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

FU Wandong, LIAO Miaofei, ZHANG Dongxu, ZHOU Yufang, and YANG Huicheng

Studies on Bioflocculant Production bysp. NUM8, a Marine Bacteria Isolated from the Circulating Seawater

FU Wandong1), 2), *, LIAO Miaofei1), ZHANG Dongxu2), ZHOU Yufang1), and YANG Huicheng1)

1) Zhejiang Marine Development Research Institute, Zhoushan 316021, China 2) Marine Fishery Institute of Zhejiang Province, Key Laboratory of Mariculture & Enhancement of Zhejiang Province, Zhoushan 316021,China

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), nucleotide sequencing, and BLAST analysis of the gene sequence that showed the bacteria have 99% similarity tosp. and deposited in GenBank assp. 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 72h (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 Ca2+was used as cation (flocculating activity 93.4%). The culture condition of inoculum size of 3% (v/v) was optimal flocculant production (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.

sp.NUM8;bioflocculant; flocculating activity; circulating seawater

1 Introduction

Flocculant plays an important role in various industrial processes, such as wastewater treatment, drinking water purification and food fermentation and downstream processing (Shih., 2001; Zaki., 2013; Liu., 2014; Luo., 2016; Tawila., 2019). There are three classes of flocculants, namely: 1) inorganic flocculants such as aluminum sulfate and polyaluminum chloride; 2) organic synthetic flocculants such as polyacrylamide derivatives and polyethylene amine; and 3) naturally occurring flocculants such as chitosan, sodium alginate, and microbial flocculants (Salehizadeh and Shojaosadati, 2001; Zhang., 2007; Xia., 2008; Yu., 2009). Inorganic and organic flocculanting agents such as those mentioned above are frequently used in water treat- ment and fermentation industries because of their strong flocculating activity and low cost (Chen., 2010).However, studies have shown that inorganic and organic flocculating substances may cause health and environmental problems (Levesque., 2000; Sharma., 2006). Recent neuropathological, epidemiological and biochemical studies suggest a possible link between the neurotoxicity of aluminum and the pathogenesis of Alzheimer’s disease (Polizzi., 2002; Banks., 2006).

Although a fully polymerized polyacrylamide is safe, they are not readily biodegradable andsome of their degraded monomers such as acrylamide monomer are neurotoxic and even show strong human carcinogenic potential and non-degradable in nature (Shih., 2001). Because of the limitations ofthese flocculants, biopolymers produced by microorganismsthrough the synthesis of extracellular polymers by livingcells are investigated as an alternative flocculant. Biopolymersare biodegradable and their degradation intermediatesare harmless towards human being and environment. Therefore, microbial flocculant is gradually widely researched.Bioflocculants are kinds of extracellular biopolymercontaining glycoprotein, polysaccharide,protein, cellulose,lipid, glycol-ipid and nucleic acid (Lian., 2008; Zheng., 2008; Okaiyeto., 2014; Ugbenyen., 2014; Giri., 2015; Yang., 2015; Okaiyeto., 2016; Essandoha., 2020). The industrial potential of bioflocculants has long been recognized because of their harmlessness, biodegradability and lack of secondary pollution from their degradative intermediates (Wang., 2011; Nwodo., 2013; Okaiyeto., 2014; Giri., 2015; Jiang., 2019). Though a number of microorganisms such assp. have been screened for their bioflocculant-producing capabilities, there is very little achieved a commercial scale.

The high-cost of production and low yield seem to be the major restricting factors in the advancement of research in developing bioflocculants for both scientific and commercial applications (Li., 2003; Yin., 2014). However, in order to reduce costs and optimize cultivation conditions, strategies such as fed-batch production processes were developed (Wu., 2010; Tang., 2014).

As part of our exploration for new and novel bioflocculants which could stand as alternatives to inorganic and synthetic flocculants, the present study reports on the bioflocculantproducing potential of a seawater bacterium identified assp. NUM8 isolated from circulating seawater of bio-filter. Due to its high flocculating activity, we have accumulated basic data and expected to apply in the purification of aquaculture wastewater and other industrial applications.

2 Materials and Methods

2.1 Sample Collection and Cultivation Media

The activated sedimentfrom the bottom of biological filter was collected from the aquatic farm of Zhoushan City and screened for bioflocculant production according to Jensen. (2005) with some modifications. The composition of the production media included (per litre):beef extract5.0g, glucose 10.0g, ferric citrate 0.2g, sodium chloride 5.0g, magnesium chloride6.0g, sodium sulfate 3.0g, calcium chloride 2.0g, sodium carbonate 0.2 g, boric acid 0.02g, strontium chloride 0.03g, disodium hydrogen phosphate 0.08g, and agar 15.0g(named M1) and were prepared using filtered seawater and sterilized by autoclaving at 121℃ for 15min.

