Screening of a Novel Bioflocculant-producing Strain and Research on Its Flocculation

[Objective] This study aimed to screen a bacterial strain capable of producing bioflocculant. [Method] A bacterial strain T-11 capable of producing bioflocculant was isolated from activated sludge. Detailed tests on the morphological, physiological and biochemical characteristics were carried out and identification was performed to identify the strain. Finally, the bioflocculant was isolated and purified, and the flocculating activity and chemical characteristics were measured. [Result] It was identified as Serratia plumuthica based on its morphological, physiological and biochemical characteristics. This strain secreted flocculant best in a culture medium which included sucrose and NaNO3. The maximal cell growth was achieved within 10 h and the flocculating activity paralleled to it. It was found to be effective for flocculation of kaolin suspension, when added at a final concentration of 0.7 mg/L, over a range of pHs （2-7）, and temperature （approximately 30-80 ℃）. Chemical analysis indicated that the bioflocculant was an acidic polysaccharide consisting of glucose, glucuronic acid and galactose, talose and altrose. Infrared spectrum analysis also revealed typical characteristics of polysaccharides. [Conclusion] The biofloccu- lants produced by strain T-11 can greatly improve the ability of activated sludge to settle.

Production and application of a novel bioflocculant by multiple-microorganism consortia using brewery wastewater as carbon source

The flocculating activity of a novel bioflocculant MMF1 produced by multiple-microorganism consortia MM1 was investigated. MM1 was composed of strain BAFRT4 identified as Staphylococcus sp. and strain CYGS1 identified as Pseudomonas sp. The flocculating activity of MMF1 isolated from the screening medium was 82.9%, which is remarkably higher than that of the bioflocculant produced by either of the strains under the same condition. Brewery wastewater was also used as the carbon source for MM1, and the cost-effective production medium for MM1 mainly comprised 1.0 L brewery water （chemical oxygen demand （COD） 5000 mg/L）, 0.5 g/L urea, 0.5 g/L yeast extract, and 0.2 g/L （NH4）2SO4. The optimal conditions for the production of MMF1 was inoculum size 2%, initial pH 6.0, cultivating temperature 30℃, and shaking speed 160 r/min, under which the flocculating activity of the MMF1 reached 96.8%. Fifteen grams of purified bioflocculant could be recovered from 1.0 L of fermentation broth. MMF1 was identified as a macromolecular substance containing both protein and polysaccharide. It showed good flocculating performance in treating indigotin printing and dyeing wastewater, and the maximal removal efficiencies of COD and chroma were 79.2% and 86.5%, respectively.

Characterization of a bioflocculant from a newly isolated Vagococcus sp. W31

Screening of microorganisms producing flocculating substances was carried out. A strain secreting a large amount of bioflocculant was isolated from wastewater samples collected from the Little Moon River in Beijing. Based on the morphological properties and 16S rDNA sequence analysis, the isolate (designated W31) was classified as Vagococcus sp. A bioflocculant (named MBFW31) produced by W31 was extracted from the culture broth by ethanol precipitation and purified by gel chroma-tography. MBFW31 was heat-stable and had strong flocculating activity in a wide range of pH with relatively low dosage re-quirement. MBFW31 was identified as a polysaccharide with molecular weight over 2×106. It contained neutral sugar and uronic acid as its major and minor components, respectively. Infrared spectra showed the presence of hydroxyl, carboxyl and methoxyl group in its molecules. The present results suggested that MBFW31 had potential application in wastewater treatment.

Optimum conditions to treat high-concentration microparticle slime water with bioflocculants

We studied how bioflocculants,produced by white-rot fungi,affect flocculation in slime water.Based on a test in an orthogonal design,flocculation conditions were optimized.The results show that flocculation activity is at its highest when the following conditions are met:slime water concentration 27.42 g/L;coagulant aid(CaCl_2) mass concentration 5.0 g/L;two-segment stirrings:the first at a stirring speed of 60 r/min for 180 s and the second 180 r/min for 60 s;a pH of 11 and a flocculant concentration of 15 mL/L.The flocculation activity can be up to 98.71%of bioflocculants at the time.Further experiments indicate that most of the flocculation active material is found outside the mycelium cells.This is the extracellular secretion produced by mycelium cells during the fermentation process.This flocculant has strong thermal stability.Many kinds of cations have a flocculation function to assist bioflocculants.This aid-flocculation effect of the divalent cation Ca^(2+) is obvious in the bioflocculant produced by the white-rot fungus.Therefore,this is of great value when applied to control engineering in the battle against water pollution.

