Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6567-4
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Characterization of a bioflocculant from potato starch wastewater and its application in sludge dewatering Junyuan Guo 1 & Yuzhe Zhang 1 & Jing Zhao 1 & Yu Zhang 1 & Xiao Xiao 1 & Bin Wang 1 & Bi Shu 1
Received: 19 February 2015 / Revised: 18 March 2015 / Accepted: 20 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract A bioflocculant was produced by using potato starch wastewater; its potential in sludge dewatering and potato starch wastewater treatment was investigated. Production of this bioflocculant was positively associated with cell growth, and a highest value of 0.81 g/L was obtained. When incubated with this bioflocculant, dry solids (DS) and specific resistance to filtration (SRF) of typical wastewater activated sludge reached 20.8 % and 3.9×1012 m/kg, respectively, which were much better than the ones obtained with conventional chemical flocculants. Sludge dewatering was further improved when both the bioflocculant and conventional polyacrylamide (PAM) were used simultaneously. With potato starch wastewater, chemical oxygen demand (COD) and turbidity removal rates could reach 52.4 and 81.7 %, respectively, at pH value of 7.5 when the bioflocculant dose was adjusted to 30 mg/L; from a practical standpoint, the removal of COD and turbidity reached 48.3 and 72.5 %, respectively, without pH value adjustment.
Keywords Bioflocculant . Potato starch wastewater . Sludge dewatering
Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6567-4) contains supplementary material, which is available to authorized users. * Junyuan Guo [email protected] 1
College of Resources and Environment, Chengdu University of Information Technology, Chengdu, Sichuan 610225, China
Introduction Microbial bioflocculant (MBF), secreted by microorganisms during their growth and cell lysis, was a kind of environmentally safe material with the character of being harmless and biodegradable. Along with the increasing requirement to environmental quality, characteristics of bioflocculants (Aljuboori et al. 2013) and their performances have been investigated in the treatment of meat processing wastewater (Gong et al. 2008), swine wastewater (Guo et al. 2013), and heavy metal ions (Guo 2015). High cultivation cost was still the impediment for the industrialized production and application of the bioflocculants (Guo et al. 2013, 2014; Zhao et al. 2012). Recently, attempts have been made to get new efficient mutant and seeking for low-cost substrates to reduce the production cost. For example, activated sludge and corn stover were always used to produce bioflocculant (Liu et al. 2013; More et al. 2012; Wang et al. 2013), which can gain both environmental and economical benefits. Potato starch wastewater, produced in the manufacturing process of potato starch and some related products, is one of the most seriously polluted wastewaters in food industry. It can be used to cultivate microorganisms to produce bioflocculant, due to its large amount of organic pollutants (Pu et al. 2014). Thus, strains that can effectively utilize the large amount of organics in potato starch wastewater to produce bioflocculants are of academic and practical interests. In this study, experiments first determined the active ingredient and characterization of a bioflocculant, which was harvested from potato starch wastewater. Subsequently, the actual application of this bioflocculant in sludge dewatering and potato starch wastewater treatment was investigated under a variety of conditions.
Appl Microbiol Biotechnol
Materials and methods Reagents CaCl2 (Hengxing Chemicals, China) was prepared at the concentration of 3.0 g/L. NaOH and HCl (Sanpu Chemicals, China) were prepared at the concentration of 1.0 mol/L. NaOH and HCl were used to adjust the pH values of sludge and potato starch wastewater samples in sludge dewatering and potato starch wastewater treatment process. Potato starch wastewater Potato starch wastewater in this study was prepared in laboratory according to the procedures of industrial potato starch production. Potatoes (1000 g) were washed and minced, and then the cell sap in potatoes was extruded and centrifuged at the speed of 5000 rpm for 10 min. The obtained potato dregs and starch were washed two times with 1.0 L tap water, respectively, and then placed still for 1 h. All these supernatants were collected and mixed with the wastewater for use. The chemical oxygen demand (COD) and total nitrogen (TN) concentrations of this water were 7836 and 25.6 mg/L; the turbidity and pH value were 695 NTU and 6.8, respectively. Bacteria strain and its bioflocculant Bioflocculant-producing strain, Paenibacillus polymyxa, was deposited in China Center for Type Culture Collection (CCTC C) (No. M206017). This strain was screened from the soil collected in Yuelu Mountain Changsha, China (Ruan et al. 2007; Yang et al. 2006, 2009; Zhang et al. 2013). Before the production of the bioflocculant, this strain was