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Recovering organic matters and ions from wastewater by genetically engineered Bacillus subtilis biomass
Journal of Environmental Management 161 (2015) 402e407
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Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Research article
Recovering organic matters and ions from wastewater by genetically engineered Bacillus subtilis biomass Wei Zhu a, *, Yujie Liu a, Xia Cao a, Sainan Zhang a, Chaoyuan Wang a, Xinli Lin b, c a
Key Laboratory for Microorganisms and Biotransformation, College of Life Science, South-Central University for Nationalities, Wuhan 430074, China Angimmune LLC, Rockville, MD, USA c Zhoushan CSP Biosciences, Inc., Zhoushan, Zhejiang, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 March 2015 Received in revised form 8 June 2015 Accepted 7 July 2015 Available online xxx
Water pollution causes substantial damage to the environment and to human health, and the current methods to treat pollution suffer from high cost and low efficiency, resulting in increased environmental damages. Using genetic modification and functional selection, we developed a novel biosorbent from Genetically Engineered Bacillus subtilis (GEBS) cells. At a ratio of biosorbent to direct blue dye of about 1:1.25 in a water solution, the dye pigments can be completely adsorbed in 40 s, decreasing COD to zero. Contrary to other biosorbents, ions such as Fe2þ and Cu2þ have significant advantages in terms of the adsorbing efficiency. The GEBS biomass can therefore capture both organics and ions from wastewater simultaneously and achieve co-precipitation in 2e10 min, which are features critical for practical applications of wastewater treatment. In addition, we used six different eluting solutions to regenerate used biomass, all resulting in renewed, highly efficient color and COD elimination capacities, with the best elution solution being NaHCO3 and Na2CO3. For practical applications, we showed a high COD elimination rate when using the GEBS biomass to treat raw water from textile enterprises, paper mill, and petrochemical industries. Compared with currently available adsorbing agents, the GEBS cells can adsorb organic and ion waste much faster and with much higher efficiency, can be regenerated and recycled efficiently, and may have broad applications in treating organic water pollution. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Promotion Organic wastewater treatment Recycling Regeneration Genetically engineered Bacillus subtilis Co-precipitation
1. Introduction Wastewater discharge constitutes one of the major environmental pollutions, especially in developing countries (Wang, 2012; Sato et al., 2013). New and affordable wastewater treatment methods are therefore urgently needed for solving water pollution problems, especially for developing countries. Although many kinds of wastewater treatment methods based on the activated sludge technique for sewage purification have been developed (Vijayaraghavan and Yun, 2008; Clouzot et al., 2013), the technique presents many problems. First, the end product of microbe degradation of organic matters is CO2, which will not only increase global warming directly, but also result in the failure of recycling organic materials for energy storage (Grant et al., 2012). In fact this process wastes useful and recyclable organic matters and artificially increases the “useless carbon depletion”
* Corresponding author. E-mail addresses: [email protected], [email protected] (W. Zhu). http://dx.doi.org/10.1016/j.jenvman.2015.07.019 0301-4797/© 2015 Elsevier Ltd. All rights reserved.
