Accepted Manuscript Preparation and Application of a Novel Bioflocculant by Two Strains of Rhizopus sp. Using Potato Starch Wastewater as Nutrilite Sheng-yan Pu, Lan-lan Qin, Jun-ping Che, Bao-rong Zhang, Mo Xu PII: DOI: Reference:

S0960-8524(14)00429-5 http://dx.doi.org/10.1016/j.biortech.2014.03.124 BITE 13255

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

31 December 2013 21 March 2014 23 March 2014

Please cite this article as: Pu, S-y., Qin, L-l., Che, J-p., Zhang, B-r., Xu, M., Preparation and Application of a Novel Bioflocculant by Two Strains of Rhizopus sp. Using Potato Starch Wastewater as Nutrilite, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.03.124

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Preparation and Application of a Novel Bioflocculant by Two Strains of Rhizopus sp. Using Potato Starch Wastewater as Nutrilite Sheng-yan Pu a,c∗, Lan-lan Qin a,b*, Jun-ping Che b, Bao-rong Zhang b and Mo Xu a,c a

State Key Laboratory of Geohazard Prevention and Geoenvironment Protection,

Chengdu University of Technology, Chengdu 610059, PR China. b

College of Earth and Environmental Science, Lanzhou University, Lanzhou 730000,

PR China. c

Sichuan Environmental Protection Key Laboratory of Groundwater Antipollution

and Resource Security, Chengdu 610059, PR China.

Abstract: A complex bioflocculant MBF917 was prepared by Rhizopus sp. M9 and M17 using potato starch wastewater (PSW) as nutrilite, and its flocculation characteristics of treating PSW were studied. Culture conditions of the two strains were optimized, and flocculating conditions of the bioflocculant for treating PSW were also investigated. The optimal and economical culture conditions were determined as COD of about 1600 mg/L, 0.3 g/L urea and 0.04 g/L potassium dihydrogen phosphate, with no need of adding carbon sources or adjusting pH. When the bioflocculant was used to flocculate PSW, the optimal dosage was 0.1 mL/L with addition of 5 mL/L 10% CaCl2 as coagulant aid, and there was no need to adjust pH. After flocculation, COD and turbidity removal rates of the PSW could reach 54.09% and 92.11% respectively, and 1.1 g/L proteic substance was recycled from the PSW as ∗

Corresponding author: Address: State Key Laboratory of Geohazard Prevention and Geoenvironment Protection, Chengdu University of Technology, Chengdu 610059, PR China. Tel./fax: +86 (0) 28 8407 3253; E-mail addresses: [email protected] (S.Y. Pu); [email protected](L.L. Qin); [email protected] (M. Xu); Notes: The authors declare that there is no conflict of interests regarding the publication of this paper.

a byproduct that could be used for animal feed. Keywords: Potato starch wastewater; Complex bioflocculant; Cheap cultivation; Flocculation efficiency; Flocculation treatment

1. Introduction Potato starch wastewater (PSW) is usually produced in the manufacturing process of potato starch and some related products. It is one of the most seriously polluted wastewaters in food industry because of the large amount of organic pollutants in it (Abeling & Seyfried, 1993). In pretreatment of wastewater, turbidity caused by protein is usually removed by flocculation treatment (Liu et al., 2013b), which is an easy and effective method to remove suspended solids and colloids. This method has been widely used in drinking water (Li et al., 2009) and wastewater treatments (Buthelezi et al., 2009). Flocculants in water treatment can generally be classified into three major groups: (1) inorganic flocculants, such as aluminum sulfate, ferric chloride and their polymers; (2) organic flocculants, such as organic synthetic flocculants (polyacrylamide), and natural polymer flocculants (chitosan, lignin); (3) bioflocculants. Bioflocculant is a kind of biodegradable macromolecular flocculants secreted by microorganisms, and its main components are glycoprotein, polysaccharide, protein, etc (Yue et al., 2006). Due to their merits of high efficiency, security, nontoxic biodegradation and no secondary pollution, bioflocculants have great potentials for industrial application and have become a research focus in water treatment at present (Salehizadeh & Shojaosadati, 2001). For its avirulence,