first inoculated in 250 mL seed medium consisting of peptone 10.0 g, beef extracts 3.0 g, and NaCl 5.0 g dissolved in 1.0 L distilled water with the pH value adjusted to 7.0 and was incubated on a reciprocal shaker at 150 rpm and 30 °C for 24 h. After the cultivation, 2.0 %v/v of the above inoculum (or seed liquid) was used to inoculate the fermentation medium of composition 1.0 L potato starch wastewater, 4 g K2HPO 4, 2 g KH2PO4, 0.2 g MgSO4, 0.1 g NaCl, and 2.0 g urea, and was incubated in the same procedure to produce bioflocculant. The bioflocculant was extracted from the whole fermentation liquor and was purified using the methods proposed (Ruan et al. 2007; Yang et al. 2009). Thermal stability of biological products depends on their activity ingredients; as known to all, the bioflocculants with sugars were thermostable, while those made of protein were generally sensitive to heat. In this study, it can be seen that the flocculating activity of the supernatant of the fermentation liquor only decreased by approximately 0.3–10 % after being heated for 30 min at 30, 40, 50, 60, 70, 80, 90, 100, and 110 °C, respectively. This fact that the flocculating activities
varied a little with the changing temperature indicated that the main backbone of this bioflocculant was a polysaccharide rather than a protein. Chemical analysis of the bioflocculant revealed that its sugar content was up to 96.2 % with the molar weight of 1.16× 106 D. The infrared spectrum of the bioflocculant displayed a broad stretching peak in the range from 3408 to 3413 cm−1 which can be assigned to –OH and N–H groups; the peak around 2937 cm−1 was probably an indication of C– H stretching vibration; the peak around 1685 cm−1 was characteristic of C=O groups, indicating the presence of carboxyl groups. The peaks at 1269, 1134, 1065, and 1016 cm−1 were characteristic of C–O groups (Ruan et al. 2007). The presence of these groups is all preferable functional groups for the flocculation process in polyelectrolyte. Sludge dewatering by the bioflocculant Sludge for dewatering tests was obtained from the secondary settling tank at Tuanjie Wastewater Treatment Co., Ltd., Sichuan province, China. Dry solids (DS), specific resistance to filtration (SRF), and pH value of the sludge are 13.2 %, 11.3 × 10 12 m/kg, and 6.5. The sludge dewatering was expressed in terms of dry solids (DS) and specific resistance to filtration (SRF). Flocculants, including the bioflocculant (1.5 g/L), Al2(SO4)3 (8.0 g/L), FeCl3 (8.0 g/L), PAC (4.0 g/ L), and PAM (0.15 g/L), were separately added into a 200-mL mixing chamber with 100 mL sludge, and the mixtures were stirred at the design agitation speed for 10 min. After agitation, all the samples were allowed to stand for 30 min and then were poured into the funnel fitted with a filter paper separately. After 2 min of gravitational drainage, a vacuum of 0.04 MPa was applied. The volume of the filtrate collected every 15 s was recorded. The DS of dewatered sludge was determined according to Eq. (1): DS ¼
W2 100 % W1
ð1Þ
Where W1 is the weight of wet filter cake and W2 is the weight of filter cake after drying at 105 °C for 8 h. The SRF was calculated by the following equation: dV PA2 ¼ dt μðαcV þ Rm AÞ
ð2Þ
Where t is the time (s), V is the filtrate volume (m3), µ is the filtrate viscosity (N s/m2), A is the filter area (m2), P is the pressure drop across filter (N/m2), c is the slurry concentration (kg/m3), α is the SRF and Rm is the resistance of filter medium (neglected). In sludge dewatering, the central composite design (CCD), which is the standard response surface methodology (RSM),
Appl Microbiol Biotechnol
was selected to investigate the interactions of parameters including bioflocculant dose (x1), PAM dose (x2), pH value (x3), CaCl2 dose (x4), and agitation speed (x5). The response variable (y) that represented DS (y1) or SRF (y2) was fitted by a second-order model in the form of quadratic polynomial equation: X m
y ¼ β0 þ
i¼1
X m
β i xi þ
i< j
X m
β i j xi x j þ
βii xi 2
ð3Þ
i¼1
where y is the response variable to be modeled; xi and xj are independent variables which determine y; and β0, βi, and βii are the offset term, the i linear coefficient, and the quadratic coefficient, respectively. βij is the term that reflects the interaction between xi and xj. The actual design ran by the statistic software, Design-expert 7.1.3 (Stat-Ease Inc., USA), is presented in Table 1.
Potato starch wastewater treatment using the bioflocculant Doses of the bioflocculant and pH values of the potato starch wastewater were used to optimize the flocculating conditions for potato starch wastewater treatment. A sample of 1.0 L wastewater was poured in a beaker, and the pH value was adjusted using 1.0 mol/L NaOH or HCl if necessary. The bioflocculant was then added, and the mixture was stirred at the design agitation speed for 10 min and then allowed to stand 30 min. The supernatant was collected and the residual COD and turbidity were determined according to the APHA Standard Methods (APHA 2005).