2.2 Isolation of Bioflocculant-Producing Bacteria

Bioflocculantproducing bacteria were originally scr- eened based on colony morphology. One gram of wet sample was diluted with 20mL of sterile seawater. The suspension was vortexed and allowed to sediment for 5 min, out of which 100µL of the suspension was inoculated onto the surface of nutrient agar plates, spread with a sterile glass rod and incubated for 72h. The distinct isolates were picked and streaked onto nutrient agar plates. Each isolated strains was grown in 100mL of M2 medium (M1 without agar) on a rotary shaker(160rmin−1) at 28℃. During incubation, the flocculating activity of fermentation broths was assayed every 6h. The fermentation broths were centrifuged at 8000×for 5min and the cell free culture supernatant were assayed at the same time for flocculating activity. Finally, one strain with high and stable flocculating activity for kaolin was selected for further study.

2.3 Measurement of Flocculating Activity

Using a suspension of kaolin clay as test material, flo- cculating activity was measured according to the method of Okaiyeto. (2014) and Yin. (2014). The kaolin clay (average diameter 4μm) wassuspended in distilled water at a concentration of 4.0gL−1at pH 7, 1.0mL of 1% CaCl2and 2.0mL of the cell-free supernatant were added into 47mL of kaolin suspension in 50mL color comparison tube. The mixture was blended by turned upside and down vigorously and then stand for 10min at room temperature.The optical density of the supernatant solution was measured with a ThermoSpectronic spectrophotometer (Helios Epsilon, USA) at 600nm. A control experiment was prepared in the same way but the cell-free supernatant was replaced with the un-inoculated production medium. The flocculating activity was estimated from the formula:

Flocculating activity={(A−B)/A}×100%,

in which A and B were optical densities of the control and samples respectively at 600nm.

2.4 Identification of Bacteria

The bacterium was identified by molecular technique based on the 16S rRNA gene amplification by polymerase chain reaction (PCR) followed by sequencing of the amplified gene(by Sangon Biotech (Shanghai) Co., Ltd.). Template DNA of the bacterium for use in the PCR was prepared using the bacterial genomic DNA extraction kit.The PCR amplification reaction was carried out as follows, in 25μL reaction volume containing 10mmol MgCl22.0 μL,10×PCR buffer 2.5μL, 10mmol dNTPs 1.0μL, 10 μmol forward primer (27F: 5’-AGAGTTTGATCCTGGC TCAG-3’) and reverse primer (1492R: 5’-GGCTACCTT GTTACGACTT-3’) 1.0μL respectively, template DNA 1.0μL, 2U Supertherm Taq polymerase, and ultrapure water 15.5μL. The primers in this study were used to amp- lify nearly 1500bp 16S rRNA sequences. The PCR programme was an initial denaturation (95℃ for 5min), 30 cycles of denaturation (94℃ for 1min), annealing (58℃ for 1min ) and extension (72℃ for 2min), and a final extension (72℃ for 10min). Gel electrophoresis of PCR products were conducted on 1.2% agarose gels to confirm that a fragment of the correct size had been amplified, and then sequenced the amplified gene.

2.5 Factors on the Flocculating Activity of Different Culture Conditions

Influences of culture conditions including time course assay, inoculum size, carbon sources, nitrogen sources and metal ions, as well as initial pH of the production medium on the bacteria NUM8 producing extracellular bioflocculant activity were investigated. In the experiments on the effects of medium composition, only the carbon or nitrogen sources and concentration were replaced while the other constituents, temperature (28℃) and shaking speed (160rmin−1) were kept constant. Different inoculum sizes ranging 0.5%–6% of the seed culture were used to inoculate the production medium and the effect of each on flocculant production was assessed according to the description of Zhang. (2007). The effectof different carbon sources (10, 20 and 40gL−1respectively) on flocculating activity were assessed included glucose, fructose, starch,sucrose, maltose, lactose, and Na2CO3. The effectof different nitrogen sources (5, 10 and 20gL−1respectively) on flocculating activity were assessed included peptone, tryptone, beef extract, yeast powder, ammonium sulfate, sodium nitrate, and urea. While the effect of pH on flocculating activity was evaluated by adjusting the initial pH of the culture media range from 3 to 10 using HCl (1.0molL−1) and NaOH (1. 0molL−1). The effect of different cations on flocculating activity were assessed by adding 1.0% NaCl, KCl, FeCl3, FeSO4, MgSO4, CaCl2, AlCl3, and MnSO4, and without adding any metal salts.