bioflocculants are widely used for the recovery of suspended solids (SS). In contrast, chemical flocculants cannot be easily degraded in nature and may result in some healthy and environmental problems (Gross & Kalra, 2002). During recent decades, flocculation characteristics of bioflocculants (Aljuboori et al., 2013) and optimization (Yang et al., 2009) of culture conditions for bioflocculant- producing bacterium have been widely investigated in the treatment of some real wastewaters such as starch wastewater (Deng et al., 2003), oil-field produced water (Yue et al., 2006), dying wastewater (Buthelezi et al., 2012; Liu et al., 2009) meat processing wastewater (Gong et al., 2008), swine wastewater (Guo et al., 2013), and landfill leachate (Zouboulis et al., 2004). Nowadays, extensive attentions have been paid to the bioflocculants produced by compound microorganisms, as well as their low cost production. Some reports show that these bioflocculants have much higher flocculation efficiency than single ones, and that the adaptability of microorganisms to environmental variation is much stronger (Wang et al., 2007a; Zhu et al., 2004). However, bioflocculants have not been widely industrialized due to the high cultivation cost (He et al., 2004; Jang et al., 2001; Zhao et al., 2011). Thus, cheap cultivation mediums including acetic and malt roots were consecutively searched in previous investigations. Besides, some nontoxic and nutrient wastewaters in food and fermentation industry (Jia & Yu, 2012) such as fish meal wastewater (Zhou et al., 2003), distillery wastewater (Wang et al., 2007b), brewery wastewater (Zhang et al., 2007), dairy wastewater (Wang et al., 2007b), and sauce wastewater (Liu et al., 2006), were also used to decrease the cultivation cost. The above reports show that

flocculation efficiency of bioflocculants produced by microorganisms in these wastewaters does not decrease obviously, but the cultivation cost and organic pollutant content in wastewater decrease significantly. Therefore, it is a good way for wastewater utilization, which can gain both environmental and economical benefits. PSW has the same characteristics as the wastewaters mentioned above, but its production is seasonal (September to March). Moreover, conventional anaerobic biological treatments are not fit for it owing to the slow start-up and high cost of investment and operation (Chan et al., 2009). However, the possibility of purifying PSW by using it as a substrate to cultivate microorganisms for some use has been reported (Chang et al., 2008; Liu et al., 2013a; Nitschke & Pastore, 2006), and the results indicate that hydrolysis and sterilization of PSW and nutrilite supplementation are both unnecessary. In this paper, two bioflocculant-producing microorganisms were isolated to produce complex bioflocculant, and the flocculation characteristics of the bioflocculant were studied. The PSW culture conditions of microorganisms were optimized, and the obtained complex bioflocculant was used to treat PSW at the end of this work. 2. Materials and Methods 2.1. Materials Microorganisms: The bioflocculant-producing microorganisms were isolated from soil, sauces, musty substance and activated sludge. Cultivation Mediums: In order to make the wastewater cultivation conditions

much simpler, potato dextrose agar (PDA) medium used to cultivate fungi was used in this study, since the pH of PSW was generally between 4 and 6 under which the fungi grew well. The composition of isolation medium was as follows: 20 g peeled off and minced potato which had been boiled in 100 mL water for 30 min, 2 g glucose, and 2 g agar, with no need to adjust pH. The screening medium contained the same component as the isolation medium except agar. Wastewater cultivation medium was PSW diluted by some times, and some carbon, nitrogen and phosphorus sources were added into it. Potato Starch Wastewater: PSW in this study was prepared in laboratory according to the procedures of industrial potato starch production. 1000 g potatoes were washed and minced, and then the cell sap in potatoes was extruded and centrifugated at the speed of 3000 rpm for 10 min. The obtained potato dregs and starch were washed twice with 1 liter tap water respectively, and then placed still for 1 h. All these supernatants were collected and mixed with the wastewater for use. The water quality of the wastewater was shown in Table 1.

2.2. Experimental details 2.2.1. Screening and identification of bioflocculant-producing microorganisms Microorganisms were isolated by method of dilution butter on plate. The diluted samples were inoculated on the sterilized plates. They were cultivated in an incubator at 28℃ for 2 days into 50 mL isolation medium. Then, different microorganisms were inoculated into 50 mL screening medium in 150 mL flasks respectively, and they were

cultivated in a rotary shaker at the speed of 150 rpm and 28℃ for 2 days. Several drops of fermentation broth were added into 100 mL kaolin suspension (5 g/L) in 250 mL beaker, and the flocculating activity of the ones that could flocculate the suspension was measured. Five bioflocculant-producing microorganisms with the highest flocculating activity were selected to construct the multiple-microorganism consortia, and then inoculated at 28℃ for 2 days into isolation slant culture-medium for further experiments. According to morphological observations, based on colonial morphology and mycelial morphology observed under a microscope, bioflocculant-producing bacteria could be identified in combination with Handbook of Fungus Identification. 2.2.2. Preparation of bacterial suspension and multiple-microorganism consortia Sterile pipette was used to drop 5 mL sterile water on two slants of each strain. After scrape the spores on the slant by inoculating loop, some water was injected into the sterile conical flask containing glass beads under aseptic conditions. Then, the same method was applied to add 15 mL sterile water on the slant in order to scrape out the remaining spores. After treated with horizontal rotating oscillation for 3min, spore suspension of the two slants was merged into the conical flask and poured into sterile funnel containing absorbent cotton. Thus, the single bacterial suspension was prepared. To construct the multiple-microorganism consortia producing bioflocculant with high flocculating activity, arbitrary two strains of the five strains were combined into1 mL (0.5 mL: 0.5 mL) suspension, and bacterial suspension of each strain in the same