Results Time course assay of cell quantity and bioflocculant production As seen from the growth curve of the strain in potato starch wastewater medium in Supplementary Fig. S1, the cells were in logarithm growth phase during 6–54 h, with a rapid growth period occurring during 12–18 h, and entered stationary phase since 60 h with a maximum cell number of 19.3×107 (mL−1). The average growth rate was about 3.2×106 (mL−1 h−1). On 78 h and onward, the cells were in death phase. Supplementary Fig. S1 also shows that the cells produced bioflocculants along with their growth, and the bioflocculant quantity was increased rapidly with cultivation time and peaked (0.81 g/L) at 60 h. Afterward, the bioflocculant quantity was decreased monotonically to 0.22 g/L at t=96 h, which may be due to cell autolysis and enzymatic activity decrease.
Table 1 Coded levels for five variables framed by the central composite design Factors
Codes
Codes levels −1
0
1
Bioflocculant (g/L)
x1
0.3
1.5
3.0
PAM (g/L) pH CaCl2 (mg/L) Agitation speed (rpm)
x2 x3 x4 x5
0.05 5.5 30 100
0.15 7.5 60 200
0.25 9.5 90 300
Application of the bioflocculant in sludge dewatering Sludge dewatering by the bioflocculant For the sludge dewatering tests, various conditioners, including 5.0 mL distilled water (blank), 5.0 mL CaCl2 (3.0 g/L), 5.0 mL fermentation liquid, and 2.0 mL fermentation liquid + 3.0 mL CaCl2 (3.0 g/L), were separately added into the sludge samples (100 mL) after the pH values were adjusted to 7.5. From Supplementary Fig. S2, the biggest filtrate volume of 50.5 mL was obtained when 2.0 mL fermentation liquid together with 3.0 mL CaCl2 was added into the sludge. It was not able to improve the sludge dewatering to such an extent by the fermentation liquid alone, so it is meaningful to enhance the sludge dewatering by the utilization of the bioflocculant in presence of Ca2+. A similar feature can also be noted in Supplementary Fig. S3; the biggest volume of the filtrate was obtained when 6.0 mg of CaCl2 (2.0 mL, 3.0 g/L) was used after the addition of 2.0 mL fermentation liquid into the sludge samples (100 mL). Zhang et al. (2010) reached a similar conclusion when the microbial flocculant TJ-F1 was used as a novel conditioner in sludge dewatering. Thus, in presence of 6.0 mg of CaCl2, the bioflocculant was applied to sludge dewatering in the following investigation to get its knowledge of flocculation characteristics. Figure 1 depicted the DS and SRF with different bioflocculant doses, the DS was increased by 2.3–57.6 % when the bioflocculant dose was adjusted in the range of 0.3–3.0 g/L at pH value of 7.5, meaning that the sludge dewatering improved, and the corresponding SRF was decreased by 13.2–65.5 %. The DS and SRF reached their optimal values of 20.8 % and 3.9 × 10 12 m/kg when the bioflocculant was adjusted to 1.5 g/L, a little different from results conducted by Yang et al. (2012), in which the DS was increased from 13.1 to 17.5 % (an increase of 33.6 %) when 6 g/(kg DS) MBF10 was added at pH value of 8.0. Increasing in sludge dewatering attributed to the formation of stabilized flocs. Higher bioflocculant than optimum increased the colloidal matter and the viscosity and produced smaller size flocs. The excessive bioflocculant prevents the small flocs to grow big, and most of the water will then remain trapped in the
Appl Microbiol Biotechnol 25
12 DS
SRF
15 3
10
0.3 0.6 0.9 1.2 1.5 1.8 2.1 2.4 2.7 3.0 Bioflocculant dose (g/L)
12
DS (%)
6
SRF (10 m/kg)
9 20
0
Fig. 1 Effects of bioflocculant dose on the sludge dewatering
interior of the small flocs, which lead to difficulties in sludge dewatering (More et al. 2012). From Fig. 2, this bioflocculant was relatively effective in a pH value range of 6.5–8.5, and the corresponding DS varies in the range of 15.5–20.8 %. The maximum of 20.8 % was achieved at pH value of 7.5 under the premise of its optimum dose of 1.5 g/L. Comparison of sludge dewatering using the bioflocculant with other flocculants From a practical standpoint, no one will adjust the pH value prior to conditioning and dewatering, but the pH value is interesting from a scientific standpoint. The optimization studies performed for pH values and doses of chemical flocculants, including FeCl3, Al2(SO4)3, PAC, and PAM, are depicted in Supplementary Figs. S4–S7. It is clearly showed that PAC and PAM worked well in a wider pH range of 5.5–9.5, while the favorable pH values for FeCl3 and Al2(SO4)3 to improve sludge dewatering was in the range of 5.5–7.5. At their optimal pH value, these four chemical flocculants got their best at 25 0.3 g/L 1.5 g/L 2.7 g/L
0.9 g/L 2.1 g/L