2.6 Time Course of NUM8 Bioflocculant Production

For the time course experiment, optimum culture conditions were used forflocculant production in accordance with the method described byLiu. (2010) and Nwodo. (2013), with some modifications. The isolate was cultured under optimal growth conditions. The composition of the medium for the bioflocculant production was as follows: sodium nitrate 5.0g, sucrose 10.0g, ferric citrate 0.2g, sodium chloride 5.0g, magnesium chloride 6.0g, sodium sulfate 3.0g, calcium chloride 2.0g, sodium carbonate 0.2g, boric acid 0.02g, strontium chloride 0.03g, disodium hydrogen phosphate 0.08g in 1L of filtered natural sea water. The standardized saline solution was used as seed culture for inoculum preparation. Seed culture (inoculum size, 1% v/v) was inoculated into 100mL of medium in 300mL flasks (prepared in duplicates) and in- cubated on a rotatory shaker (160rmin−1) at 28℃. Sam- ples were drawn every 12h for a period of 192h. Two milliliters of culture broth was centrifuged at 8000×for 5min and the cell free supernatant was used to determine the flocculating activity. The growth of the bacteria was monitored by optical density OD600.The pH of the broth samples was also measured.

2.7 Extraction, Purification and Characterization of the Bioflocculant

Extraction and Purification of bioflocculantwere in accordance with the method described by Mabinya. (2012), with some modifications.After 72h of fermentation, the culture broth was centrifuged (8000×, 10min) to remove bacterial cells. One volume of distilled water was added to the supernatant phase and centrifuged (8000×, 15min) to remove insoluble substances. The supernatant was then mixed with 3 volumes of ethanol, stirred and left standing at 4℃ for 8h. The supernatant was decanted and the precipitate vacuum-dried to obtain the crude biopolymer. The crude product was then dissolved in distilled water and mixed with 1 volume of chloroform/n-butyl alcohol (5:2, v/v). After stirring, the mixture was left standing at room temperature for 12h. The upper phase was separated, centrifuged (4000×, 10min) and the supernatant concentrated at 45℃. Two volumes of ethanol were added, the precipitate recovered, vacuum-dried and re-dissolved in distilled water. Thereafter, the protein content was measured using the Folin-Lowry method, and total carbohydrate was assayed using Phenol-Sulphuric acid protocol.

2.8 Statistical Analysis

Triplicate values were obtained, averaged, andstatistically subjected to One-way Analysis of Variance (ANO VA) followed by Tukey HSD test to determine differences in the mean values among the treatments. Significance was concluded at<0.05. Statistical analysis was performed using SPSS version 22.0 for Windows. The error barsrepresent the standard deviation (SD) of the data.

3 Results

3.1 Screening and Identification of Bioflocculant-Producing Strain

There were 48 different strains isolated from the activated sediment. Among these strains, strain NUM8 show- ed the strongest flocculating activity to Kaolin suspension. Therefore, we chose the strain NUM8 as the test strain to produce bioflocculant. The colonies of strain NUM8 were milk white in color, circular, smooth, humid, and regular edges.

At the same time, the 16S rRNA of strain NUM8 was sequenced following PCR amplification. The result of PCR product was identified in 1.2% agarose gel electrophoresis. The 16S rRNA sequences of strain NUM8 were determined to be 1437bp. The 16S rRNA sequence of strain NUM8 was registered in GenBank (Accession number JX435820).

According to the 16S rRNA sequence and the biochemical and physiological characteristics, the strain NUM8 could be identified assp., and namedsp. NUM8. The strain NUM8 16S rRNA sequence in NCBI blast analysis was to find the most similar to the known sequences then chose the high level similar sequence for phylogenetic analysis. As shown in Fig.1, Maximum Likelihood tree (HKY+I+G model) based on 16S rRNA sequence was constructed by using MEGA. The phylogenetic position of strainNUM8 in relation to representatives of the main lineagesinThe strain NUM8 showed the highest degrees of relativity tosp.sharing a 99% 16S rRNA similarity.

Fig.1 ML tree (HKY+I+G model) based on 16S rRNA sequence was constructed by using MEGA.