consortia was inoculated in 50 mL screening medium in a rotary shaker with the speed of 150 rpm at 28℃ for 2 days. The flocculating activity of the fermentation broth was measured. The combination with highest flocculating activity was inoculated at 28℃ for 7 days into isolation slant culture-medium and preserved in the fridge for further study. 2.2.3. Distribution of the flocculating activity Flocculating rate was used as a measurement of the flocculating activity. Kaolin suspension was formed by suspending 0.5 g amount of kaolin clay (200 meshes) into 100 mL deionized water and certain amount of bioflocculant (fermentation broth) was added in the reactor. The mixture was stirred at 300 rpm for 30 s and then 150 rpm for 5 min. Then, the mixture was kept still for 10 min. Hanna HI93703-11 turbidity meter (Pallarès et al., 2011) was used to measure the turbidity of the liquid at the depth of 0.1 m beneath the supernatant liquid level, which was sucked up by syringe. The turbidity of blank control with no bioflocculant but distilled water was also measured. The flocculating rate can be calculated as follows: Flocculating rate (%) =(A-B)/A×100%, where A and B are the turbidity of the blank control and the supernatant, respectively. The fermentation broth was centrifugated at 3000 rpm for 10 min, and the flocculating rates of the supernatant (A), cell suspended in the same volume of distilled water (B), cell suspended in the same volume of distilled water after being washed (C) and the fermentation broth without centrifugation (D) were measured, respectively.

2.2.4. Purification of the complex bioflocculant The fermentation broth was centrifugated at 3000 rpm for 10 min, and then the supernatant was poured into two volumes of cold ethanol and centrifugated at 3000 rpm for 10 min again to precipitate the crude bioflocculant. 2.2.5. Composition and thermal stability analysis of the complex bioflocculant The flocculating rate of the supernatant was measured after being heated in water bath at different temperatures for 10 min and in boiled water for different times to show the thermal stability. The saccharide in the bioflocculant was determined by Molish reaction and anthrone reaction, while the protein and amino acid were determined by ninhydrin reaction, biuret reaction and protein yellow reaction. The outlines of these reactions are as follows (Zhang et al., 2012): Molish reaction: Furfural and its derivatives are synthesized by dehydration reaction of carbohydrate with concentrated sulfuric acid, and then the resultant reacts with alpha–naphthol to form amaranth compound. Anthrone reaction: Anthrone reacts with saccharides to produce cyan compound under the treatment of concentrated sulfuric acid. Biuret reaction: Polypeptide in protein with similar structure to biuret combines with Cu 2+ to generate amaranth compound under alkaline condition. Ninhydrin reaction: Alpha–amino acid and protein can react with ninhydrin to produce bluish violet compound, which can be used to determine whether the bioflocculant contains protein and amino acid.

Protein yellow reaction: With concentrated nitric acid, yellow compound can be produced by heating the mixture of protein containing benzene ring. 2.2.6. Optimization of wastewater cultivation conditions The sterilization of wastewater, kind of carbon sources, COD concentration of wastewater, concentration of nutrilite, compound proportion of two strains, pH of wastewater, inoculation amount, cultivation time and proportion of wastewater medium volume and vessel cubage were used as main factors to optimize the culture conditions, and the flocculating rate of kaolin suspension was used as optimizing index. The optimization experiments were carried out in 50 mL wastewater culture medium which was prepared by diluting PSW to different multiple in 150 mL flakes which would be put in a rotary shaker with the speed of 150 rpm at 28℃ for 2 days. 2.2.7. PSW treatment using the novel bioflocculant Dosage of bioflocculant, pH of wastewater, kind of added cations, dosage of coagulant aid and sedimentation time were used to optimize the flocculating conditions of the complex bioflocculant for PSW. 2.3. Instruments. Olympus CH20 Biomicroscopy; DZKW-D-4 Electro-Thermostatic Water Bath; YX 280 B stainless and portable pressure-stream sterilizer; HZQ-X100A constant temperature incubator; DHG-9145A Electric constant temperature drying oven; JJ-4A digital motor stirrer; Beijing Jingli LD5-2A centrifuge.

3. Results and Discussion 3.1. Preparation of the complex bioflocculant by cultivating two strains of Rhizopus sp. 3.1.1. Screening and identification of microorganisms 28 strains of mould and 15 strains of microzyme which could produce bioflocculant with flocculating rate over 80% were isolated from samples in this study. Among the 10 combination modes with the best five strains, 7 combination modes could improve the flocculating rates because of the synbiosis between two strains (Kurane & Matsuyama, 1994), and the flocculating activity of the bioflocculant produced by multiple-microorganism consortia M9+M17 was the highest one, which could reach a flocculating rate of 95.46% at the dosage of 0.2 mL fermentation broth per liter kaolin suspension. M9 and M17 are both mould. The colony of M9 is circular, and the mycelium is offwhite, flocculent and long. The colony of M17 is also circular, while the mycelium is white, flat and much shorter. According to microscopic examination, rhizoid and stolon were found while no diaphragm appeared. The sporangium was round and sporangiospore could be found. Based on the Handbook of Fungus identification by Jingchao Wei (Wei, 1979), M9 and M17 were identified as Rhizopus sp., and the produced bioflocculant was named as MBF917. 3.1.2. Distribution of flocculating activity and purification of the complex bioflocculant The flocculating rate of each part of the fermentation broth from multiple-