8.0, 8.0, 4.0, and 0.15 g/L, respectively. The declines in DS were observed when moving away from this point, because the excessive flocculant leads to the stabilization of the colloidal system again. Theoretically, both Fe and Al salts will lower the pH value, in this study, when the sludge was without pH value adjustment, after addition of the Fe and Al salts, the pH values of sludge samples were dropped from 6.5 to 6.3 and 6.1, respectively. Likewise, when Ca2+ and the bioflocculant were used, the pH value was still 6.5. Table 2 presented a comparison of sludge dewatering using the bioflocculant with FeCl3, Al2(SO4)3, PAC, and PAM at their optimal doses and pH values. After conditioning, DS and SRF of the sludge treated by the bioflocculant were 20.8 % and 3.9×1012 m/kg, respectively, which were much better than FeCl3, Al2(SO4)3, and PAC, but poorer than PAM. Yang et al. (2009) has reported the fact that the composite flocculants can reduce the risk brought by the synthetic chemical flocculants, since their doses were decreased to the least. Composite flocculants is the combinations of two or more single component flocculant, which can overcome the shortcomings of single flocculant and can improve the flocculating activity. In this study, despite the effective performance of PAM alone, its degraded monomers have been reported to be toxic and non-readily degradable and always carry serious health and environmental concerns; on contrary, bioflocculant has been considered to be biodegradable and environmentfriendly. Thus, in order to achieve the same or better performance by using less dose of PAM, the aid of bioflocculant was selected to be composited with PAM to reduce the risk. When sludge pH value was adjusted to 7.5, 1.5 g/L of the bioflocculant was added into sludge with FeCl3 (8.0 g/L), Al2(SO4)3 (8.0 g/L), PAC (4.0 g/L), and PAM (0.15 g/L), respectively. It was found that there was an evident improvement of sludge dewatering for the combined use of the bioflocculant with PAM. For single bioflocculant and PAM treatment, SRF decreased by 65.5 and 71.7 %, and DS increased to 20.8 and 24.2 %, respectively. The sludge dewatering by the component of the bioflocculant and PAM was improved, with the SRF decreased by 81.4 % and DS increased to 28.4 % under the optimal flocculant conditions.
20 DS (%)
Sludge dewatering by the composite of the bioflocculant and PAM 15
Statistical analysis
10
To understand the influence of bioflocculant dose, PAM dose, pH value, CaCl2 dose, and agitation speed on the corresponding responses (SRF and DS, y1 and y2), the experiments were designed using RSM based on the performance of the bioflocculant and PAM in sludge dewatering. The following equations represent empirical relationship in the form of
3
4
5
6
7
8
9
10
11
12
13
pH values Fig. 2 Effects of pH values on the sludge dewatering by the bioflocculant
Appl Microbiol Biotechnol Table 2 Results of sludge dewatering with different flocculants
Flocculants
Optimal dose (g/L)
Optimal pH
DS (%)
SRF (1012 m/kg)
Blank FeCl3 Al2(SO4)3 PAC PAM
– 8.0 8.0 4.0 0.15
6.5 6.5 7.5 7.5
13.2 16.4 15.9 20.6 24.2
11.3 4.5 4.7 3.8 3.2
Bioflocculant in this study
1.5
7.5
20.8
3.9
DS dry solids, SRF specific resistance to filtration
quadratic polynomial between the two responses and the five factors (x1–x5): y1 ¼ 3:31 þ 0:15x1 þ 0:16x2 −1:22x3 þ 0:19x4 −0:48x5 þ1:45x1 x2 þ 1:50x1 x3 þ 1:80x1 x4 −1:60x1 x5 þ 0:18x2 x3 ð4Þ −0:52x2 x4 þ 0:65x2 x5 −0:98x3 x4 þ 0:13x3 x5 þ 0:35x4 x5 2 2 2 2 2 þ2:92x1 þ 4:64x2 þ 1:19x3 þ 0:19x4 þ 0:58x5 y2 ¼ 26:54 þ 0:03x1 þ 0:21x2 þ 2:38x3 −0:62x4 þ 1:94x5 − 2:97 x1 x2 − 1:78 x1 x3 − 2:33 x1 x4 þ 3:25 x1 x5 − 0:55 x2 x3 þ 0:30 x2 x4 − 2:50 x2 x5 þ 1:47 x3 x4 þ 0:75 x3 x5 − 0:67x4 x5 − 5:52 x1 2 − 7:75 x2 2 − 1:74 x3 2 − 0:30 x4 2 − 1:37 x5 2 ð5Þ Statistical testing of these two models was performed with the Fisher’s statistical method for analysis of variance (ANOVA) (Guo et al. 2013). Results of ANOVA analysis in terms of coded variables for SRF and DS indicated that all the final models were significant at 95 % confidence level with values of ‘Prob>F’ (F
SRF
x3 x1x2 x1x3 x1x4 x1x5 x12 x22 x52 x3 x1x2
–1.22 1.45 1.50 1.80 –1.60 2.92 4.64 0.58 2.38 –2.97
1 1 1 1 1 1 1 1 1 1
0.26 0.52 0.52 0.52 0.52 0.36 0.36 0.36 0.40 0.80
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Characterization of a bioflocculant from potato starch wastewater and its application in sludge dewatering Junyuan Guo 1 & Yuzhe Zhang 1 & Jing Zhao 1 & Yu Zhang 1 & Xiao Xiao 1 & Bin Wang 1 & Bi Shu 1
Received: 19 February 2015 / Revised: 18 March 2015 / Accepted: 20 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract A bioflocculant was produced by using potato starch wastewater; its potential in sludge dewatering and potato starch wastewater treatment was investigated. Production of this bioflocculant was positively associated with cell growth, and a highest value of 0.81 g/L was obtained. When incubated with this bioflocculant, dry solids (DS) and specific resistance to filtration (SRF) of typical wastewater activated sludge reached 20.8 % and 3.9×1012 m/kg, respectively, which were much better than the ones obtained with conventional chemical flocculants. Sludge dewatering was further improved when both the bioflocculant and conventional polyacrylamide (PAM) were used simultaneously. With potato starch wastewater, chemical oxygen demand (COD) and turbidity removal rates could reach 52.4 and 81.7 %, respectively, at pH value of 7.5 when the bioflocculant dose was adjusted to 30 mg/L; from a practical standpoint, the removal of COD and turbidity reached 48.3 and 72.5 %, respectively, without pH value adjustment.
