enzyme activity increased to 163 U/mL by adding mannose (2 mg/mL) to the medium. Such a high enzyme activity has not been achieved without mutation or genetic manipulation. The K_m and V_max of the enzyme in culture medium were determined to be 0.092 mM and 35.54 U/mg, respectively, with p-nitrophenyl β-d-glucopyranoside as substrate, confirming its high catalytic activity. The enzyme displayed optimum activity at pH 5.4 and 45°C. The enzyme was fairly stable between acidic to alkaline pH and retained about 75 ∼ 65% residual activities between pH 4 and 10.6 and demonstrated full activity at 45°C for 3 days. The enzyme was also stable in the presence of Zn²⁺ and Mg²⁺ and 80% of the residual activity was observed in the presence of Mn²⁺, Ca²⁺, K⁺, Cu²⁺, EDTA, and sodium azide. Around 70% of the activity was retained in the presence of 2 M guanidium HCl and 3 M urea, whereas the activity was 5 and 2 times higher in the presence of 4 mM beta-mercaptoethanol and 50 mM DTT, respectively. The enzyme obtained from the culture filtrate showed potential cellulose saccharifying ability which increased further when supplemented with commercial cellulase. Thus, this enzyme could be used without any additional downstream processing for commercial cellulase preparation and production of bioethanol or for other biotechnological applications.     

Flocculation properties of xanthan produced by Xanthomonas campestris

Summary Xanthan had a flocculating activity in a kaolin suspension and high flocculating activity was obtained in the suspension (pH 7.0) adding Al³⁺, Fe³⁺ or Fe²⁺. Xanthan had high flocculating activity not only in other inorganic suspensions such as active carbon and acid clay but also in organic suspensions of cellulose and yeast. From these flocculation properties, xanthan is anticipated to be utilized in wide areas as a new biodegradable, harmless biopolymer flocculant.     

Purification and characterization of SDG-β-d-glucosidase hydrolyzing secoisolariciresinol diglucoside to secoisolariciresinol from Aspergillus oryzae

The SDG-β-d-glucosidase that hydrolyzes the glucopyranoside bond of secoisolariciresinol diglucoside (SDG) to release secoisolariciresinol (SECO) was isolated from Aspergillus oryzae 39 strain and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 64.9 kDa. The optimum temperature of the SDG-β-d-glucosidase was 40 °C, and the optimum pH was 5.0. The SDG-β-d-glucosidase was stable at less than 65 °C, and pH 4.0–6.0. Ca²⁺, K⁺, Mg²⁺ and Na⁺ ions have no significant effect on enzyme activity, Zn²⁺ and Cu²⁺ ions have weakly effect on enzyme activity, but Fe³⁺ ion inhibits enzyme activity strongly. The K_m value of SDG-β-d-glucosidase was 0.14 mM for SDG.     

Comparison of Extracellular Cellulase Activities of Clostridium thermocellum LQRI and Trichoderma reesei QM9414

The crude extracellular cellulase of Clostridium thermocellum LQRI (virgin strain) was very active and solubilized microcrystalline cellulose at one-half the rate observed for the extracellular cellulase of Trichoderma reesei QM9414 (mutant strain). C. thermocellum cellulase activity differed considerably from that of T. reesei as follows: higher endoglucanase/exoglucanase activity ratio; absence of extracellular cellobiase or β-xylosidase activity; long-chain oligosaccharides instead of short-chain oligosaccharides as initial (15-min) hydrolytic products on microcrystalline cellulose; mainly cellobiose or xylobiose as long-term (24-h) hydrolysis products of Avicel and MN300 or xylan; and high activity and stability at 60 to 70°C. Under optimized reaction conditions, the kinetic properties (V_max, 0.4 μmol/min per mg of protein; energy of activation, 33 kJ; temperature coefficient, 1.8) of C. thermocellum cellulose-solubilizing activity were comparable to those reported for T. reesei, except that the dyed Avicel concentration at half-maximal velocity was twofold higher (182 μM). The cellulose-solubilizing activity of the two crude cellulases differed considerably in response to various enzyme inhibitors. Most notably, Ag²⁺ and Hg²⁺ effectively inhibited C. thermocellum but not T. reesei cellulase at <20 μM, whereas Ca²⁺, Mg²⁺, and Mn²⁺ inhibited T. reesei but not C. thermocellum cellulase at >10 mM. Both enzymes were inhibited by Cu²⁺ (>20 mM), Zn²⁺ (>1.0 mM), and ethylene glycol-bis(β-aminoethyl ether)- N,N-tetraacetic acid (>10 mM). T. reesei but not C. thermocellum cellulose-solubilizing activity was 20% inhibited by glucose (73 mM) and cellobiose (29 mM). Both cellulases preferentially cleaved the internal glycosidic bonds of cellooligosaccharides. The overall rates of cellooligosaccharide degradation were higher for T. reesei than for C. thermocellum cellulase, except that the rates of conversion of cellohexaose to cellotriose were equivalent.     

Purification and properties of dipeptidyl peptidase IV from Streptococcus mitis ATCC 9811

Dipeptidyl peptidase IV (EC 3.4.14.—) from Streptococcus mitis ATCC 9811 was purified to a specific activity of 56.2 units/mg protein by a series of column chromatographic techniques. The purified enzyme was apparently homogeneous as judged by disc gel electrophoresis. Gel filtration on a calibrated column indicated an apparent molecular weight of 120,000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate in a constant acrylamide concentration resulted in the appearance of a single component for which a molecular weight of 53,000 was calculated. The purified enzyme has an optimum pH between 6.0 and 8.7 and an isoelectric point of 4.0. The K_m value toward glycylprolyl-p-nitroanilide is about 6.0 × 10^?5m. Substrate specificity studies indicated that the purified enzyme hydrolyzes specifically N-terminal X-proline from X-Pro-p-nitroanilides. Inhibition of this enzyme was achieved with Hg²⁺, Pb²⁺, Zn²⁺, EDTA, and diisopropyl phosphorofluoridate, but not with N-ethyl-maleimide and sulfhydryl inhibitors.