(Xie et al., 2013). Second, the proliferation of microbes can actually generate additional sludge, which can be difficult to recycle and reuse (Yan et al., 2013). Third, the present water treatment system based on activated sludge results in high residual nitrogen and phosphorus, which can cause eutrophication of discharged water and further environmental pollution (Kartal et al., 2010). Furthermore, the current wastewater processing has disadvantages such as high energy consumption (McCarty et al., 2011; Logan and Rabaey, 2012) and inefficient use of time (Pathak et al., 2014); also the sewage treatment plants often necessitate large capital investments (Vijayaraghavan and Yun, 2008; Nidheesh et al., 2013). The inefficient utilization of land and energy are a heavy burden to the global energy crisis and urgently need improvement (Logan and Rabaey, 2012). On the other hand, when adsorption technologies are used, the adsorption efficiency of the reported adsorbent and flocculant is quite low, and neither the organic matter nor treatment agents can be easily recycled (Baoe et al., 2011; Won et al., 2009, 2013; Han and Yun, 2007; Xi et al., 2013). Although it has been proposed that bacterial biomass can potentially be used as an efficient biosorbent
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for the removal of both dyes and metal ions, in practice, the difficulties in long adsorption time, reapplication of the microbial biomass, as well as poor selectivity, have hindered their application (Vijayaraghavan and Yun, 2008; Pathak et al., 2014). Even there have been commercialization attempts to use biosorption for wastewater treatment, the progress has been slow after more than a decade of research and development (Vijayaraghavan and Yun, ra and Aicha, 2006; Sivasamy and Sundarabal, 2011; 2008; Nace Xiong et al., 2010; Inoue et al., 2013; Kalpana et al., 2011; Umpuch et al., 2015). Thus a new area of global emission reduction and energy conservation can be created if the organic pollutants in wastewater can be recovered and reused with new treatment technologies. Here we report a new type of recyclable wastewater treatment agent with the ability to recover organic material and ions that can be used to solve the aforementioned sewage treatment problems such as high energy consumption, inefficient use of time, CO2 production, and other defects such as eutrophication (Vijayaraghavan and Yun, 2008; Kartal et al., 2010). The agent is a selected mutant bacterial cell (Genetically Engineered Bacillus subtilis, GEBS), with the yqfY and spo0A genes knocked-out. By knocking-out the yqfY gene, the acetylation reaction during cell wall peptidoglycan synthesis is eliminated, and the resulting cells become more polar and positively charged due to the increased abundance of free amines and hydroxyl groups on the cell wall. Additionally, knocking-out the spo0A gene prevents the formation of spores, which has no organic waste adsorbing capability. The resulting GEBS clone has better affinity and enhanced adsorbing capacity for organic molecules (details of the construction and characterization of GEBS clone will be published in a separate paper). Our results show that GEBS can rapidly adsorb Direct Blue dye type organic molecules and ions in waste water, and can rapidly lower the chemical oxygen demand (COD) level to the required standard, and in some cases even to zero. In addition, the cells can be regenerated and reused in a short time (3e5 min). Using GEBS biomass to rapidly and efficiently recover organic matter and ions in wastewater is an important method towards reducing carbon emissions (Xie et al., 2013). The application of this new biosorbent can overcome the defects of the traditional biochemical treatment method by improving the energy efficiency and greatly shortening the process flow and time of organic wastewater treatment. The new application can also reduce the land required for wastewater treatment, and will furthermore recycle water resources and reduce carbon emission. The novel agent and application process may serve as an important contribution for both global energy and pollution reduction. 2. Materials and methods 2.1. GEBS selection and culture A bacterial strain B. Subtilis (Accession: JN165753) (isolated from the South Lake, Wuhan, China) was irradiated by UV (power 15 W, irradiation distance 30 cm, irradiation time 70 s) to induce random mutations. A mutant ZN0871v11 was selected by the highest capacity of adsorbing Direct Blue 2B dye. Mutant ZN0871v11 was further genetically engineered by reforming the cell wall peptidoglycan synthesis pathway (to knock out the yqfY and spo0A genes). The clone is called GEBS. The GEBS clone was inoculated in LB medium (Tryptone 1.0 g, yeast extract 0.5 g, NaCl 0.5 g, H2O 100 mL), and grown at 37 C for 48 h. The resulting culture was centrifuged at 7280 g for 5 min, and the supernatant was discarded. Cell pellets were resuspended and boiled for 5 min, washed 3 times with distilled water, and then added to the samples to be treated in predetermined proportions. The ratio of wet weight (WT)