Keywords Bioflocculant . Potato starch wastewater . Sludge dewatering
Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6567-4) contains supplementary material, which is available to authorized users. * Junyuan Guo [email protected] 1
College of Resources and Environment, Chengdu University of Information Technology, Chengdu, Sichuan 610225, China
Introduction Microbial bioflocculant (MBF), secreted by microorganisms during their growth and cell lysis, was a kind of environmentally safe material with the character of being harmless and biodegradable. Along with the increasing requirement to environmental quality, characteristics of bioflocculants (Aljuboori et al. 2013) and their performances have been investigated in the treatment of meat processing wastewater (Gong et al. 2008), swine wastewater (Guo et al. 2013), and heavy metal ions (Guo 2015). High cultivation cost was still the impediment for the industrialized production and application of the bioflocculants (Guo et al. 2013, 2014; Zhao et al. 2012). Recently, attempts have been made to get new efficient mutant and seeking for low-cost substrates to reduce the production cost. For example, activated sludge and corn stover were always used to produce bioflocculant (Liu et al. 2013; More et al. 2012; Wang et al. 2013), which can gain both environmental and economical benefits. Potato starch wastewater, produced in the manufacturing process of potato starch and some related products, is one of the most seriously polluted wastewaters in food industry. It can be used to cultivate microorganisms to produce bioflocculant, due to its large amount of organic pollutants (Pu et al. 2014). Thus, strains that can effectively utilize the large amount of organics in potato starch wastewater to produce bioflocculants are of academic and practical interests. In this study, experiments first determined the active ingredient and characterization of a bioflocculant, which was harvested from potato starch wastewater. Subsequently, the actual application of this bioflocculant in sludge dewatering and potato starch wastewater treatment was investigated under a variety of conditions.
Appl Microbiol Biotechnol
Materials and methods Reagents CaCl2 (Hengxing Chemicals, China) was prepared at the concentration of 3.0 g/L. NaOH and HCl (Sanpu Chemicals, China) were prepared at the concentration of 1.0 mol/L. NaOH and HCl were used to adjust the pH values of sludge and potato starch wastewater samples in sludge dewatering and potato starch wastewater treatment process. Potato starch wastewater Potato starch wastewater in this study was prepared in laboratory according to the procedures of industrial potato starch production. Potatoes (1000 g) were washed and minced, and then the cell sap in potatoes was extruded and centrifuged at the speed of 5000 rpm for 10 min. The obtained potato dregs and starch were washed two times with 1.0 L tap water, respectively, and then placed still for 1 h. All these supernatants were collected and mixed with the wastewater for use. The chemical oxygen demand (COD) and total nitrogen (TN) concentrations of this water were 7836 and 25.6 mg/L; the turbidity and pH value were 695 NTU and 6.8, respectively. Bacteria strain and its bioflocculant Bioflocculant-producing strain, Paenibacillus polymyxa, was deposited in China Center for Type Culture Collection (CCTC C) (No. M206017). This strain was screened from the soil collected in Yuelu Mountain Changsha, China (Ruan et al. 2007; Yang et al. 2006, 2009; Zhang et al. 2013). Before the production of the bioflocculant, this strain was first inoculated in 250 mL seed medium consisting of peptone 10.0 g, beef extracts 3.0 g, and NaCl 5.0 g dissolved in 1.0 L distilled water with the pH value adjusted to 7.0 and was incubated on a reciprocal shaker at 150 rpm and 30 °C for 24 h. After the cultivation, 2.0 %v/v of the above inoculum (or seed liquid) was used to inoculate the fermentation medium of composition 1.0 L potato starch wastewater, 4 g K2HPO 4, 2 g KH2PO4, 0.2 g MgSO4, 0.1 g NaCl, and 2.0 g urea, and was incubated in the same procedure to produce bioflocculant. The bioflocculant was extracted from the whole fermentation liquor and was purified using the methods proposed (Ruan et al. 2007; Yang et al. 2009). Thermal stability of biological products depends on their activity ingredients; as known to all, the bioflocculants with sugars were thermostable, while those made of protein were generally sensitive to heat. In this study, it can be seen that the flocculating activity of the supernatant of the fermentation liquor only decreased by approximately 0.3–10 % after being heated for 30 min at 30, 40, 50, 60, 70, 80, 90, 100, and 110 °C, respectively. This fact that the flocculating activities
