A novel bioflocculant MBF057 produced by a salt-tolerant Haloplanus vescus HW0579 was investigated in this study. The effects of culture conditions such as initial pH, inoculum size, and chemical oxygen demand (COD) of K-acid wastewater on MBF0579 production were studied. The result showed that 8.09 g/L purified MBF0579 was extracted with the following optimized conditions: 780 mg/L COD of K-acid wastewater as carbon source, inoculum size 12.5%, and initial pH 7.0. The biopolymer contained 78.6% polysaccharides and 21.1% proteins. The highest flocculating rate of 81.86 and 95.07% for the COD and chroma of acid brilliant scarlet gelb rot (yellow/red, GR) dye wastewater were achieved at a dosage of 150 mg/L, pH 2.0 and contact time 100 min. Overall, these findings indicate bioflocculation offers an effective alternative method of decreasing acid brilliant scarlet GR during dye wastewater treatment.
Azo-dye-contaminated wastewater represents a great threat to the environment and human health. Accordingly, it is necessary to develop feasible efficient methods to diminish azo dye concentration from effluents. To date, a substantial number of methods have been developed to remove dye (Lloret et al. 2011; Blanco-Flores et al. 2014; Hou et al. 2014a, b; Harruddin et al. 2015), among which flocculation is recognized as one of the most effective and inexpensive (Mishra et al. 2014). However, in the past few decades, several synthetic high polymer flocculants that are frequently used owing to their facile operation, high flocculating efficiency, and low cost have been found to be strong carcinogens capable of exerting neurotoxic effects on the pup rabbits brain (Robinson & Bishop 2002). Accordingly, bioflocculation has attracted increasing scientific and technological attention in the wastewater treatment field because it is not dangerous to humans, uses easily biodegraded materials, and is free of secondary pollution by degradation intermediates (Zhong et al. 2014).
High-costs are the major bottleneck of microbial flocculants development for commercial use in wastewater treatment (Zhong et al. 2014). Hence, the industrial-scale production and application of microbial flocculants as potential alternatives to the synthetic ones have yet to be achieved. Although several investigations using inexpensive substrates for microbial flocculants production have been conducted (Wang et al. 2013; Zaki et al. 2013), there have been no studies on the production of microbial flocculants from K-acid (1-amino-8-naphthol-4, 6-disulfonic acid) wastewater.
K-acid is a xenobiotic compound used in the production of several acid, azo-based reactive dyes, and organic pigment. Although K-acid wastewater is very harmful to ecological systems and human health, it is a potentially inexpensive medium and a rich source of carbon and other nutrients that have the potential for use as bioflocculants. Hence, microorganisms that use wastes as substrates for the production of interesting materials not only contribute to the production of these value-added compounds, but also focus on the minimization of waste disposal.
As far as we know, there have been no reports on biodegradation of K-acid. Moreover, to the best of our knowledge, this work is the first report dealing with the production of bioflocculants from archaeal strain. Therefore, the present study was conducted to: (1) isolate and identify bioflocculant-producing archaeons from K-acid wastewater sludge; (2) produce bioflocculant using archaeons isolated from K-acid wastewater; (3) evaluate the performance of this bioflocculant and its application to acid brilliant scarlet GR dye removal.
EXPERIMENTS AND METHODS
Isolation and cultural conditions
Bioflocculant-producing archaeons were isolated from activated sludge samples (pH 7.2–7.5) taken from a K-acid wastewater treatment plant located in Jiangsu, China. Each isolated archaeon was cultivated in screening medium (50 mL) containing 0.25 g/L K-acid, 5.0 g/L (NH4)2SO4, 5.0 g/L K2HPO4, 2.0 g/L KH2PO4, 0.5 g/L MgSO4, and 0.5 g/L NaCl with oscillation (200 rpm) at 35 °C for 3 d. These procedures were the same as previously described (Bala Subramanian et al. 2010).
Next, 1 mL of fermentation broth was added into 100 mL kaolin suspension (4 g/L) in a 250-mL beaker and the flocculating activities of the suspensions were measured. Culture broths propitious to flocculating rate were further explored. Five strains were found to produce flocculants, among which HW0579 exhibited the excellent flocculating activity in kaolin suspension. Therefore, HW0579 was inoculated onto an isolation slant culture-medium and cultivated at 30°C for 4 d, after which it was preserved at 4°C for further study. The determination of morphology, growth characteristics, nutrition, miscellaneous biochemical tests, and sensitivity to antimicrobial agents were conducted for all species in the same medium, according to the proposed minimal standards for description of new taxa in the order Halobacteriales (Shimoshige et al. 2013; Han & Cui 2014). Polymerase chain reaction (PCR) amplification of the 16S rDNA was conducted by Takara Biotechnology Co., Ltd (Shimoshige et al. 2013).