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and dry weight of the cells is 6.88:1. 2.2. Adsorbing efficiency at different temperatures, pH, and with different ions The concentration of Direct Blue 2B solution was 250 mg/L. The effect of temperature on the adsorbing ability of GEBS biomass was tested from 20 C to 42 C. The pH range used in this study was from 4.0 to 10.3 at room temperature. The following buffer systems were used: 0.1 M potassium acetate buffer solution (pH 4.0e5.5), 0.1 M potassium phosphate buffer (pH 6.0e7.0), 0.1 M TriseHCl buffer solution (pH 7.5e8.5) and CAPS (3-(cyclohexylamino) -1-propane sulphonic acid) (Amresco, Solon, OH) buffer (pH 10e13). The concentrations of Fe2þ, Cu2þ, Zn2þ, Ca2þ, Mg2þ, Kþ, Naþ tested were 20, 50, 100, 150, 200 mg/L respectively. 3. Results 3.1. Effects of temperature and pH on adsorbing efficiency The adsorbing efficiency of GEBS biomass at different pH is shown in Fig. 1a. The figure shows significant differences in adsorbing efficiency at the measured pH range between 5.0 and 10.3. The efficiency is the highest at the low pH, which is 93% at pH 5.0, and at pH 5.5 it has already decreased to 60%, with further decreased adsorbing efficiency at higher pH. The optimal pH of the adsorbing efficiency of GEBS biomass is significantly higher than other reported biosorbents. In reported literatures, when various bacterial cells were used as biosorbents, the required pH was very low (e.g. pH1.0), and the adsorption time was quite long (e.g. 12e24 h) (Baoe and Xiu, 2011; Won et al., 2009; Vijayaraghavan and Yun, 2007; Won and Yun, 2008). Fig. 1b shows that the adsorbing efficiency of GEBS biomass for Direct Blue dye is over 91% in a range of temperatures from 20 C to 42 C. The efficiency reached a maximum of 100% when measured at 37 C and 42 C. In general, the results showed that temperature has no obvious effect on the adsorbing efficiency (Fig. 1b). 3.2. Adsorbing efficiency of GEBS biomass in the presence of different ions As shown in Fig. 1c, we studied the effects of 7 common metal ions on the adsorbing efficiency of GEBS cells. It is important to note that in our adsorption system, the tested metal ions don't interfere with dye biosorption as it does in other systems; on the contrary, the ions enhanced the absorbing rate and increased the adsorbing efficiency. Results show that at concentrations of 20, 50, 100, 150, and 200 mg/L, the metal ions significantly promoted the adsorbing efficiency. The efficiency increases as the concentration of ions increased. Among different ions, the influence of Fe2þ, Cu2þ, and Zn2þ are the most significant (Fig. 1c). In the testing conditions, when the concentration of Fe2þ was present in the low range of 20 mg/L, the adsorbing efficiency was only 28%, but the adsorbing efficiency increases to 50% at 50 mg/L, and to 100% at 150 mg/L. The adsorbing efficiency profile of Cu2þ is similar to that of Fe2þ: at low concentration (20e100 mg/L), the efficiency is about 12e38%, but when the concentration reached 150 mg/L and 200 mg/L, the efficiency increased to the maximum of 97% and 100%, respectively. At low concentrations (20 mg/L and 50 mg/L), Zn2þ had a small positive effect of 11e37% on adsorbing efficiency, but the effect reached to 90% at a high concentration of 200 mg/L. The adsorbing efficiency was not enhanced significantly by Ca2þand Mg2þ in the tested concentrations, with a moderate adsorbing efficiency increase of 53e66% for Ca2þ and 44e55% for Mg2þ, respectively. Monovalent ions (Naþ and Kþ) have limited effects on the adsorbing efficiency of
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Fig. 1. The adsorbing efficiency at different temperature, pH and ions using Direct Blue dye solution. The reaction volume is 100 mL; the GEBS concentration is 1200 mg/L; and the concentration of Direct Blue solution was 250 mg/L. (a) The adsorbing efficiency of pH at room temperature for 30 min. (b) The adsorbing efficiency of temperature at pH5.0 for 30 min. (c) The adsorbing efficiency of metal ions on the efficiency of GEBS biomass (metal ions concentration: 20, 50, 100, 150, 200 mg/L, temperature: 25 C, pH 5.0, time: 3e5 min) to adsorb Direct Blue.
GEBS cells for Direct Blue dye with adsorbing efficiency of 15e29% for Kþ and 11e18% for Naþ. Among the common ions tested, the most efficient adsorption promoters are Fe2þ and Cu2þ, followed by Zn2þ. We then tested the adsorbing efficiency of GEBS cells at an optimal condition of pH 5.0, 37 C, and Fe2þ or Cu2þ at concentration of 150e200 mg/L. Precipitation appeared immediately after the GEBS cells and Direct Blue dye solution was mixed. The time of incubation for biosorption was shortened from about 50 min to 2e10 min when ions were added. Results showed that the adsorbing efficiency was 100% either by measuring at OD545 or COD in the supernatant. The residual Fe2þ and Cl in the supernatant decreased from 73.68 and 532.5 mg/L to 4.01 and 0.7 mg/L respectively. The adsorbing efficiency were 94.56% (Fe2þ) and 99.83% (Cl) respectively, which indicated that the GEBS cells can capture organics and ions from wastewater simultaneously and achieve co-precipitation within several minutes. The results also demonstrated the superior adsorbing efficiency and high adsorbing rate of GEBS cells compared with other biosorbents reported in the literatures (Baoe et al., 2011; Won et al., 2009, 2013; Xi et al., 2013; Vijayaraghavan and Yun, 2007; Won and Yun, 2008). 3.3. The uptake of organic molecules and adsorbing equilibration The GEBS biomass was added to five bottles containing Direct Blue dye solution according to the arithmetic progression method. After mixing evenly, the mixtures were placed at room temperature. The adsorbing phenomenon of Direct Blue dye can be observed visibly by appearance of coagulation and sedimentation, resulting in GEBS cells turning blue and the simultaneous fading of the color of the dye solution. Fig. 2a shows the equilibrium of a linear decrease of supernatant at 545 nm at increasing amount of GEBS cells. The linear equation is given as: y ¼ 0.1208x, where y (mg) is the adsorbed organics and x (mg) is the amount of the biosorbent (WT).