Screening of flocculant-producing strains by NTG mutagenesis

Screening of new microorganism being able to produce efficiently flocculants was carried out. A new model for screening efficient flocculant-producing strains was designed and tested. The results showed that this model for screening efficient flocculant-producing strains is very reliable and can greatly shorten the screening period. 13 flocculant-producing strains were isolated from activated sludge by conventional method. A strain, designated as HHE6, produced the bioflocculant with the turbidity removal 98% for kaolin suspension. Six of 13 strains selected as the original strains were treated with NTG as mutagen, and five mutant strains(HHE-P7, HHE-A8, HHE-P21, HHE-P24, HHE-A26) with high flocculation efficiency was obtained by selection, which exhibited the flocculation rate for kaolin suspension above 90%. Strains HHE6, HHE-P7, and HHE-P24 were classified as Penicillium purpurogenum, HHE-P21 as Penicillium cyclopium, HHE-A26 as Aspergillus versicolor and HHE-A8 as Aspergillus fumigatus, and it is hitherto unreported for biofloccutant-producing strains of Penicillium. The growth of the six strains(HHE6, HHE-P7, HHE-A8, HHE-P21, HHE-P24, HHE-A26) had similar curves, i.e. firstly increasing rapidly, keeping relatively constant then and finally decreasing gradually with cultivation time. The production of bioflocculants by strains showed the similar pattern to strain growth.

Characteristics and Culture Conditions of a Bioflocculant Produced by Penicillium sp.

Objective To study the characteristics of a bioflocculant named MBF7 produced by Penicillum strain HHE-P7 and the effects of cultivation conditions on bioflocculant production. Methods The chemical group in the bioflocculant molecules was shown by Fourier transform infrared （FTIR） spectra, and the average molecular weight of MBF7 was estimated by gel permeation chromatography. The effects of medium components on bioflocculant production and flocculating activity were studied. Results Phospho-, amino-, hydroxyl, and carboxyl groups were the major fractions of MBF7, and the molecule weight was about 3.0 × 10^5 Da. In addition, the carbon and nitrogen sources favorable for the bioflocculant production were glucose and yeast extract respectively. When the initial pH of the medium was adjusted to 5.0, high flocculant efficiency could be achieved. Conclusion The bioflocculant MBF7 is a new macromolecule with high flocculating efficiency for Kaolin suspension, and could be produced under appropriate culture conditions.

Bioflocculant Produced by Klebsiella sp. MYC and Its Appli-cation in the Treatment of Oil-field Produced Water

Seventy-nine strains of bioflocculant-producing bacteria were isolated from 3 activated sludge samples. Among them, strain MYC was found to have the highest and stable flocculating rate for both kaolin clay suspension and oil-field produced water. The bacterial strain was identified as Klebsiella sp. MYC according to its morphological and biochemical characteristics and 16SrDNA sequence. The optimal medium for bioflocculant production by this bacterial strain was composed of cane sugar 20 g L^-1, KH2PO4 2 g L^-1, K2HPO4 5 g L^- 1, （ NH4）2SO4 0.2 g L^-1, urea 0.5 g L^- 1 and yeast extract 0.5 g L^- 1, the initial pH being 5.5. When the suspension of kaolin clay was treated with0.5% of Klebsiella sp. MYC culture broth, the flocculating rate reached more than 90.0% in the presence of 500mg L^-1 CaCI2, while the flocculating rate for oil-field produced water was near 80.0% in a pH range of 7.0 - 9.0 with the separation of oil and suspended particles from the oil-field produced water under similar conditions. The environment-friendly nature of the bioflocculant and high flocculating rate of the strain make the bioflocculant produced by Klebsiella sp. MYC an attractive bioflocculant in oil-field produced water treatment.

Pilot scale treatment of low turbidity water using compound bioflocculant and polymerized aluminium ferrum chloride

To investigate the application of compound bioflocculant （CBF） in drinking water treatment at pilot plant, CBF and polymerized aluminium ferrum chloride （PAFC） coagulant were used to treat raw water taken from Longhupao Reservoir in Heilongjiang Province for the removal of turbidity, COl）, UV254 and residual Al. Coagulation test shows that the coagulation enhanced by CBF and PAFC exhibits more effective performance than that enhanced by the individual of them, and the total combination dosage is lower than that of the individual. The residual Al from PAFC can be removed efficiently by CBF. The removal efficiency of turbidity reaches 76.6% by combining CBF of 2 mg/L and PAFC of 15 mg/L, COl） is decreased from 3.80 mg/L to 1.62 mg/ L, and the concentration of residual Al is only 0. 033 mg/L in the product water. It can be speculated that adsorption-bridging and sweep-coagulation processes are predominant in the flocculation process by the combination of CBF and PAFC.