3.2 Factors on the Flocculating Activity of Different Culture Conditions

3.2.1 Effect of carbon source and concentration on flocculant production

The effect of carbon sources and different concentrations were investigated by cultivating the strain in the same medium. As shown in Fig.2, the strain NUM8 showed preference for sucrose as an effective carbon source for flocculant production with the highest flocculating activity (94.8%), followed by glucose, fructose, maltose, lactose and starch. The medium containing inorganic carbon source Na2CO3supported flocculant production with low flocculating activity. The variance of flocculating activity were observed in different carbon source concentration gradient 1.0%, 2.0%, and 4.0%, nevertheless the difference in the different concentration were not significant (>0.05). The results showed that different carbon source had different effects on the floc- culation activity, which confirmed the strain NUM8 had the different utilization degree on carbon source in the same culture condition, and the ability of utilization the sucrose was the highest. Sucrose is cheaper than fructose in the industrial application. Additionally, there was no significant difference with carbon source concentrations, so the lowest concentration 1.0% was chose.

Fig.2 Effect of carbon source on the flocculating activity.

3.2.2 Effect of nitrogen source and concentration on flocculant production

The effect of nitrogen source and different concentration were also investigated by cultivating the strain in the same medium, except that nitrogen source and concentration were changed. The results were shown in Fig.3. The beef extract, peptone and sodium nitrate had higher flocculation rate than tryptone, yeast powder, ammonium sulfate and urea. The variance of flocculating activity were observed in different nitrogen source concentration gradient 0.5%, 1%, and 2%, nevertheless the difference in the different concentrations were not significant (> 0.05). Through the analysis of variance, the strain NUM8 was able to utilize both organic and inorganic nitrogen sources. Relative to other nitrogen sources, sodium nitrate is the most cost-effective nitrogen source and was chose as the nitrogen source in the following experiments. Additionally, there was no significant difference with different nitrogen source concentrations, so the lowest concentration 0.5% was chose.

Fig.3 Effect of nitrogen source on the flocculating activity.

3.2.3 Effect of initial pH of medium on flocculant production

The effect of the initial pH of the culture medium on strain NUM8 production was evaluatedat pH values ranging from 3 to 10.The results are represented in Table 1. The flocculating activity was found to be distinctly higher in acidicpH conditions than in neutral and alkaline conditions. When the initial pH was 5.0, the flocculating activity of NUM8 reached a peak (93.2%) after cultured 72 h. The flocculating activity decreased steadily as the pH tended towards alkalinity. It would appear that pH of the natural habitat of the test bacteria has no bearing on its bioflocculant production potential as the habitat had an alkaline pH of about 10.0. When pH was 3.0 or 10.0,the test bacteria only have a very little amount of growth. When the initial pH was reach 11.0, the bacteria almost cannot grow. So we did not assay the data.

Table 1 Effect of pH, cations and inoculum size on the flocculating activity

3.2.4 Effect of inoculum size on flocculant production

The relationship between inoculum size and flocculant production was investigated and the results are presented in Table 1. It was observed that the increase in inoculums size of the seed culture from 0.5% to % 3 (v/v) resulted in an increase in flocculating activity. However, a further increase in inoculum size led to a steady decrease in flocculating activity culminating at 83.7% observed at 6% (v/v). Inoculum size of 3% (v/v), which resulted in optimum flocculant production and was used in all subsequent experiments.

3.2.5 Effect of cations on the flocculating activity

The effect of various cations on flocculating activity produced by strain NUM8 was depicted in Table 1. CaCl2, AlCl3, and MnSO4could obviously enhance flocculating activity. The highest flocculating activity of 93.4% was observed with CaCl2, followed by MnSO4at 91.1% and AlCl3at 90.3%. FeCl3could seriously inhibit the flocculating activity.

3.3 Time Course of the Strain NUM8 Flocculant Production

The time course of flocculant production produced by the strain NUM8 was shown in Fig.4. During the course of culture, the bioflocculant production of strain NUM8 was almost in parallel with bacteria growth and positive correlation with the biomass, which indicated the production of flocculants. The substantial parallel between the flocculating rate and growth process suggested that flocculating substance was caused by the bacterial synthesis then secreted into the extracellular, rather than cell auto- lysis causes.

Fig.4 Time course assay of bioflocculant production.

At the beginning of culture, the flocculating activity was almost zero, which showed medium itself has no flocculation. In the initial phase, the cell grew up slowly, which was the bacteria cell adaptive phase, with the new cell mass formation and growth. The cell metabolic activity was the strongest and new synthesized materials were the fastest. At the same time, flocculating activity sustained rise and reached the highest 94.5% at 72h. When the bacteria growth was into the stationary phase, at this stage, the bacteria new proliferation of cells and the old cells was almost equal in the culture liquid, and the quantities were relatively stable. As the extension of culture time, flocculating rate reduced slowly. Possibly after stationary phase, the growth of bacteria was into decline phase and nutrients were depleted. Due to lack of nutrition, bacteria begin to decompose polymer material which secreted into the culture medium by itself before. So the 72 h was as the optimal incubation time.