microorganism consortia M9+M17 was shown in Fig. 1. From Fig. 1, the supernatant had the highest flocculating rate, while the washed cells had the lowest, which confirmed that the flocculating substance mainly distributed in the supernatant. It was the viscous secretion produced by M9 and M17 but not their cells that had the flocculating activity. This is similar to most strains such as Klebsiella sp. MYC (Yue et al., 2006) and Serratia ficaria (Gong et al., 2008). After adding 20 mL pre-cooling ethanol of 4℃ into 10 mL supernatant containing multiple-microorganism consortia M9+M17 (0.5 mL: 0.5 mL), 0.0123g crude bioflocculant (MBF917) was obtained through centrifugation and drying process, which was a kind of colourless and transparent crystal. 3.1.3. Thermal stability and composition analysis of the novel bioflocculant Thermal stability of the bioflocculant was shown in Fig.2. It can be seen that the flocculating rate of the supernatant of multiple-microorganism consortia M9+M17 only decreased by approximately 4% after being heated in boiled water for 60 min at a dosage of 0.2 mL (the supernatant) /1 L (kaolin suspension), which apparently confirmed the thermal stability of bioflocculant similar to MBFA9 (Deng et al., 2003). The chemical composition of MBF917 was analyzed by chromogenic reactions, and the results were shown in Table 2. The main component of MBF917 was identified as polysaccharide rather than protein, which could be attributed to the thermal stability of the bioflocculant because protein denatured easily in boiled water.

3.2. Optimization of wastewater cultivation conditions for bioflocculant production

by compound microorganisms 3.2.1. Effect of sterilization on flocculating activity There are large amounts of microorganisms in PSW (Suyu, 2003). In order to learn the relation between the bioflocculant-producing microorganisms and indigenous microorganisms, sterilization of wastewater (diluted by twice) or not was investigated. Specifically, bacterial suspension of 2 mL M9 and 2 mL M17 was cultivated in 50 mL sterile and non-sterilized PSW diluted by twice respectively, and the flocculating rate of the fermentation broth after cultivation was respectively measured. The results showed that M9 and M17 grew much better in sterilized wastewater and the flocculating rate of fermentation broth after microorganism cultivation was much higher (87.27%), which meant that there was no synbiosis between them. Notably, the flocculating rate of fermentation broth produced by microorganisms cultivated in non-sterilized wastewater without any nutrilite could also reach 81.15%. Considering the industrial reality and the cost of sterilization, wastewater would not be sterilized in this experiment. 3.2.2. Effect of carbon source addition on flocculating activity Experimentally, bacterial suspension of 2 mL M9 and 2 mL M17 was cultivated in 50 mL PSW diluted by twice, and 0.5 mL 10 g/L urea, 0.5 mL 10 g/L potassium dihydrogen phosphate and 2% of various carbon sources including 1 g glucose, 1 g starch, 1 mL methanol and 1 mL 95% ethanol were added respectively. The flocculating rates of fermentation broth were shown in Fig. 3.

During the experiment, it was found that microorganisms grew best and gave off alcohol aroma in wastewater with addition of glucose, but that the flocculating rate was very low. This meant that glucose could promote the growth of other microorganisms without bioflocculant production ability in wastewater, but it was not a good carbon source for M9 and M17. In contrast, starch, methanol and ethanol were all better carbon source for M9 and M17 growth and bioflocculant production, among which ethanol was the best. 3.2.3. Effect of nutrilite addition on flocculating activity The effect of COD concentration of PSW and the concentration of carbon (95% ethanol), nitrogen (10 g/L urea) and phosphorus (10 g/L potassium dihydrogen phosphate) sources on the flocculating rate was investigated by orthogonal experiments (Table 3). 50 mL wastewater culture medium was prepared by diluting PSW to different multiple, and the carbon, nitrogen and phosphorus sources were added into the culture medium. Under the optimal dosage of fermentation broth after cultivation, the flocculating rates were measured. Here, the inoculation amount of M9 and M17 were both 2 mL bacterial suspension.