Two-stage pH Control Mode in Batch Fermentation of a Novel Bioflocculant from Corynebacterium Glutamicum

The effect of pH of the fermentation medium on cell growth and the production of a novel bioflocculant(named REA-11) by Corynebacterium glutamicum CCTCC M201005 were investigated. The maximum biomass(2.23 g/L) and flocculating activity(142.2 U/mL) were simultaneously obtained at the 14th hour when the pH value of the culture medium was maintained at 7.0 during the whole fermentation process. The production of REA-11 kept on a trend of increase till the later phase of fermentation process, which resulted in the ultimate flocculating activity of the culture broth to enhance to nearly 100 U/mL at pH 6.0. A two-stage pH control mode was adopted in REA-11 production in which the pH value of the culture medium was controlled at 7.0 during the first 14 h, then decreased to 6.0 that was maintained until the end of the fermentation process. With the two-stage pH control mode, the maximum flocculating activity reached 178.8 U/mL which was 30% higher than that obtained under the condition of pH 7.0 and the biomass enhanced about 15%. Compared with the fermentation process without pH control, REA-11 production and cell growth via the two-stage pH control mode increased 80% and 25%, respectively.

Three-stage fermentation and kinetic modeling of bioflocculant by Corynebacterium glutamicum

Fermentation of bioflocculant with Corynebacterium glutamicum was studied by way of kinetic modeling.Lorentzian modified Logistic model, time-corrected Luedeking–Piret and Luedeking–Piret type models were proposed and applied to describe the cell growth, bioflocculant synthesis and consumption of substrates, with the correlation of initial biomass concentration and initial glucose concentration, respectively. The results showed that these models could well characterize the batch culture process of C. glutamicum at various initial glucose concentrations from 10.0 to 17.5 g·L-1. The initial biomass concentration could shorten the lag time of cell growth,while the maximum biomass concentration was achieved only at the optimal initial glucose concentration of16.22 g·L-1. A novel three-stage fed-batch strategy for bioflocculant production was developed based on the model prediction, in which the lag phase, quick biomass growth and bioflocculant production stages were sequentially proceeded with the adjustment of glucose concentration and dissolved oxygen. Biomass of2.23 g·L-1was obtained and bioflocculant concentration was enhanced to 176.32 mg·L-1, 18.62% and403.63% higher than those in the batch process, respectively, indicating an efficient fed-batch culture strategy for bioflocculant production.

The flocculation efficiency of compound bioflocculant by flocculant-producing bacteria

The flucculation efficiency of compound bioflocculant produced by flocculant-producing bacteria was investigated in this study. Cheap cellulose was selected as the substrate for the production of a lower cost bioflocculant. The end product of cellulose decomposing bacteria was utilized as substrate for flocculant-producing bacteria. The optimum fermentation conditions were determined as follows： the initial fermentation and fermentation time was 5 d and 1 d respectively, the temperature was 30 ℃, the rotation speed was 120 r/min, the amount of CaCl2 solution （ 10% ） was 1.5 ml/L. The flocculation test indicated that the bioflocculant had high efficiency in the removal of the turbldity raw water from Songhua River.

Screening and flocculating properties of bioflocculant-producing microorganisms

Screening of bioflocculant-producing microorganisms was carded out. A strain that secreted excellent bioflocculant was isolated from municipal sewage using the spread plate technique, identified as Klebsiella sp. by the analytical profile index （API） identification system, and named A9. Several important factors that had an effect on A9＇s bioflocculant-producing and flocculating activity were studied. A total of 4 g/L Kaolin suspension was used to measure the flocculating activity of the bioflocculant from A9. It was found that maltose and urea were Ag＇s best carbon and nitrogen sources, respectively, and the flocculating activity of the flocculating agent from A9 was markedly increased by the addition of trivalent cations such as Fe^3＋ and Al^3＋; furthermore, the bioflocculant produced by A9 was most effective when the pH value was 6.0.