Fig. 2. Adsorbing equilibrium of GEBS cells. (a) Capacity of biosorbent capturing Direct Blue dye. Y: adsorbed organics (mg); X: GEBS cells (mg, wet weight). (b) Kinetic curve using Lagergren models applied to biosorption of Direct Blue 2B on GEBS biomass. Y: Dye adsorbed (mg)/Biomass(g,wt); X: Time (s).
The equation shows that the dose of the biosorbent added is inversely proportional to the color of the supernatant. When the GEBS cells added was 77.98 mg (WT), the color of the supernatant was reduced to 0. At the ratio of GEBS cell mass (dry weight) and dye mass of 1:1.25 or more, the color and COD of the treated dye solution were reduced to “0”. The adsorbing efficiency is obviously higher than the adsorption capacity of agricultural solid wastes
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and microorganisms for the removal of dyes (Guo et al., 2013; Won et al., 2013; Xi et al., 2013; Won and Yun, 2008; Pajot et al., 2011). The kinetic curve for biosorption of Direct Blue 2B by GEBS biomass followed by the Lagergren and Ho et al. models is shown in Fig. 2b. From the curve we derived the following equation: y ¼ 2.3354Ln(x) þ 24.411, where y is the milligram dye adsorbed per gram GEBS biomass (WT), and x is the time in seconds. The figure shows that the capturing or biosorption was very quick, almost reaching the maximum in 5 s. In addition, the biosorption was irreversible; therefore the curve shown in Fig. 2b significantly differs from the curves of the reported biosorbents, though the equilibrium equation used was the same (Chu and Chen, 2002).
3.4. Recovery efficiency of the adsorbed organic molecules To test possible regeneration and reuse, GEBS cells with adsorbed Direct Blue 2B dye (Org-GEBS) were mixed with six different regeneration eluents, incubated for 2 min, and centrifuged at 7280 g for 2 min. The resulting supernatants turned blue, which were quantified by OD545 measurement. The results showed that the captured dye molecules could be eluted from the biosorbent by the tested regeneration eluents, thereby recovering the used GEBS cells. The total amount of dye eluted into the supernatant increased with increasing volume of regeneration eluents, and the dye color on GEBS cells gradually disappeared (Fig. 3a, b). When the added regeneration eluent reaches a certain volume, the captured dye can be washed away completely, and GEBS cells gradually became white and finally returned to their original state. At room temperature, the elution formula for each eluent can be expressed as the following, as shown in Fig. 3.