3.4 Chemical Analysis of Bioflocculant Composition

The composition analyses indicated the bioflocculant of strain NUM8 to be principally a glycoprotein made up of about 34.3% protein and 63.4% total carbohydrate.

4 Discussion

The bioflocculant production is affected by many factors,such as the constituents of the culture medium andculture conditions (He., 2004; Liu., 2015a). The effects of the key factors affecting bioflocculant production bystrain NUM8, wereinvestigated with an aim to identify the cost-optimal culture conditions.

4.1 Effect of Carbon Source and Nitrogen Source on Flocculant Production

Different bacteria have the different capability of using carbon source and nitrogen source, even if the same carbon source and nitrogen source. Contrary to the present studies with strain NUM8, in addition to peptone, yeast extract was found to be able to effectively use, which was a key parameter influencing optimal production of the bioflocculant produced bysp. LC13 (Su., 2012). Xia. (2008) repotted thatTJ-1 was able to effectively use not only beef extract, peptone but also yeast extract as nitrogen source. A number of strains have been reported to optimally produce bioflocculant in the presence of organic nitrogen sources and sometimes combined organic and inorganic sources (Gong., 2008; Xia., 2008).

With respect to sucrose, these results seem to support the findings reported by Li. (2009) in which sucrose was favorable carbon sources for the bioflocculant production byX14. Studies carried out by He. (2009) and Patil. (2010) also showed a better bioflocculant recovery rate with sucrose as a carbon source on bioflocculant production bysp. V3a’ andrespectively. Similarly, sucrose was utilized as the most favorable carbon source for the production of a bioflocculant produced bysp.Gilbert (Nontembiso., 2011).

4.2 Effect of Initial pH of Medium on Flocculant Production

The initial pH of the culture medium determines the electric charge of the cells and the oxidation reduction potential which can affect nutrient absorption and enzymatic reaction (Salehizadeh and Shojaosadati, 2001). The initial pH of the production medium is one of the factors affecting the production and flocculating activity of the bioflocculant (Zhang., 2007; Xia., 2008; Cosa., 2011; Liu., 2015b; Kunle., 2016).

So the pH of the solution is also a key factor in flocculation and thus effectively influences the flocculation process (Tang., 2014). For different strains, their requirement for the initial pH varied greatly. Similar pH values reported bioflocculant produced byKG03 to have maximum activity at acidic pH 4.0 (Yim., 2007). There were two other studies reported the lower pH by Zheng. (2008) in which the maximum flocculating activities produced bysp. F19 were observed at pH 2.0. There was also at acidic initial pH of 3.0, the bioflocculant production and activity ofsp. Gilbert was at peak (Nontembiso., 2011). Whileproduced a bioflocculant effective over a weakly acidic pH range of 5.0 to 7.0 (Gong., 2008).

However, these observations are different from other studies, where the initial pH from neutral to alkaline the bioflocculant produced were also at peak. Bioflocculant produced bysp. Rob preferred alkaline conditions (Cosa., 2011). The alkaline pH, especially pH 10.2, effectively stimulated the flocculant production ofUTEX2341 (Liu., 2015a). The bioflocculant produced byTJ-1 (Xia., 2008),sp. nov. (Bouchotroch., 2001),sp. W31 (Gao., 2006) and the mutantsp. V3a’ (He., 2010) attained the highest flocculating activity at pH neutral.

4.3 Effect of Inoculum Size on Flocculant Production

It has been articulated in previous studies that small inoculum sizes prolong the stagnant phase of bacteria growth and a large inoculum size forms niche and inhibit bioflocculant production (Su., 2012). The production of bioflocculant was significantly enhanced when 3% inoculum size was used and a slight decrease in bioflocculant production was noticed with further increase in inoculums sizes (Ugbenyen., 2014; Kunle., 2016). Gong. (2008) and Okaiyeto. (2014) found that 1% and 2% inoculum size was favourable for bioflocculant production byand. Leo respectively. On the other hand, Wang. (2007) observed that 5% inoculum size was found to be preferable for bioflocculant production by.

4.4 Effect of Cations on the Flocculating Activity

Cations play a vital role in flocculation inneutralizing and stabilising the residual negative chargeof both functional groups of bioflocculant and the surfacecharge of the suspended particles, and consequentlyweaken electrostatic repulsion between particles, thusenhancing floc- culation (Okaiyeto., 2014; Ugbenyen., 2014; Kunle., 2016). From our findings, thehighest flocculating activity was observed with CaCl2andfollowed by MnSO4and AlCl3. However, many previous studies havereported on human health being implicated in aluminumresidual water. As a result, calcium chloride waschosen as a cation of choice in this study.