varied a little with the changing temperature indicated that the main backbone of this bioflocculant was a polysaccharide rather than a protein. Chemical analysis of the bioflocculant revealed that its sugar content was up to 96.2 % with the molar weight of 1.16× 106 D. The infrared spectrum of the bioflocculant displayed a broad stretching peak in the range from 3408 to 3413 cm−1 which can be assigned to –OH and N–H groups; the peak around 2937 cm−1 was probably an indication of C– H stretching vibration; the peak around 1685 cm−1 was characteristic of C=O groups, indicating the presence of carboxyl groups. The peaks at 1269, 1134, 1065, and 1016 cm−1 were characteristic of C–O groups (Ruan et al. 2007). The presence of these groups is all preferable functional groups for the flocculation process in polyelectrolyte. Sludge dewatering by the bioflocculant Sludge for dewatering tests was obtained from the secondary settling tank at Tuanjie Wastewater Treatment Co., Ltd., Sichuan province, China. Dry solids (DS), specific resistance to filtration (SRF), and pH value of the sludge are 13.2 %, 11.3 × 10 12 m/kg, and 6.5. The sludge dewatering was expressed in terms of dry solids (DS) and specific resistance to filtration (SRF). Flocculants, including the bioflocculant (1.5 g/L), Al2(SO4)3 (8.0 g/L), FeCl3 (8.0 g/L), PAC (4.0 g/ L), and PAM (0.15 g/L), were separately added into a 200-mL mixing chamber with 100 mL sludge, and the mixtures were stirred at the design agitation speed for 10 min. After agitation, all the samples were allowed to stand for 30 min and then were poured into the funnel fitted with a filter paper separately. After 2 min of gravitational drainage, a vacuum of 0.04 MPa was applied. The volume of the filtrate collected every 15 s was recorded. The DS of dewatered sludge was determined according to Eq. (1): DS ¼
W2 100 % W1
ð1Þ
Where W1 is the weight of wet filter cake and W2 is the weight of filter cake after drying at 105 °C for 8 h. The SRF was calculated by the following equation: dV PA2 ¼ dt μðαcV þ Rm AÞ
ð2Þ
Where t is the time (s), V is the filtrate volume (m3), µ is the filtrate viscosity (N s/m2), A is the filter area (m2), P is the pressure drop across filter (N/m2), c is the slurry concentration (kg/m3), α is the SRF and Rm is the resistance of filter medium (neglected). In sludge dewatering, the central composite design (CCD), which is the standard response surface methodology (RSM),
Appl Microbiol Biotechnol
was selected to investigate the interactions of parameters including bioflocculant dose (x1), PAM dose (x2), pH value (x3), CaCl2 dose (x4), and agitation speed (x5). The response variable (y) that represented DS (y1) or SRF (y2) was fitted by a second-order model in the form of quadratic polynomial equation: X m
y ¼ β0 þ
i¼1
X m
β i xi þ
i< j
X m
β i j xi x j þ
βii xi 2
ð3Þ
i¼1
where y is the response variable to be modeled; xi and xj are independent variables which determine y; and β0, βi, and βii are the offset term, the i linear coefficient, and the quadratic coefficient, respectively. βij is the term that reflects the interaction between xi and xj. The actual design ran by the statistic software, Design-expert 7.1.3 (Stat-Ease Inc., USA), is presented in Table 1.
Potato starch wastewater treatment using the bioflocculant Doses of the bioflocculant and pH values of the potato starch wastewater were used to optimize the flocculating conditions for potato starch wastewater treatment. A sample of 1.0 L wastewater was poured in a beaker, and the pH value was adjusted using 1.0 mol/L NaOH or HCl if necessary. The bioflocculant was then added, and the mixture was stirred at the design agitation speed for 10 min and then allowed to stand 30 min. The supernatant was collected and the residual COD and turbidity were determined according to the APHA Standard Methods (APHA 2005).