It can be seen from Table 3 that microorganisms did not grow very well in high COD concentration wastewater with high osmotic pressure, and that the addition of nutrilite restrained their growth. The more the nutrilite was added, the more strongly their growth was restrained. In contrast, when the wastewater was diluted to lower COD concentration, nutrilite in it was not enough for microorganisms’ growth and the

addition of nutrilite would increase the cultivation cost. Different addition amounts of nutrilite would cause different results. The R values in Table 3 indicated that the COD concentration of wastewater and the concentration of carbon source addition had greater effects on the flocculating rate than the concentration of phosphorus source addition, whereas the concentration of nitrogen source addition had little effect. According to the flocculating rates, carbon source in wastewater was enough for microorganisms’ growth while nitrogen source was deficient. In view of this, urea was needed to make the wastewater more suitable for microorganisms’ growth. The optimal cultivation conditions for the compound microorganisms producing bioflocculant were as follows: COD of about 1600 mg/L, 0.3 g/L urea and 0.04 g/L potassium dihydrogen phosphate. The flocculating rate of fermentation broth produced under these conditions for 2 days could reach 90.43% at the dosage of 1 mL broth per liter kaolin suspension. 3.2.4. Effect of compound proportion of two strains on flocculating activity Different proportions of the two strains (total amount was 5 mL) were inoculated into 50 mL wastewater culture medium under the optimal cultivation conditions, and the flocculating rates of fermentation broth on kaolin suspension were investigated. Apparently, there was good synergistic effect between M9 and M17, and the flocculating rate would drop if decreasing the amount of any one. The bioflocculant production ability of M17 was much stronger than that of M9, so that less amount of bacterial suspension was needed under the highest flocculating rate. The compound proportion of 3:2 (M9:M17) was determined as the best one for further study.

3.2.5. Effect of initial pH on flocculating activity M9 and M17 in optimum compound proportion (3 mL:2 mL) were inoculated into 50 mL wastewater culture medium in different pH values under the optimal cultivation conditions. The effect of initial pH of the wastewater culture medium on the flocculating activity of the complex bioflocculant was investigated. When the initial pH was over the range of neutral and weak acidic conditions (pH 4.5 - pH 7.5), the flocculating rates of fermentation broth were all above 85%, with the highest flocculating rate of 92.14% at pH = 5.5. This pH range just covered the pH range of PSW and mould growth, so that pH adjustment was not needed in this experiment. 3.2.6. Effect of total inoculation amount, cultivation time and dosage of bioflocculant on flocculating activity Different total amounts of compound microorganisms were inoculated in 50 mL wastewater culture medium under the optimal cultivation conditions and pH values, respectively. The flocculating rates of fermentation broth after cultivation for different time at different dosages were investigated and the results were shown in Table 4. The results indicated that the total inoculation amount, cultivation time and dosage of bioflocculant should be considered together. There was an optimal dosage for each inoculation amount for different cultivation times. The flocculating activity would be the highest under optimal dosage, but decrease when the dosage was more or less than the optimal one. The flocculating efficiency would be poor not only under lower inoculation amount because of too low concentration of bioflocculant in the

fermentation broth, but also under higher inoculation amount because of too high bioflocculant concentration in the fermentation broth. When bioflocculant was insufficient, the bridging phenomenon could not effectively form. In contrast, excessive addition of negatively charged bioflocculant would cause the competition and repulsion of negatively charged kaolin particles, which led to the poor settling ability (Gong et al., 2008). Under this situation, colloid particles would be surrounded by large amount of polymers in wastewater and keep stable due to absence of bridging possibility.

3.2.7. Effect of proportion of wastewater medium volume and vessel cubage on flocculating activity Microorganisms used in this study were both aerobic ones and large amount of air should be supplied during their growth. Wastewater medium volume in vessel can affect the air quantity directly, i.e., large amount of wastewater in vessel makes less air left, so that microorganisms will grow badly under this condition, thus causing the decrease of the flocculating rate. Fig. 4 showed the flocculating rates of fermentation broth produced by cultivating M9 (60 mL/L) and M17 (40 mL/L) under the optimal cultivation conditions and pH values in different amount of wastewater in 250 mL flasks. The results indicated that when the proportion of wastewater medium volume and vessel cubage was under 4/5, the air quantity in vessels would maintain the growth of microorganisms under stirring function, which had little effect on flocculating activity of the produced bioflocculant.

3.3. Effect analysis of bioflocculant produced by microorganisms cultivated in PSW The cultivation of bioflocculant-producing microorganisms using PSW attained both the environmental and economic benefits. The COD concentration of PSW after microorganisms’ cultivation was 510 mg/L with a removal rate of 93.60% and the turbidity was 122 NTU with a removal rate of 82.87%. After treatment with aerobic method, it could be used to irrigate potato plant field without any other treatment or be discharged directly up to standard. Meanwhile, the bioflocculant production cost decreased significantly when cultivating the microorganisms in PSW, but the flocculating efficiency had no sharp decrease. Thus, the high cost problem of bioflocculant production could be solved. The comparison of microorganisms cultivated in PDA and PSW medium for 2 days was shown in Table 5. The more microorganisms were inoculated, the higher concentration of bioflocculant in fermentation broth and the less optimal dosage were. Under the optimal dosages, the fewer microorganisms were inoculated, the lower flocculating rate was. The flocculating rate could reach 92.71% after cultivation for 48 h at the dosage of 0.5 mL/L, which was just 0.3 mL higher per liter than that produced in PDA medium. In order to save microorganisms and cultivation time, the following cultivation conditions were chosen in this study: 3 mL (60 mL/L) M9 and 2mL (40 mL/L) M17 were inoculated in 50 mL wastewater culture medium for cultivation of 35 h, and then 5 mL fermentation broth per liter was dosed to flocculate kaolin suspension. Under these conditions, the flocculating rate of kaolin suspension could reach 92.67%, and the COD and turbidity removal rates of PSW after cultivation of microorganisms