For For For For For For
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Eluent A: y ¼ 0.0097x. Eluent B: y ¼ 0.0205x Eluent C: y ¼ 0.0218x detergent solution (2 g/L): y ¼ 4.5141x Na2CO3 (5 mM): y ¼ 8.6897x NaHCO3 (10 mM): y ¼ 5.6145x
According to the formula, in the experimental range shown in Fig. 3, the amount of dye released is directly proportional to the volume of the eluent used. The elution efficiency of the first three eluents is eluent B > eluent C > eluent A. One mL of eluent A, B, and C can release 9.7 mg, 21.8 mg, and 20.5 mg of Direct Blue dye from GEBS cells, respectively. Also, 1 mg of Na2CO3, NaHCO3, and detergent powder could release 8.69 mg, 5.61 mg, and 4.51 mg of Direct Blue dye from the GEBS cells, respectively. Accordingly, the elution capability of Na2CO3 is about two times that of detergent powder's. For all of the six eluents tested, at a certain range, the elution capacity is also inversely proportional to the elution concentration: the higher the elution concentration, the less elution volume will be required for GEBS cell recovery. 3.5. Efficiency of recycling and reuse of GEBS biomass The regenerated GEBS biomass can be used to adsorb the organic dye again with high efficiency. Among the 6 regeneration eluents tested, Na2CO3 and NaHCO3 solution produced the best recycling results, which showed that the regenerated GEBS cells can still efficiently eliminate color of the tested dye solution (Table 1). We recycled 26.7 mg of wet weight Org-GEBS 3 times using the above 6 eluents. After regenerating with Eluent A for total of 3 times, the recovered GEBS cells still have over 85% of the original color elimination ability and 93.6e90.3% of the original COD elimination ability, after each round of regeneration respectively. The results did show that the color and COD elimination ability decrease with each round of regeneration. For eluent B, the same experiments showed over 88.65% of the original color elimination ability and 98.08e91.52% of the original COD elimination ability. For Eluent C, the data were over 95.25% for color elimination and 97.31%e92.18% for COD elimination, respectively. The reasons of decreased color and COD elimination efficiency could be that some GEBS cells were lost due to cohering on the vessel wall following each elution, or/and the alkaline eluents may cause permanent structural damage to the cells. The adsorbing efficiency of the GEBS cells recycled with 2 g/L detergent powder solution for 3 times were 90.1 e 87% for color elimination and 93.4e86.9% for COD elimination, respectively. The data for 5 mM Na2CO3 solution was 100 e 95.2% for color elimination, and 96.7e92.1% for COD elimination; while for 10 mM NaHCO3 solution was 100% for color and 97.4e95.6% for COD. 3.6. Industrial wastewater treatment with GEBS cells
Fig. 3. Recovery efficiency of six different eluents. GEBS cells with adsorbed Direct Blue dye were mixed with six different regeneration eluents, incubated for 2 min, and centrifuged at 7280 g for 2 min. The resulting supernatants turned blue, which were quantified at 545 nm after diluting 80 times. (a) The recovery efficiency of eluent A, B and C to Direct Blue adsorbed by GEBS cells. (b) The recovery efficiency of Na2CO3, NaHCO3, and detergent powder to Direct Blue adsorbed by GEBS cells.
Raw industrial wastewater samples (1 sample each from a textile and paper mill industry, and 2 samples from a petrochemical enterprise) were treated with GEBS cells at pH 5.0, with an addition of 200 mg/L of Fe2þ. The mixtures were incubated for 3e5 min at room temperature after 2 g of wet weight GEBS cells were added to 1 L wastewater. The resulting GEBS cells were then regenerated three times with 10 mM NaHCO3 eluent and their adsorbing ability tested each time after regeneration (Table 1). The color (OD545) elimination rate of all samples was 100% for the regenerated GEBS cells. The COD elimination rate of the sample from textile enterprise were between 99.39 and 86%, while data for sample from the paper mill were 97.55%e89.07%. The data for the 2 samples from
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petrochemical enterprise were 95.88 e 90.2%, and 97.57e91.65%, respectively. 4. Discussion This study shows that at a fixed ratio, our adsorbing agent can adsorb organic and ions pollutants in wastewater, and can essentially eliminate dye color and COD in less than a minute. The ratio of the biosorbent to the Direct Blue 2B organic dyes can be amounted to 1:1.25, demonstrating a very high adsorbing capability. Importantly, this biosorbent can be regenerated within 3e5 min, and therefore has high regeneration efficiency and presents low cost for future industrial application. The pH has a significant effect on the capturing capacity. One of the reasons may be that at acidic conditions (pH 5.0e5.5), the polarity of the organic molecules undergoes changes leading to different ionic states and binding profiles. Results showed that metal ions, especially Fe2þ and Cu2þ can consolidate the polarity and ionic state of organic molecules to promote the capturing capacity and rate of GEBS biomass. The residue of cations, anions and organics in the supernatant decreased to very low concentration indicates that the cations and anions not only may affect the dye molecules and the surface of GEBS biomass, but also achieve coprecipitation with the biosorbent and organics in a very short time. Both the cations and anions take part in the interaction between the surface of GEBS cells and dye molecules during the biosorption. Compared with many reported biosorbents (Xiong et al., 2010), the GEBS cells differentiate themselves with their ability to capture both organics and ions from wastewater, which is very important in wastewater treatment. As described above, our biosorbent