Results Time course assay of cell quantity and bioflocculant production As seen from the growth curve of the strain in potato starch wastewater medium in Supplementary Fig. S1, the cells were in logarithm growth phase during 6–54 h, with a rapid growth period occurring during 12–18 h, and entered stationary phase since 60 h with a maximum cell number of 19.3×107 (mL−1). The average growth rate was about 3.2×106 (mL−1 h−1). On 78 h and onward, the cells were in death phase. Supplementary Fig. S1 also shows that the cells produced bioflocculants along with their growth, and the bioflocculant quantity was increased rapidly with cultivation time and peaked (0.81 g/L) at 60 h. Afterward, the bioflocculant quantity was decreased monotonically to 0.22 g/L at t=96 h, which may be due to cell autolysis and enzymatic activity decrease.
Table 1 Coded levels for five variables framed by the central composite design Factors
Codes
Codes levels −1
0
1
Bioflocculant (g/L)
x1
0.3
1.5
3.0
PAM (g/L) pH CaCl2 (mg/L) Agitation speed (rpm)
x2 x3 x4 x5
0.05 5.5 30 100
0.15 7.5 60 200
0.25 9.5 90 300
Application of the bioflocculant in sludge dewatering Sludge dewatering by the bioflocculant For the sludge dewatering tests, various conditioners, including 5.0 mL distilled water (blank), 5.0 mL CaCl2 (3.0 g/L), 5.0 mL fermentation liquid, and 2.0 mL fermentation liquid + 3.0 mL CaCl2 (3.0 g/L), were separately added into the sludge samples (100 mL) after the pH values were adjusted to 7.5. From Supplementary Fig. S2, the biggest filtrate volume of 50.5 mL was obtained when 2.0 mL fermentation liquid together with 3.0 mL CaCl2 was added into the sludge. It was not able to improve the sludge dewatering to such an extent by the fermentation liquid alone, so it is meaningful to enhance the sludge dewatering by the utilization of the bioflocculant in presence of Ca2+. A similar feature can also be noted in Supplementary Fig. S3; the biggest volume of the filtrate was obtained when 6.0 mg of CaCl2 (2.0 mL, 3.0 g/L) was used after the addition of 2.0 mL fermentation liquid into the sludge samples (100 mL). Zhang et al. (2010) reached a similar conclusion when the microbial flocculant TJ-F1 was used as a novel conditioner in sludge dewatering. Thus, in presence of 6.0 mg of CaCl2, the bioflocculant was applied to sludge dewatering in the following investigation to get its knowledge of flocculation characteristics. Figure 1 depicted the DS and SRF with different bioflocculant doses, the DS was increased by 2.3–57.6 % when the bioflocculant dose was adjusted in the range of 0.3–3.0 g/L at pH value of 7.5, meaning that the sludge dewatering improved, and the corresponding SRF was decreased by 13.2–65.5 %. The DS and SRF reached their optimal values of 20.8 % and 3.9 × 10 12 m/kg when the bioflocculant was adjusted to 1.5 g/L, a little different from results conducted by Yang et al. (2012), in which the DS was increased from 13.1 to 17.5 % (an increase of 33.6 %) when 6 g/(kg DS) MBF10 was added at pH value of 8.0. Increasing in sludge dewatering attributed to the formation of stabilized flocs. Higher bioflocculant than optimum increased the colloidal matter and the viscosity and produced smaller size flocs. The excessive bioflocculant prevents the small flocs to grow big, and most of the water will then remain trapped in the
Appl Microbiol Biotechnol 25
12 DS
SRF
15 3
10
0.3 0.6 0.9 1.2 1.5 1.8 2.1 2.4 2.7 3.0 Bioflocculant dose (g/L)
12
DS (%)
6
SRF (10 m/kg)
9 20
0
Fig. 1 Effects of bioflocculant dose on the sludge dewatering
interior of the small flocs, which lead to difficulties in sludge dewatering (More et al. 2012). From Fig. 2, this bioflocculant was relatively effective in a pH value range of 6.5–8.5, and the corresponding DS varies in the range of 15.5–20.8 %. The maximum of 20.8 % was achieved at pH value of 7.5 under the premise of its optimum dose of 1.5 g/L. Comparison of sludge dewatering using the bioflocculant with other flocculants From a practical standpoint, no one will adjust the pH value prior to conditioning and dewatering, but the pH value is interesting from a scientific standpoint. The optimization studies performed for pH values and doses of chemical flocculants, including FeCl3, Al2(SO4)3, PAC, and PAM, are depicted in Supplementary Figs. S4–S7. It is clearly showed that PAC and PAM worked well in a wider pH range of 5.5–9.5, while the favorable pH values for FeCl3 and Al2(SO4)3 to improve sludge dewatering was in the range of 5.5–7.5. At their optimal pH value, these four chemical flocculants got their best at 25 0.3 g/L 1.5 g/L 2.7 g/L