4.5 Time Course of the Strain NUM8 Flocculant Production

The result of time course of the strain NUM8 flocculant production is similar with the results of Shih. (2001), Lu. (2005), and Xia. (2008), which bioflocculants were collected in the latter logarithmic growth phase and the early stationary phase and decreased after those stages. Similar trendswere observed that bioflocculants attained maximum flocculating activityat 72h, after which flocculating activity declined withcultivation time (Gong.,2008;Elkady., 2011; Ugbenyen.,2012). Contrary to the above, Zheng.(2008) found that the production of bioflocculant bysp.F19 increased with an increase in cultivation time with themaximum flocculating activity reached after 36h. While the flocculating activity produced bysp. W31 increasedwith cultivation time and rea- ched maximum at 60h (Gao.,2006).

4.6 Chemical Analysis of Bioflocculant Composition

A number of other organisms have been reported to produce different kinds of glycoprotein bioflocculants (Xia., 2008; Okaiyeto.,2014; Yin.,2014; Giri.,2015; Okaiyeto.,2016; Jiang.,2019). While a number of other organisms were reported to pro- duce polysaccharide where no amino acids were detected (Wang., 2007; Ugbenyen.,2014; Yang.,2015).

5 Conclusions

The bioflocculant produced bysp. NUM8 showed good flocculating activity for kaolin suspension. Sucrose and sodium nitrate were used as sole carbon and nitrogen sources for optimal bioflocculant production by the bacteria, respectively. The divalent cation (Ca2+) as well as initial medium pH 5.0 resulted in optimal production of bioflocculant. The flocculating activity sustained rise and reached the highest 94.5% at culture time of 72h. The chemical analyses indicated the bioflocculant to be a glycoprotein made up of about 34.3% protein and 63.4% total carbohydrate. The high flocculating activity of the strain NUM8 under different physicochemical conditions indicates that the strain has the potential to be utilized on an industrial scale for bioflocculant production, which could serve as a possible substitute for hazardous chemical flocculants commonly utilized in water treatment processes.

Acknowledgements

This work was supported by the Public Welfare Projects in Zhejiang Province (No. LGN21C200001), the Public Welfare Projects in Zhoushan city (Nos. 2021C 41005 and 2021C41007) and the Natural Science Foundation of Marine Fishery Institute of Zhejiang Province (No. 2020KF 010).

Banks, W. A., Niehoff, M. L., Drago, D., and Zatta, P., 2006. Aluminum complexing enhances amyloid protein penetration of blood-brain barrier., 1116 (1): 215-221.

Bouchotroch, S., Quesada, E., Del Moral, A., Llamas, I., and Béjar, V., 2001.sp. nov., a novel moderately halophilic, exopolysaccharide-producing bacterium., 51 (5): 1625-1632.

Chen, T., Gao, B., and Yue, Q., 2010. Effect of dosing method and pH on color removal performance and floc aggregation of polyferric chloride-polyamine dual coagulant in synthetic dyeing wastewater treatment., 355 (1): 121-129.

Cosa, S., Mabinya, L. V., Olaniran, A. O., Okoh, O. O., and Bernard, K., 2011. Bioflocculant production bysp. Rob isolated from the bottom sediment of Algoa Bay in the Eastern Cape, South Africa., 16 (3): 2431- 2442.

Elkady, M. F., Soha, F., Sahar, Z., Gadallah, A., and Desouky, A., 2011.strain 32A, a bioflocculant- produing bacteria isolated from an Egyptian salt production pond., 102 (17): 8143-8151.

Essandoha, M., Garcia, R. A., Nieman, C. M., and Strahanb, G. D., 2020. Influence of methylation on the effectiveness of meat and bone meal protein as a bioflocculant., 122: 55-61.

Gao, J., Bao, H. Y., Xin, M. X., Liu, Y. X., Li, Q., and Zhang, Y. F., 2006. Characterization of a bioflocculant from a newly isolatedsp. W31., 7 (3): 186-192.

Giri, S. S., Harshiny, M., Sen, S. S., Sukumaran, V., and Park, S. C., 2015. Production and characterization of a thermostable bio-flocculant fromF9, isolated from wastewater sludge., 121: 45-50.

Gong, W. X., Wang, S. G., Sun, X. F., Liu, X. W., Yue, Q. Y., and Gao, B. Y., 2008. Bioflocculant production by culture ofand its application in wastewater treatment., 99 (11): 4668-4674.

He, J., Zhen, Q., Qiu, N., Liu, Z., Wang, B., Shao, Z., and Yu, Z., 2009. Medium optimization for the production of a novel bioflocculant fromsp. V3a’ using response surface methodology., 100: 5922-5927.