were 93.60% and 82.87%, respectively. Evidently, the cultivated PSW can be used either to discharge to reach standard after aerobic treatment or to irrigate potato plant field after mixed with fresh water.

3.4. PSW treatment using complex bioflocculant Because of its avirulence, complex bioflocculant produced in this study can be used in the flocculating treatment of food industry wastewater to recover protein. The fermentation broth of multiple-microorganism consortia M9+M17 (0.5 mL: 0.5 mL) was applied to treat PSW under different conditions, and the turbidity of the supernatant was measured to calculate the flocculating rates after standing for 10 min. Fig. 5A showed that the flocculating rate decreased when too much (more than 0.1 mL/100 mL) bioflocculant was added, and the reason has been discussed above. According to Fig. 5B, the flocculating rates were all over 80% in the range of pH 4 to 6, which was just the pH of the wastewater, so pH adjustment was not necessary any more. As shown in Fig. 5C, the flocculating effect could be promoted with the addition of divalent cations especially the addition of Mg2+ and Ca2+. However, monovalent cations (Na+, K+) and trivalent cations (Al3+, Fe3+), which were usually used as chemical flocculants in wastewater treatment, had inhibitory effect on the flocculability of the bioflocculant. During the experiment, it was found that the flocculating rate was decreased to 70% below when the bioflocculant was compounded with either AlCl3 or Fe2(SO4)3. Hence, the compound between the bioflocculant and chemical flocculants was not considered.

Besides, the effects of coagulant aid dosage and sedimentation time on flocculating rate of PSW were also investigated and the results were shown in Fig. 5D and 5E. The optimal process for removing turbidity was as follows: 0.1 mL crude bioflocculant and 5 mL 10% CaCl2 were added into 1 liter PSW, with no need to adjust pH. Under the optimal flocculating conditions, the COD of PSW was 3420 mg/L with a removal rate of 54.09% and the turbidity was 56 NTU with a removal rate of 92.11%. Meanwhile, 1.1 g/L protein could be recycled from PSW, which could be used for animal feed because both the bioflocculant and the wastewater were innocuous. 3.5. Flocculating efficiency of bioflocculants and chemical flocculants In order to compare the flocculating efficiency of MBF917 and some chemical flocculants, AlCl3, Fe2(SO4)3 and PAM were employed to treat PSW respectively, and the results (including some other bioflocculants) were shown in Table 6. Apparently, MBF917 had advantages in treating PSW because of the lowest dosage, unnecessary pH adjustment, lowest cost and much higher turbidity and COD removal rates.

4. Conclusions Complex bioflocculant MBF917 with higher flocculating efficiency and lower dosage was produced by compound microorganisms Rhizopus sp. M9 and M17 in this research. PSW was used as the culture medium and the optimal cultivating conditions were determined as: COD of about 1600 mg/L, 0.3 g/L urea and 0.04 g/L potassium dihydrogen phosphate, with no need of pH adjustment and sterilization. As an industrial trial, MBF917 was used to treat PSW, and the optimal condition for

removing turbidity was 0.1 mL/L crude bioflocculant and 5 ml/L 10% CaCl2, with no need to adjust pH.

Acknowledgments The authors thank Prof. You-le Wang for a helpful discussion. This work was supported by the National Natural Science Foundation of China and the Research Fund of State Key Laboratory of Geohazard Prevention and Geoenvironment Protection (No. SKLGP2013Z009, No. SKLGP2014Z006). Dr. S. Y. Pu is grateful for support from the Cultivating Program of Middle-Aged Key Teachers of Chengdu University of Technology (NO. KYGG201305) and the Project Funded by China Postdoctoral Science Foundation.