Screening of Bioflocculant-Producing Strain by Ion Implantation and Flocculating Characteristics of Bioflocculants

A bioflocculant-producing mutator strain, NIM-192, was screened out through nitrogen ion implanting into F J-7 strain. The results showed that NIM-192 had good genetic stability and high flocculating activity, and the flocculating rate increased by 34.26% than that of the original. Sucrose, complex nitrogen source contained yeast extract, urea and pH 7.0～ 9.0 were chosen as the best carbon source, nitrogen source and initial solution pH for bioflocculant production, respectively. The bioflocculant kept high and stable flocculating activity at alkalinous reaction mixture with a pH beyond 7.0, while the flocculating activity was remarkably reduced when the reaction pH was lower than 7.0. Addition of many cations could obviously increase the flocculating rate, among which Ca^2＋ demonstrated the best effect. The bioflocculant had very strong acid-base stability and thermo-stability.The flocculating rate kept over 86% when pH of the bioflocculant was in a range of 3.0 ～ 12.0, and the change of flocculating activity was not great when heated at 100℃ for 60 rain.

Flocculating Properties and Production of the Compound Bioflocculant by Rhizobium Radiobacter F2 and Bacillus Sphaeicus F6

A compound bioflocculant CBF,produced by mixed culture of Rhizobium radiobacter F2 and Bacillus sphaeicus F6, was investigated with regard to its production and flocculating properties. The optimization of the culture medium constituents including carbon source,nitrogen source and C / N ratio,metal ions and ionic strength on CBF production were studied. Flocculating properties of CBF were examined by a series of experiments and CBF had good flocculating activities in kaolin suspension with divalent cations and stable over wide range of p H. Studies of the flocculating properties revealed that the flocculation could be stimulated by cations Ca2+,Mg2+,Fe2+,Al3+and Fe3+. In addition,it was stable at 4-30 ℃ in the presence of Ca Cl2. It was found to be effective for flocculation of a kaolin suspension under neutral and weak alkaline conditions( p H 7. 0- 9. 0),and flocculating activities of higher than 95% were obtained when the CBF concentrations among 6- 14 mg / L at p H 8. 0. The results of this study indicate that CBF is a potential replacement of conventional synthetic flocculants and is widely applied in water treatment and downstream processing of food and fermentation industries.

Screening and Characterization of a Bioflocculant Produced by Aeromonas sp

To isolate the bioflocculant-producing bacteria from activated sludge and investigate the flocculating characteristics of the newly isolated bioflocculant. Methods Bacteria were screened from activated sludge samples to isolate bioflocculant-producing bacteria. Flocculating activity was used as a measure of the flocculating capability of the bioflocculant. Results A novel bioflocculant-producing bacterium was isolated, which was identified to belong to genus Aeromonas and named as Aeromonas sp. Nil. Flocculating activity increased in the presence of K, Na^＋, or Ca^2＋. The highest flocculating activities for kaolin suspension were obtained in acidic pH ranges, and optimum pHs for it were 3.0, 4.0, and 5.0 with 1 mmol/L K^＋, Ca^＋, and Na^＋ present, respectively. The highest flocculating activities for soil suspension were observed at pH 8.0. The bioflocculant had a good flocculating activity and could achieve a flocculating activity of 92.4% for kaolin suspension at a dosage of only 1 mg.L-1, and its activity in kaolin suspension was decreased by only 9.2% after heating at 100℃ for 60 min. Conclusion The bioflocculant produced by Aeromonas sp. Nil has strong flocculating activity and high stability, which affords high possibility of its practical use.

Characterization of an extracellular polysaccharide produced by Bacillus sp.RL-2

A strain secreting a strongly acidic polysaccharide flocculating agent was isolated from activated sludge, and identified as Bacillus brevis. The bioflocculant was produced by RL-2 during the late logarithmic growth in the batch culture and was recovered from supernatant by ethanol precipitation. The bioflocculant is thermo-stable as its activity remains stable after heated at 100 °C for 45 min. Its flocculating activity with kaolin suspensions was stimulated by the addition of Ca2+, Al3+ and Cu2+. The flocculant consists of glucose, mannose, and galacturonic acid. Its average molecular mass was estimated to be approximately 2.86×105 by the method of viscosity. The flocculant aggregates various inorganic and organic compounds in solution.