has significant practical advantages. Many reported adsorbents have relatively low adsorbing capability and some need lengthy times (e.g. 5 d) to reach a high adsorption ratio, in addition to difficulties in their recycling ra and Aicha, 2006; Sivasamy and reuse (Pathak et al., 2014; Nace and Sundarabal, 2011; Xiong et al., 2010; Inoue et al., 2013; Kalpana et al., 2011; Umpuch et al., 2015) (Table 2). Among the general applied absorbents, active carbon is used the most because it can be regenerated and reused. However, the used active carbon needs to be regenerated in a factory, demanding sophisticated equipment, consuming high energy, and resulting in costly recycling (Chen et al., 2012). One of the unique aspects of the GEBS cells is that it does not obey the complex Lagergren equation (Won and Yun, 2008; Pajot et al., 2011; Chen et al., 2012), but rather a simple linear proportional relationship. The biosorbent can complete adsorption reaction in a short time, and has overwhelming capturing capabilities for organic molecules. Another feature is that GEBS cells can overcome many weakness and difficulties inherent in the existing water treatment systems because they can adsorb organic pollutants and ions in short time (5e10 min) and do not require special equipment and conditions. Moreover, the biosorbent can be recycled and reused many times, requiring only small volumes of eluent to achieve regeneration, and thus greatly reduces the cost for treating wastewater and polluted water. The adsorbed organic pollutants during the regeneration process can also be recovered and used, making complete reservation of energy and environment possible. In this study, we also compared the regeneration efficiency of 6 different eluents (Table 1). Results showed that the biosorbent can be regenerated with high efficiency by all 6 of the eluents. The principle of each of the above 6 eluents on the regeneration of the biosorbent, however, is different. For eluents A, B, and C, a strong electrostatic force and affinity to the adsorbed organic molecules may be the main regeneration forces. Eluent A and C also contain
SDS, which is a strong surfactant and may add to the regeneration. The regeneration ability of Na2CO3 and NaHCO3 could be due to ion exchange interactions. The biosorbent captures organic molecules at acidic conditions, and when the pH shifts to strongly alkaline in Na2CO3 and NaHCO3 solutions, the captured molecules could be released by the changes in charge. The elution principle of the detergent used in this study may be the same as it is in laundry: the detergent helps to “wash out” the adsorbed pollutants and regenerates the biosorbent. For industrial applications in sewage treatment, optimal eluent selection may depend on the type of pollutant. Compared with the existing organic wastewater processing techniques (Table 2), treating water pollution with the biosorbent described in this study is faster, more efficient, and overcomes many practical problems facing the classical sewage processing ra and Aicha, 2006; Xiong et al., 2010). In addition, the GEBS (Nace cells used in this study are easy to produce and regenerate, and the released organic pollutants from the regeneration process can be used in energy generation such as methane fermentation, further reducing the environmental impact (Kalpana et al., 2011). In summary, the method described in this study is a new technology based upon a highly efficient wastewater treatment agent, which has high adsorption rate, low producing cost, low energy consumption, reduced land use, low demand for equipment and environment, no secondary pollution, and is both recyclable and reusable. Although not tested, theoretically, the biosorbent may even be used to enrich and clean nuclear pollutants released into the environment in emergent situations. 5. Conclusions In this study, we used direct blue dye solution as a model for polluted water treatment with the biosorbent GEBS cells because the dye is easy to monitor by visible color change, and easy to quantify by spectrophotometric methods. The results show that, compared with the existing flocculent and adsorbents, the GEBS biomass offers significant advantages in several aspects. First, because of the genetic selection and engineering, the capturing capacity is a perfect combination of electrostatic, hydrophobic, and affinity interactions, and therefore has a much higher capturing capacity than the existing flocculent and adsorbents do. Second, the GEBS cells can adsorb organics and cations and anions simultaneously from wastewater and achieve co-precipitation within several minutes. Third, the capturing and regeneration rate is much faster, in the range of 3e5 min; this property may enable more efficient and practical industrial applications. In summary, from GEBS cell production to recycling, the whole process is simple to use, with low energy consumption, and without toxic secondary pollution. Fourth, the results of raw industrial wastewater samples show that the GEBS cells can capture many kinds of complex organic molecules in raw industrial pollution waters and can be easily and rapidly regenerated in several minutes and reused in a simulated “real world” situation. These results demonstrate that GEBS cells can have broad application in water pollution treatment. This is therefore a new environment-friendly technology for treating water pollution in general, and for treating difficult to treat organic water pollution in particular. Acknowledgments We thank Professors Buhai Li and Xiaomei Sun for reagents. This study is supported by the Fundamental Research Funds for the Central Universities of China (Grant No. CZY12023; ZZY10009), the Natural Science Key Foundation of South-Central University for Nationalities (Grant No. YZZ07014).