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12. Jang, J.-H., Ike, M., Kim, S.M., Fujita, M. 2001. Production of a novel bioflocculant by fed-batch culture of Citrobacter sp. Biotechnology letters, 23(8), 593-597. 13. Jia, B.-j., Yu, J.-m. 2012. The research status and development trend of microbial flocculant. Physics Procedia, 24, 425-428. 14. Kurane, R., Matsuyama, H. 1994. Production of a bioflocculant by mixed culture. Bioscience Biotechnology Biochemistry, 58, 1589-1594. 15. Li, Z., Zhong, S., Lei, H.-y., Chen, R.-w., Yu, Q., Li, H.-L. 2009. Production of a novel bioflocculant by Bacillus licheniformis X14 and its application to low temperature drinking water treatment. Bioresource technology, 100(14), 3650-3656. 16. Liu, H., Zhou, K., Hu, Y., Cheng, W., Liu, J., Zhou, Y. 2006. Study on the production of bioflocculant by Penicillium sp. using sauce wastewater. Techniques and Equipment for Environmental Pollution, 7, 40-44;. 17. Liu, J.-X., Yue, Q.-Y., Gao, B.-Y., Wang, Y., Li, Q., Zhang, P.-D. 2013a. Research on microbial lipid production from potato starch wastewater as culture medium by Lipomyces starkeyi. Water Science & Technology, 67(8). 18. Liu, W., Yuan, H., Yang, J., Li, B. 2009. Characterization of bioflocculants from biologically aerated filter backwashed sludge and its application in dying wastewater treatment. Bioresource technology, 100(9), 2629-2632. 19. Liu, Z.-y., Hu, Z.-q., Wang, T., Chen, Y.-y., Zhang, J., Yu, J.-r., Zhang, T., Zhang, Y.-f., Li, Y.-l. 2013b. Production of novel microbial flocculants by Klebsiella sp. TG-1 using waste residue from the food industry and its use in defecating the trona

suspension. Bioresource technology, 139, 265-271. 20. Nitschke, M., Pastore, G.M. 2006. Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava wastewater. Bioresource Technology, 97(2), 336-341. 21. Pallarès, A., François, P., Pons, M.-N., Schmitt, P. 2011. Suspended particles in wastewater: their optical, sedimentation and acoustical characterization and modeling. Water Science & Technology, 63(2). 22. Salehizadeh, H., Shojaosadati, S. 2001. Extracellular biopolymeric flocculants: recent trends and biotechnological importance. Biotechnology advances, 19(5), 371-385. 23. Suyu, L. 2003. Cooperative Effect of Varied Microorganism in Purifying Maize Starch Wastewater. Environmental Protection, 1, 008. 24. Wang, S.-G., Gong, W.-X., Liu, X.-W., Tian, L., Yue, Q.-Y., Gao, B.-Y. 2007a. Production of a novel bioflocculant by culture of Klebsiella mobilis using dairy wastewater. Biochemical engineering journal, 36(2), 81-86. 25. Wang, Y.-n., Wang, X., Zhang, W., Chen, J. 2007b. Study on Culture of Compound Bioflocculant-producing Bacteria by Distillery Wastewater. China Water & Wastewater, 23(1), 38-42. 26. Wei, J. 1979. Hand Book of Fungus Identification. Technology press of Shanghai, Shanghai. PR China. 27. Yang, Z.-H., Huang, J., Zeng, G.-M., Ruan, M., Zhou, C.-S., Li, L., Rong, Z.-G. 2009. Optimization of flocculation conditions for kaolin suspension using the

composite flocculant of MBFGA1 and PAC by response surface methodology. Bioresource technology, 100(18), 4233-4239. 28. Yue, L., Ma, C., Chi, Z. 2006. Bioflocculant produced by Klebsiella sp. MYC and its application in the treatment of oil-field produced water. Journal of Ocean University of China, 5(4), 333-338. 29. Zhang, C.-L., Cui, Y.-N., Wang, Y. 2012. Bioflocculant produced from bacteria for decolorization, Cr removal and swine wastewater application. Sustainable Environment Research, 22(2), 129-134. 30. Zhang, Z.-q., Lin, B., Xia, S.-q., Wang, X.-j., Yang, A.-m. 2007. Production and application of a novel bioflocculant by multiple-microorganism consortia using brewery wastewater as carbon source. Journal of Environmental Sciences, 19(6), 667-673. 31. Zhao, X., Zhao, Y.L., Chen, Z.L., Luo, H.M., Feng, H.X., Jiang, F. 2011. Study on Potato Starch Wastewater Pretreatment Using Flocculation. Applied Mechanics and Materials, 71, 2644-2648. 32. Zhou, X., Wang, J., Zhou, J. 2003. Studies on Properties of a Bioflocculant Produced by Pseudomonas sp. GX4-1 in Fish Meal Wastewater. Research of Environmental Sciences, 16(3), 31-34. 33. Zhu, Y.-b., Feng, M., Yang, J.-x., Ma, F., Wu, B., Li, S.-g., Huang, J.-l. 2004. Screening of complex bioflocculant producing bacteriumand their flocculating mechanism. Journal of Harbin Institute of Technology, 6, 017. 34. Zouboulis, A.I., Chai, X.-L., Katsoyiannis, I.A. 2004. The application of

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Fig.1 Flocculating activity distribution

Fig.2 Effect of heating temperature (A)and heating time (B) on Flocculating Activity

Fig.3 Effect of carbon source addition on flocculating activity

Fig.4 Effect of proportion of wastewater medium volume and vessel cubage on flocculating activity

Fig.5 Effects of flocculating conditions on flocculating rate: A) Dosage of bioflocculant; B) pH; C) Cation species; D) Dosage of coagulant aid; E) Sedimentation time.