Bioflocculant production and flocculating activity assay
K-acid wastewater (CODCr = 19,094 mg/L, BOD5 = 170 mg/L, pH 4.5, K-acid 2.44 g/L, chromotropic acid 1.23 g/L, H-acid 0.98 g/L, color 4,200, Na2SO4 270 g/L) was obtained from the secondary sedimentation tank of the JiHua Chemical Plant in Jiangsu, China. The concentrations of K-acid, chromotrophic-acid and H-acid were determined by high pressure liquid chromotography (HPLC) techniques (Zarabadi-Poor & Barroso-Flores 2014). The culture medium consisted of 1 L diluted K-acid wastewater (CODCr = 750 mg/L) containing 0.5 g urea and 0.5 g yeast extract. Prior to cultivation, K-acid wastewater was diluted to the desired CODCr, after which the initial pH of the K-acid wastewater medium was adjusted to the determined value. Batch fermentations were conducted in a 5-L stirred tank reactor (Model KF5L, KoBio-Tech Co., Inchon, Korea) at 30 °C for 96 h with agitation at 200 rpm. Samples were drawn and monitored for pH, biomass, chemical oxygen demand (COD), biochemical oxygen demand (BOD) and flocculation properties. Purification of the bioflocculant was conducted as previously described (Zaki et al. 2013), and the flocculating activity was measured according to the previously described kaolin suspension method (Nwodo et al. 2014). The zeta potential of bioflocculant and kaolin suspension were measured using a Zetaphoremeter (Zetaphoremeter IV, Zetacompact Z8000, CAD Instrumentation, France) (Nwodo et al. 2014).
This work optimized the culture conditions of bioflocculant-producing archaeon in K-acid wastewater using response surface methodology (RSM), aiming to enhance bioflocculant production. Three significant independent process variables: the CODCr of the K-acid wastewater, inoculum size, and initial pH, were proposed in the RSM model. Statistical analyses were performed using Design Expert Version 8.0. A total of 20 experiments were conducted using a face central composite design to investigate the optimum factors which contributed to the bioflocculant production (Table 1). Each of these three significant variables was assessed at five different levels (−1.682, −1, 0, +1, +1.682). The average yield which was achieved in these experiments was used as the response variable (Y) and all the experiments were conducted in triplicate.
|COD wastewater (mg/L)||330||500||750||1000||1170|
|Inoculum size (%)||8.3||10.0||12.5||15.0||16.7|
|COD wastewater (mg/L)||330||500||750||1000||1170|
|Inoculum size (%)||8.3||10.0||12.5||15.0||16.7|
Physical and chemical analysis of the bioflocculant
The polysaccharide concentration of the purified biopolymer was determined by the phenol–sulfuric method (Nwodo et al. 2014). The protein concentration of the purified biopolymer was determined by the Coomassie brilliant blue G-250 dye binding method using bovine serum albumin as the protein standard (Aljuboori et al. 2015). Neutral sugar, amino sugar, and uronic acid content were determined using the standard methods (Ghosh et al. 2009). The monosaccharide composition of the purified biopolymer was analyzed after hydrolysis with 3 M trifluoroacetic acid at 100°C for 4 h using cellulose TLC with ethyl acetate, pyridine, acetic acid, and water (5:5:1:3, v/v) as a solvent. Monosaccharides were detected by spraying with aniline phthalic acid reagent and heating at 110°C for 5 min (Yokoi et al. 1997). Gel permeation chromatography equipped with RID-10A detector (Shimadzu, Japan) was conducted in a glass column (900 × 10 mm) to determine the molecular weight of the bioflocculant. A sample solution (20 μL) was injected, and the column was eluted with 0.05 M NaCl solution at a flow activity of 0.7 mL/min. Characterization of the chromatographic system was achieved using a mixture containing protein urease (480 kDa), ovalbumin (45 kDa), cytochrome C (12.3 kDa), and blue dextran (2,000 kDa).