Studies on Bioflocculant Production by Pseudoalteromonas sp. NUM8, a Marine Bacteria Isolated from the Circulating Seawater

A bioflocculant producing potential bacteria was isolated from the circulating seawater of bio-filter using streak plate methods.The bacteria was identified through biochemical characteristics,partial 16S ribosomal ribonucleic acids(rRNA),nucleo-tide sequencing,and BLAST analysis of the gene sequence that showed the bacteria have 99%similarity to Pseudoalteromonas sp.and deposited in GenBank as Pseudoalteromonas sp.NUM8 with accession number JX435820.Influences of time course assay,carbon sources,nitrogen sources,inoculum size,as well as initial pH on the bacteria producing extracellular bioflocculant activity were investigated.The results showed that the strain optimal production period of microbial bioflocculant was at 72 h(flocculating activity of 94.5%),then dropped slowly.The bacteria optimally produced the bioflocculant when 1.0%sucrose and 0.5%sodium nitrate were used as sole sources of carbon and nitrogen with flocculating activities of 92.8%and 93.8%respectively.Also,the bacteria produced the bioflocculant optimally when initial pH of the medium was 5.0(flocculating activity 93.2%),and when Ca^(2+)was used as cation(flocculating activity 93.4%).The culture condition of inoculum size of 3%(v/v)was optimal flocculant pro-duction(flocculating activity 94.4%).Composition analyses indicated the bioflocculant to be principally a glycoprotein made up of about 34.3%protein and 63.4%total carbohydrate.

On an ecology-friendly treatment reagent for oily wastewater

Bioflocculants have received increasing attention in research because they are low cost,non-toxic,biodegradable,operationally simple and useful in many water and wastewater treatment applications.In this study,we focused mainly on the screening of the flocculant-producing strain and its application in oily wastewater treatment.Bacteria strain B-6-1 was isolated by the pyridine screening method.UPC-1 reagent produced by B-6-1 had efficient flocculating activity.It was deduced that the main component of UPC-1 reagent was polysaccharide through purification and analysis.The influence of dosage of reagent,pH value,temperature and oil concentration on the flocculation effect of UPC-1 reagent was investigated.

Screening of Seawater Compound Bioflocculant Producing Bacterial Strains and Optimization of Their Culture Medium

Compared with single bioflocculant,compound bioflocculant(CBF)normally has more stable and higher flocculation efficiencies.In order to produce a new compound bioflocculant,four bacterial strains with relative high rate of flocculation were selected from the coastal zone sediment,all of which were determined as Pseudomonas sp.by 16S rRNA sequencing.Through compounding,the ideal proportion of compound bioflocculant consisting of two bacterial strains(F5∶M5)was determined as 1∶2,resulting in 6%increase in terms of flocculation rate.The significant fermentation factors in culture media and their limitations were investigated via single factor experiments and Plackett⁃Burman design,obtaining four key variables,MgSO_(4),(NH_(4))_(2)SO_(4),sucrose and peptone,which were further optimized by response surface methodology(RSM)for their optimal concentrations.Finally,the ideal culture medium was determined,containing(NH_(4))_(2) SO_(4)0.523 g,MgSO_(4)3.7 g,sucrose 12.82 g,K_(2)HPO_(4)4 g,KH_(2)PO_(4)3 g,NaCl 33 g,and deionized water 1 L.The flocculation rate of experimental data(88.92)agreed well with the model prediction,which was 16.60 higher than original rate.

Surface properties of bacteria from activated sludge in relation to bioflocculation

Two bacterial stains were isolated from the activated sludge and identified as Leucobacter sp. and Alcaligenesfaecalis by 16S rDNA sequencing. Pure cultures of these two strains, representing well or poorly settled bacteria, were used to investigate the mechanism of bioflocculation in activated sludge. Based on the analyses of the characteristics of cells hydrophobicity, ζ-potential, flocculation ability and extracellular polymeric substance （EPS） composition under different growth stages, it was found that the ratio of cell EPS protein had the highly influence on ζ-potential and hydrophobicity, which were important factors to bioflocculation. Cellulase and Proteinase K could destroy the extracellular biopolymer and resulted in a decrease in the hydrophobicity and ζ-potential. However, in our study, the flocculation characteristics exhibited differently in relation to cellulase and Proteinase K. Flocculation of cells treated with cellulase and Proteinase K decreased sharply, and then recovered quickly in cellulase treatment, while cells treated with Proteinase K showed no sign of recovery. This reveals that the presence of protein in extracellular biopolymer plays an important role to the bioflocculation of cells.