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Chem. Eng. J. 119, 121e125. Nidheesh, P.V., Gandhimathi, R., Ramesh, S.T., 2013. Degradation of dyes from aqueous solution by Fenton processes: a review. Environ. Sci. Pollut. Res. Int. 20, 2099e2132. ~ a, J.I., 2011. Unraveling the decolPajot, H.F., Delgado, O.D., de Figueroa, L.I., Farin ourizing ability of yeast isolates from dye-polluted and virgin environments: an ecological and taxonomical overview. Ant. Van Leeuwenhoek 99 (3), 443e456. Pathak, H., Soni, D., Chauhan, K., 2014. Evaluation of in vitro efficacy for decolorization and degradation of commercial azo dye RB-B by Morganella sp. HK-1 isolated from dye contaminated industrial landfill. Chemosphere 105, 126e132. Sato, T., Qadir, M., Yamamoto, S., Endo, T., Zahoor, A., 2013. Global, regional, and country level need for data on wastewater generation, treatment, and use. Agr. Water Manage. 130, 1e13. Sivasamy, A., Sundarabal, N., 2011. Biosorption of an Azo dye by Aspergillus niger and Trichoderma sp. fungal biomasses. Curr. Microbiol. 62 (2), 351e357. Umpuch, C., Sakaew, S., 2015. Adsorption characteristics of reactive black 5 onto chitosan-intercalated montmorillonite. Desalin. Water Treat. 53 (11), 2962e2969. Vijayaraghavan, K., Yun, Y.S., 2007. Utilization of fermentation waste (Corynebacterium glutamicum) for biosorption of reactive black 5 from aqueous solution. J. Hazard Mater 141, 45e52. Vijayaraghavan, K., Yun, Y.S., 2008. Bacterial biosorbents and biosorption. Biotechnol. Adv. 26, 266e291. Won, S.W., Yun, H.J., Yun, Y.S., 2009. Effect of pH on the binding mechanisms in biosorption of reactive orange 16 by Corynebacterium glutamicum. J. Colloid Interface Sci. 331 (1), 83e89. Won, S.W., Yun, Y.S., 2008. Biosorptive removal of reactive yellow 2 using waste biomass from lysine fermentation process. Dyes Pigm. 76, 502e507. Won, S.W., Choi, S.B., Mao, J., Yun, Y.S., 2013. Removal of 1-ethyl-3-methylimidazolium cations with bacterial biosorbents from aqueous media. J. Hazard. Mater. 244e245, 130e134. Wang, Z., 2012. China's wastewater treatment goals. Science 338, 604. Xi, Y., Shen, Y., Yang, F., Yang, G., Liu, C., Zhang, Z., Zhu, D., 2013. Removal of azo dye from aqueous solution by a new biosorbent prepared with Aspergillus nidulans cultured in tobacco wastewater. J. Taiwan Inst. Chem. E 44, 815e820. Xie, X., Ye, M., Hsu, P.C., Liu, N., Criddle, C.S., Cui, Y., 2013. Microbial battery for efficient energy recovery. Proc. Natl. Acad. Sci. U.S.A. 110, 15925e15930. Xiong, X.J., Meng, X.J., Zheng, T.L., 2010. Biosorption of C.I. Direct Blue 199 from aqueous solution by nonviable Aspergillus niger. J. Hazard. Mater. 175 (1e3), 241e246. Yan, P., Ji, F., Wang, J., Fan, J., Guan, W., Chen, Q., 2013. Evaluation of sludge reduction and carbon source recovery from excess sludge by the advanced sludge reduction, inorganic solids separation, phosphorus recovery, and enhanced nutrient removal (SIPER) wastewater treatment process. Bioresour. Technol. 150, 344e351.