The functional groups of the bioflocculant were determined with a Spectrum One Fourier transform infrared spectroscopy (FT-IR) spectrometer (PerkinElmer, USA). The spectrum of the sample was recorded on the spectrophotometer over a wavelength range of 400–4,000 cm−1 under ambient conditions.
Acid brilliant scarlet GR wastewater flocculation test
A standard jar tester was used for the flocculation tests in acid brilliant scarlet wastewater (CODCr = 1,450 mg/L, pH 5.5, color = 2,200) dosed with MBF0579. CaCl2 and MBF0579 (prepared as a solution of 1.0 g/L using the fermentation broth) were added into 1.0 L of acid brilliant scarlet wastewater and then fixed on a jar testing device (TA2–2, Wuhan Hengling Co. Ltd) at room temperature 20 ± 1°C.
The jar testing procedure involved a 2 min rapid mixing stage at 200 rpm followed by a slow stir phase at 40 rpm for 30 min to promote the collision of particles and hence floc growth, which resulted in a 120 min settlement period. Sodium hydroxide and hydrochloric acid were employed to adjust the pH of the solutions to the predetermined level before the jar test and keep the pH constant during the flocculation process. All experiments were performed in triplicate.
RESULTS AND DISCUSSION
Isolation and identification of bioflocculant-producing archaeon HW0579
Five promising isolates were selected from K-acid wastewater sludge for bioflocculant production. Strain HW0579, which demonstrated the highest flocculating efficiency of 94.7% in 4 g/L kaolin suspensions, was selected for further studies. This archaeon was motile, pleomorphic, aerobic, and Gram-negative. On fermentation agar plates, the colonies were red, convex, and circular with a diameter of 0.4–0.5 cm. Nitrate reduction, citrate use, and hydrolysis of starch were observed, while H2S and indole were not produced. Tween-80, casein and gelatin were not hydrolyzed. Galactose, glucose, lactose, fructose, maltose, mannose, ribose, and sucrose were used, but mannitol growth was not observed. The archaeon was sensitive to novobiocin, bacitracin, and nitrofurantoin while was resistant to rifampin, mycostatin, trimethoprim, erythromycin, penicillin G, ampicillin, chloramphenicol, neomycin, norfloxacin, ciprofloxacin, streptomycin, kanamycin, tetracycline, vancomycin, gentamicin and nalidixic acid. The 16S rDNA of strain HW0579 was sequenced and deposited in the GenBank database under the accession number KT000266. According to its biochemical, morphological, and physiological properties, and 16S rDNA BLAST result, the isolated strain HW0579 was identified as an archaeon of Haloplanus vescus. The bioﬂocculant produced by this archaeon was named MBF0579.
OPTIMIZATION OF CULTURE CONDITIONS FOR FERMENTATION
Effect of K-acid wastewater strength on MBF0579 yield
Effect of the inoculum size on MBF0579 yield
Inoculum size is known to play a key role in the production of biomass and cell growth. MBF0579 yield increased remarkably from 1.86 to 8.04 g/L with increasing inoculum size from 5 to 12.5%, and reached its maximum when inoculum size was 12.5% (Figure 1(b)). It decreased progressively, to 6.89 g/L, when inoculum size was continuously extended to 20.0%. Similar results were observed in the production of bioflocculant in Klebsiella mobilis KLE-1 (Wang et al. 2007). Thus, an inoculum size of 12.5% was selected as the inoculum size in the following experiments.
Effects of pH on MBF0579 yield in fermentation
The initial pH of the culture can determine the electric charge of the microbial cells, oxidation-reduction potential, microbial nutrient assimilation, and enzyme reaction (Patil et al. 2011); therefore, the production of the biopolymer at pH 3.0–9.0 in the K-acid wastewater was investigated. The results demonstrated that the initial pH of the culture medium conspicuously affected the MBF0579 production (Figure 1(c)). MBF0579 yield increased from 2.84 to 8.09 g/L when the initial pH increased from 3.0 to 7.0, but the biopolymer yield decreased as pH moved towards 9.0. Further increase or decrease in the initial pH resulted in a decrease of MBF0579 yield, which is in accordance with the results of previous studies (Patil et al. 2011).