0.9 g/L 2.1 g/L
8.0, 8.0, 4.0, and 0.15 g/L, respectively. The declines in DS were observed when moving away from this point, because the excessive flocculant leads to the stabilization of the colloidal system again. Theoretically, both Fe and Al salts will lower the pH value, in this study, when the sludge was without pH value adjustment, after addition of the Fe and Al salts, the pH values of sludge samples were dropped from 6.5 to 6.3 and 6.1, respectively. Likewise, when Ca2+ and the bioflocculant were used, the pH value was still 6.5. Table 2 presented a comparison of sludge dewatering using the bioflocculant with FeCl3, Al2(SO4)3, PAC, and PAM at their optimal doses and pH values. After conditioning, DS and SRF of the sludge treated by the bioflocculant were 20.8 % and 3.9×1012 m/kg, respectively, which were much better than FeCl3, Al2(SO4)3, and PAC, but poorer than PAM. Yang et al. (2009) has reported the fact that the composite flocculants can reduce the risk brought by the synthetic chemical flocculants, since their doses were decreased to the least. Composite flocculants is the combinations of two or more single component flocculant, which can overcome the shortcomings of single flocculant and can improve the flocculating activity. In this study, despite the effective performance of PAM alone, its degraded monomers have been reported to be toxic and non-readily degradable and always carry serious health and environmental concerns; on contrary, bioflocculant has been considered to be biodegradable and environmentfriendly. Thus, in order to achieve the same or better performance by using less dose of PAM, the aid of bioflocculant was selected to be composited with PAM to reduce the risk. When sludge pH value was adjusted to 7.5, 1.5 g/L of the bioflocculant was added into sludge with FeCl3 (8.0 g/L), Al2(SO4)3 (8.0 g/L), PAC (4.0 g/L), and PAM (0.15 g/L), respectively. It was found that there was an evident improvement of sludge dewatering for the combined use of the bioflocculant with PAM. For single bioflocculant and PAM treatment, SRF decreased by 65.5 and 71.7 %, and DS increased to 20.8 and 24.2 %, respectively. The sludge dewatering by the component of the bioflocculant and PAM was improved, with the SRF decreased by 81.4 % and DS increased to 28.4 % under the optimal flocculant conditions.
20 DS (%)
Sludge dewatering by the composite of the bioflocculant and PAM 15
Statistical analysis
10
To understand the influence of bioflocculant dose, PAM dose, pH value, CaCl2 dose, and agitation speed on the corresponding responses (SRF and DS, y1 and y2), the experiments were designed using RSM based on the performance of the bioflocculant and PAM in sludge dewatering. The following equations represent empirical relationship in the form of
3
4
5
6
7
8
9
10
11
12
13
pH values Fig. 2 Effects of pH values on the sludge dewatering by the bioflocculant
Appl Microbiol Biotechnol Table 2 Results of sludge dewatering with different flocculants
Flocculants
Optimal dose (g/L)
Optimal pH
DS (%)
SRF (1012 m/kg)
Blank FeCl3 Al2(SO4)3 PAC PAM
– 8.0 8.0 4.0 0.15
6.5 6.5 7.5 7.5
13.2 16.4 15.9 20.6 24.2
11.3 4.5 4.7 3.8 3.2
Bioflocculant in this study
1.5
7.5
20.8
3.9
DS dry solids, SRF specific resistance to filtration
quadratic polynomial between the two responses and the five factors (x1–x5): y1 ¼ 3:31 þ 0:15x1 þ 0:16x2 −1:22x3 þ 0:19x4 −0:48x5 þ1:45x1 x2 þ 1:50x1 x3 þ 1:80x1 x4 −1:60x1 x5 þ 0:18x2 x3 ð4Þ −0:52x2 x4 þ 0:65x2 x5 −0:98x3 x4 þ 0:13x3 x5 þ 0:35x4 x5 2 2 2 2 2 þ2:92x1 þ 4:64x2 þ 1:19x3 þ 0:19x4 þ 0:58x5 y2 ¼ 26:54 þ 0:03x1 þ 0:21x2 þ 2:38x3 −0:62x4 þ 1:94x5 − 2:97 x1 x2 − 1:78 x1 x3 − 2:33 x1 x4 þ 3:25 x1 x5 − 0:55 x2 x3 þ 0:30 x2 x4 − 2:50 x2 x5 þ 1:47 x3 x4 þ 0:75 x3 x5 − 0:67x4 x5 − 5:52 x1 2 − 7:75 x2 2 − 1:74 x3 2 − 0:30 x4 2 − 1:37 x5 2 ð5Þ Statistical testing of these two models was performed with the Fisher’s statistical method for analysis of variance (ANOVA) (Guo et al. 2013). Results of ANOVA analysis in terms of coded variables for SRF and DS indicated that all the final models were significant at 95 % confidence level with values of ‘Prob>F’ (F
SRF
x3 x1x2 x1x3 x1x4 x1x5 x12 x22 x52 x3 x1x2
–1.22 1.45 1.50 1.80 –1.60 2.92 4.64 0.58 2.38 –2.97
1 1 1 1 1 1 1 1 1 1
0.26 0.52 0.52 0.52 0.52 0.36 0.36 0.36 0.40 0.80