Table 1 Water quality of Potato Starch Wastewater CODCr (mg/L)

BOD5 (mg/L)

Total Nitrogen (mg/L)

Total Phosphorus (mg/L)

pH

7965

2360

26.82

26.5

6.0

Turbidity Chroma (NTU) (times) 712

64

Table 2 The chemical composition of MBF917 Experiment item 1

Molish reaction

2 3 4 5

Anthrone reaction Biuret reaction Ninhydrin reaction Protein yellow reaction

Experiment phenomena Clear purple ring was found on the interface of concentrated sulfuric acid and sample. The liquid presented cyan. The color of liquid did not change. There was no phenomenon. It did not present any change.

Experimental conclusion Carbohydrate Polysaccharide. Without polypeptide. Without protein. Without protein.

Table 3 Arrangement of the orthogonal experiments on nutrilite addition

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Ⅰ Ⅱ Ⅲ Ⅳ R

CODCr (mg/L) 1593 1593 1593 1593 1991 1991 1991 1991 3983 3983 3983 3983 7965 7965 7965 7965 281.4 289.1 328.4 329.9 48.5

CO(NH2)2 (g/L) 0.0 0.1 0.2 0.3 0.0 0.1 0.2 0.3 0.0 0.1 0.2 0.3 0.0 0.1 0.2 0.3 303.9 310.1 310.3 304.6 6.4

KH2 PO4 (g/L) 0.1 0.2 0.0 0.04 0.04 0.0 0.2 0.1 0.2 0.1 0.04 0.0 0.0 0.04 0.1 0.2 314.9 316.7 307.2 290.1 26.6

C2H5OH Flocculating Rate (mL/L) (%) 30 79.88 20 77.71 10 81.86 0.0 90.43 20 79.71 30 79.00 0.0 88.86 10 80.85 10 62.85 0.0 80.28 30 73.43 20 72.57 0.0 81.43 10 73.14 20 66.14 30 60.71 341.0 298.7 296.1 293.0 48.0

Table 4 Effect of total inoculation amount, cultivation time and dosage of bioflocculant on flocculating activity Cultivation

Time

Flocculating Rates (%) Total Inoculation

48 h

40 h

35 h

30 h

25 h

20 h

10 h

87.28 87.85 87.57 89.00 90.28 90.57 92.28 92.71

80.28 80.57 82.42 82.85 85.42 85.42 86.14 86.42

88.28 88.63 89.86 91.35 92.67 91.58 89.72 87.21

86.14 86.71 87.85 88.57 90.42 91.71 92.71 93.54

82.00 82.71 82.85 85.57 87.00 88.28 91.28 91.85

65.28 65.57 67.71 70.86 72.14 75.43 84.00 86.57

60.43 62.14 66.71 69.28 75.14 79.00 81.71 82.86

0.5

0.5

2

5

10

10

40

Amount(M9:M17) 1.0(0.6︰0.4) 2.0(1.2︰0.8) 3.0(1.8︰1.2) 4.0(2.4︰1.6) 5.0( (3.0︰ ︰ 2.0) ) 7.5(4.5︰3.0) 10.0(6.0︰4.0) 15.0(9.0︰6.0) Optimal Dosage of Bioflocculant (mL/L)

Table 5 Comparison of microorganisms cultivated in PDA and PSW Culture Medium PDA PSW

Bioflocculant Yield (g/L) 1.23 0.69

Cost (yuan/g rude bioflocculant) 0.862 0.014

Optimal Dosage (mL/L) 0.2 0.5

Flocculating Rate (%) 95.46 90.28

Table 6 Comparison of bioflocculants and chemical flocculants on wastewater treatment Removal Rate (%) Turbidity COD 91.12 44.81 95.69 40.25 90.67 38.90 92.11 54.09

Cost (yuan/ton) 32.05 34.50 0.88 0.001

Flocculant

Dosage

pH

Wastewater

Reference

AlCl3 Fe2(SO4)3 PAM MBF917

600 mg/l 590 mg/l 7 mg/l 0.1 ml/l

7.0 11.5 3.5 4.5

potato starch

this research

MBFA9

0.2 ml/l

9.0

85.55

68.47

/

starch

MMF1

17 ml/l

9.0

/

64.4

/

indigotin

Deng et al.,2003 Zhang et al.,2007

Highlights

Novel complex bioflocculant; High flocculation efficiency; Low cultivation cost;

Preparation and application of a novel bioflocculant by two strains of Rhizopus sp. using potato starch wastewater as nutrilite.

A complex bioflocculant MBF917 was prepared by Rhizopus sp. M9 and M17 using potato starch wastewater (PSW) as nutrilite, and its flocculation charact...
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