Production of MBF0579
Time course assay of bioflocculant production
The BOD5 concentration of K-acid wastewater before cultivation was 73.5 mg/L. The BOD5/COD ratio of K-acid wastewater was 0.094, showing the strong resistance property of K-acid to biodegradation. Figure 2(b) presents the variations of BOD5/COD ratios from 0 to 96 h. The biodegradability of solutions as measured by BOD5/COD values increased during fermentation test. At the end of the fermentation test, the concentrations of K-acid, chromotropic-acid, and H-acid were 13, 17 and 16 mg/L, respectively. As a result, the strain consumed more than 87.2% of K-acid, part of chromotropic-acid (66.2%) and H-acid (60.0%) to produce MBF0579.
The cultivation of bioflocculant-producing archaeon using K-acid wastewater had both environmental and economic benefits. The CODCr concentration of K-acid wastewater after archaeon cultivation was 176 mg/L with a removal activity of 76.53%. Additionally, the bioflocculant production cost decreased significantly when the archaeon were cultivated in K-acid wastewater, but the flocculating activity did not decrease. Thus, this procedure can reduce the cost of bioflocculant production.
Characteristics of purified MBF0579
Composition analysis of MBF0579
MBF0579 was found to be a proteoglycan comprised of 78.6% (w/w) carbohydrate and 21.1% (w/w) protein. Further analysis of the hydrolyzed MBF0579 revealed that the mass proportion of neutral sugar, amino sugar, and uronic acid was 4:3:4. The neutral sugars obtained by hydrolysis of the purified biopolymer were D-Glc, D-Xyl, D-Gal, and D-GlcA, which were in an approximate molar ratio of 4.2:1.4:2.6:3.1. The molecular weight of the biopolymer determined by size exclusion chromatography was found to be approximately 1.9 × 106 Da. Elemental analysis of purified MBF0579 revealed that the mass proportion of C, H, O, N, and S was 38.57:6.34:34.89:8.51:1.06 (w/w), respectively.
MBF0579 functional groups analysis
Flocculation performance of purified bioflocculant
Factors impacting removal efficiency of dye
pH was a key factor influencing the flocculation of dye based on both the chemical speciation of dye and surface charge of MBF0579. One reason for this is that dye can have different speciation forms at different pH. The distribution diagrams of dye species as a function of pH showed that COD and chroma were present in solution at the pH values of most natural waters. pH can also strongly modify the redox potential of dye and MBF0579, as well as induce dissolution of MBF0579. As a result, the dye removal was strongly pH dependent in the textile dyeing wastewaters. As shown in Figure 5(b), the influence of pH on dye removal by MBF0579 was investigated by adding 15.0 mg MBF0579 into 100.0 mL acid brilliant scarlet GR wastewater. The removal efficiency of dye was lowest at pH 10.0, then increased with decreasing pH to nearly 2.0, at which point a maximum uptake of 81.7 and 94.6% was achieved for COD and chroma, respectively. These results demonstrated that MBF0579 had higher removal efficiency for dye under acidic pH, which was consistent with the results of a previously conducted study (Huang et al. 2014). Overall, these findings indicate that MBF0579 has the potential for the removal of dye from aqueous solution.
Figure 5(c) illustrates the effects of contact time on the dye removal efficiency of the MBF0579 in response to the addition of 15.0 mg MBF0579 into 100.0 mL acid brilliant scarlet GR wastewater with a fixed pH of 7.0. The removal efficiency of dye increased obviously during the initial flocculation stage, then continued to increase at a relatively slow speed with contact time until flocculation equilibrium was reached. Interesting, the equilibrium time was 100 min for COD and chroma. Thus, 100 min was selected as the optimum contact time for dye flocculation from the aqueous solution by MBF0579. This is similar to the contact time reported for earlier studies investigating the flocculation of dye on various biomasses (El-Bindary et al. 2014).
This research revealed the potential for use of K-acid wastewater for the production of MBF0579. The optimized production conditions were an inoculum size of 12.5%, initial pH of 7.0, and CODCr of K-acid wastewater of 780 mg/L. A maximum yield of 8.09 g/L MBF059 was obtained in the optimized medium. In addition, the maximum removal efficiencies of acid brilliant scarlet dye wastewater were 81.86 and 95.07% for COD and chroma, respectively. Therefore, HW0579 showed great potential in removing acid brilliant scarlet dye or strengthening the treatment of acid dye wastewater.
This work was supported by National Key Technology Research and Development Program of the Ministry of Science and Technology of China (Grant No. 2012BAD36B03).