He, J., Zou, J., Shao, Z., Zhang, J., Liu, Z., and Yu, Z., 2010. Characteristics and flocculating mechanism of a novel bioflo- cculant HBF-3 produced by deep-sea bacterium mutantsp. V3a’., 26 (6): 1135-1141.

He, N., Li, Y., and Chen, J., 2004. Production of a polygalacturonic acid bioflocculant REA-11 by., 94 (1): 99-105.

Jiang, J. P., Liu, L. H., Nie, W., Chen, Y. B., and Wang, Z., 2019. Screening of a high bioflocculant-producing bacterial strain from an intensive fish pond and comparison of the bioflocculation effects with., 50 (4): 1047-1056.

Jensen, P. R., Erin, G., Chrisy, M., Tracy, J. M., and William, F., 2005. Culturable marine actinomycete diversity from tropical Pacific Ocean sediments., 7 (7): 1039-1048.

Kunle, O., Uchechukwu, U. N., Arinze, S. O., Leonard, V. M., and Anthony, I. O., 2016. Studies on bioflocculant production bysp. AEMREG7., 25 (1): 236-245.

Levesque, L., Mizzen, C. A., Mclachlan, D. R., and Fraser, P. E., 2000. Ligand specific effects on aluminium incorporation and toxicity in neurons and astrocytes., 877 (2): 191-202.

Li, Y., He, N., Guan, H., Du, G., and Chen, J., 2003. A novel polygalacturonic acid bioflocculant REA-11 produced by: A proposed biosynthetic path- way and experimental confirmation., 63 (2): 200-206.

Li, Z., Zhong, S., Lei, H. Y., Chen, R. W., Yu, Q., and Li, H. L., 2009. Production of a novel bioflocculant byX14 and its application to low temperature drinking water treatment., 100 (4): 3650- 3656.

Lian, B., Chen, Y., Zhao, J., Teng, H. H., Zhu, L., and Yuan, S., 2008. Microbial flocculation by: Applications and mechanisms., 99 (11): 4825-4831.

Liu, C., Wang, K., Jiang, J. H., Liu, W. J., and Wang, J. Y., 2015a. A novel bioflocculant produced by a salt-tolerant, alkaliphilic and biofilm-forming strainC9 and its application in harvestingUTEX2341., 93: 166-172.

Liu, H., Chen, G., and Wang, G., 2015b. Characteristics for production of hydrogen and bioflocculant bysp. XF-56 from marine intertidal sludge., 40 (3): 1414-1419.

Liu, J. W., Ma, J. W., Liu, Y. Z., Yang, Y., Yue, D. B., and Wang, H. T., 2014. Optimized cultivation of a bioflocculant M-C11 produced byand its application in sludge dewatering., 35 (3): 1183-1189.

Liu, W. J., Wang, K., Li, B. Z., Yuan, H. L., and Yang, J. S., 2010. Production and characterization of an intracellular bioflocculant byW6 cultured in low nutrition medium., 101 (3): 1044-1048.

Lu, W. Y., Zhang, T., Zhang, D. Y., Li, C. H., Wen, J. P., and Du, L. X., 2005. A bioflocculant produced byand its use in defecating the trona suspension., 27 (1): 1-7.

Luo, Y., Li, C., Abbasi, A. M., Hou, Y., and Fu, X., 2016. Screening of bioflocculant-producing bacteria and its application in clarification process of sugarcane juice., 118: 34-40.

Mabinya, L. V., Cosa, S., Nwodo, U., and Okoh, A. I., 2012. Studies on bioflocculant production bysp. Raats, a fresh water bacteria isolated from Tyume River, South Africa., 13: 1054- 1065.

Nontembiso, P., Sekelwa, C., Leonard, M. V., and Anth, O. I., 2011. Assessment of bioflocculant production bysp. Gilbert, a marine bacterium isolated from the bottom sediment of Algoa Bay., 9 (7): 1232- 1242.

Nwod, U. U., Agunbiade, M. O., Green, E., Nwamadi, M., Rumbold, H., and Okoh, A. I., 2013. Characterization of an exopolymeric flocculant produced by asp.., 6 (4): 1237-1254.

Okaiyeto, K., Nwodo, U. U., Mabinya, L. V., and Okoh, A. I., 2014. Evaluation of the flocculation potential and characterization of bioflocculant produced bysp. Leo., 50 (6): 601-608.

Okaiyeto, K., Nwodo, U. U., Mabinya, L. V., Okoli, A. S., and Okoh, A. I., 2016. Evaluation of flocculating performance of a thermostable bioflocculant produced by marinesp., 138 (2): 1-34.

Patil, S. V., Salunkhe, R. B., Patil, C. D., Patil, D. M., and Salunke, B. K., 2010. Bioflocculant exopolysaccharide production byusing flower extract ofL., 162 (4): 1095-1108.

Polizzi, S., Pira, E., Ferrara, M., Buginani, M., Papaleo, A., Al- bera, R.,., 2002. Neurotoxic effects of aluminum among foundry workers and Alzheimer’s disease., 23: 761-774.

Salehizadeh, H., and Shojaosadati, S. A., 2001. Extracellular biopolymeric flocculants recent trends and biotechnological importance., 19 (5): 371-385.

Sharma, B. R., Dhuldhoya, N. C., and Merchant, U. C., 2006. Flocculants-ecofriendly approach., 14 (2): 195-202.

Shih, I. L., Van, Y. T., Yeh, L. C., Lin, H. G., and Chang, Y. N., 2001. Production of a biopolymer flocculant fromand its flocculation properties., 78 (3): 267-272.

Su, X., Shen, X., Ding, L., and Yokota, A., 2012. Study on the flocculability of thesp., an actinomycete resuscitated from the VBNC state., 28 (1): 91-97.

Tang, J. Y., Qi, S. J., Li, Z. G., An, Q., Xie, M. Q., Yang, B.,., 2014. Production, purification and application of polysaccharide-based bioflocculant by., 113C: 463-470.

Tawila, Z., Ismail, S., Amrc, S. A., and Elkhairb, E. A., 2019. A novel efficient bioflocculant QZ-7 for the removal of heavy metals from industrial wastewater., 15: 27825- 27834.

Ugbenyen, A., Sekelwa, C., Mabinya, L. V., Olubukola, O. B., Farhad, A., and Okoh, A. I., 2012. Thermostable bacterial bioflocculant produced bysp. isolated from Algoa Bay (South Africa)., 9 (6): 2108-2120.

Ugbenyen, A. M., Cosa, S., Mabinya, L. V., and Okoh, A. I., 2014. Bioflocculant production bysp. Gilbert isolated from a marine environment in South Africa., 50 (1): 49-54.

Wang, L., Ma, F., Qu, Y., Sun, D., Li, A., Guo, J.,., 2011. Characterization of a compound bioflocculant produced by mixed culture ofF2 andF6., 27 (11): 2559-2565.

Wang, S. G., Gong, W. X., Liu, X. W., Tian, L., Yue, Q. Y., and Gao, B. Y., 2007. Production of a novel bioflocculant by cul- ture ofusing dairy wastewater., 36 (2): 1-6.

Wu, H., Li, Q., Lu, R., Wang, Y., Zhuang, X., and He, N., 2010. Fed-batch production of a bioflocculant from., 37 (11): 1203-1209.

Xia, S., Zhang, Z., Wang, X., Yang, A., Chen, L., Zhao, J.,., 2008. Production and characterization of bioflocculant byTJ-1., 99 (14): 6520-6527.

Yang, M., Liang, Y., Dou, Y, Jia X, Che, H., 2015. Isolation and identification of a bioflocculant-producing strain and optimisation of cultural conditions via a response surface model., 31 (7): 650-660.

Yim, J. H., Kim, S. J., Ahn, S. H., and Hong, K. L., 2007. Characterization of novel bioflocculant, p-KG03, from a marine dinoflagellate,KG03., 98 (2): 361-367.

Yin, Y. J., Tian, Z. M., Tang, W., Li, L., and Song, L. Y., 2014. Production and characterization of high efficiency bioflocculant isolated fromsp. ZZ-3., 171: 336-342.

Yu, G. H., He, P. J., and Shao, L. M., 2009. Characteristics of extracellular polymeric substances (EPS) fractions from excess sludges and their effects on bioflocculability., 100 (13): 3193-3198.

Zaki, S. A., Elkady, M. F., Farag, S., and Abdelhaleem, D., 2013. Characterization and flocculation properties of a carbohydrate bioflocculant from a newly isolated40B., 34 (1): 51-58.

Zhang, Z. Q., Lin, B., Xia, S. Q., Wang, X. J., and Yang, A. M., 2007. Production and application of a novel bio-flocculant by multiple microorganism consortia using brewery wastewater as carbon source., 19 (6): 242-243.

Zheng, Y., Ye, Z. L., Fang, X. L., Li, Y. H., and Cai, W. M., 2008. Production and characteristics of a bioflocculant produced bysp. F19., 99 (16): 7686-7691.

. E-mail: fuwandong@126.com

September 2, 2020;

January 21, 2021;

March 13, 2021

© Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2021

(Edited by Ji Dechun)