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Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Research article
Recovering organic matters and ions from wastewater by genetically engineered Bacillus subtilis biomass Wei Zhu a, *, Yujie Liu a, Xia Cao a, Sainan Zhang a, Chaoyuan Wang a, Xinli Lin b, c a
Key Laboratory for Microorganisms and Biotransformation, College of Life Science, South-Central University for Nationalities, Wuhan 430074, China Angimmune LLC, Rockville, MD, USA c Zhoushan CSP Biosciences, Inc., Zhoushan, Zhejiang, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 March 2015 Received in revised form 8 June 2015 Accepted 7 July 2015 Available online xxx
Water pollution causes substantial damage to the environment and to human health, and the current methods to treat pollution suffer from high cost and low efficiency, resulting in increased environmental damages. Using genetic modification and functional selection, we developed a novel biosorbent from Genetically Engineered Bacillus subtilis (GEBS) cells. At a ratio of biosorbent to direct blue dye of about 1:1.25 in a water solution, the dye pigments can be completely adsorbed in 40 s, decreasing COD to zero. Contrary to other biosorbents, ions such as Fe2þ and Cu2þ have significant advantages in terms of the adsorbing efficiency. The GEBS biomass can therefore capture both organics and ions from wastewater simultaneously and achieve co-precipitation in 2e10 min, which are features critical for practical applications of wastewater treatment. In addition, we used six different eluting solutions to regenerate used biomass, all resulting in renewed, highly efficient color and COD elimination capacities, with the best elution solution being NaHCO3 and Na2CO3. For practical applications, we showed a high COD elimination rate when using the GEBS biomass to treat raw water from textile enterprises, paper mill, and petrochemical industries. Compared with currently available adsorbing agents, the GEBS cells can adsorb organic and ion waste much faster and with much higher efficiency, can be regenerated and recycled efficiently, and may have broad applications in treating organic water pollution. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Promotion Organic wastewater treatment Recycling Regeneration Genetically engineered Bacillus subtilis Co-precipitation
1. Introduction Wastewater discharge constitutes one of the major environmental pollutions, especially in developing countries (Wang, 2012; Sato et al., 2013). New and affordable wastewater treatment methods are therefore urgently needed for solving water pollution problems, especially for developing countries. Although many kinds of wastewater treatment methods based on the activated sludge technique for sewage purification have been developed (Vijayaraghavan and Yun, 2008; Clouzot et al., 2013), the technique presents many problems. First, the end product of microbe degradation of organic matters is CO2, which will not only increase global warming directly, but also result in the failure of recycling organic materials for energy storage (Grant et al., 2012). In fact this process wastes useful and recyclable organic matters and artificially increases the “useless carbon depletion”
* Corresponding author. E-mail addresses: [email protected], [email protected] (W. Zhu). http://dx.doi.org/10.1016/j.jenvman.2015.07.019 0301-4797/© 2015 Elsevier Ltd. All rights reserved.
(Xie et al., 2013). Second, the proliferation of microbes can actually generate additional sludge, which can be difficult to recycle and reuse (Yan et al., 2013). Third, the present water treatment system based on activated sludge results in high residual nitrogen and phosphorus, which can cause eutrophication of discharged water and further environmental pollution (Kartal et al., 2010). Furthermore, the current wastewater processing has disadvantages such as high energy consumption (McCarty et al., 2011; Logan and Rabaey, 2012) and inefficient use of time (Pathak et al., 2014); also the sewage treatment plants often necessitate large capital investments (Vijayaraghavan and Yun, 2008; Nidheesh et al., 2013). The inefficient utilization of land and energy are a heavy burden to the global energy crisis and urgently need improvement (Logan and Rabaey, 2012). On the other hand, when adsorption technologies are used, the adsorption efficiency of the reported adsorbent and flocculant is quite low, and neither the organic matter nor treatment agents can be easily recycled (Baoe et al., 2011; Won et al., 2009, 2013; Han and Yun, 2007; Xi et al., 2013). Although it has been proposed that bacterial biomass can potentially be used as an efficient biosorbent
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for the removal of both dyes and metal ions, in practice, the difficulties in long adsorption time, reapplication of the microbial biomass, as well as poor selectivity, have hindered their application (Vijayaraghavan and Yun, 2008; Pathak et al., 2014). Even there have been commercialization attempts to use biosorption for wastewater treatment, the progress has been slow after more than a decade of research and development (Vijayaraghavan and Yun, ra and Aicha, 2006; Sivasamy and Sundarabal, 2011; 2008; Nace Xiong et al., 2010; Inoue et al., 2013; Kalpana et al., 2011; Umpuch et al., 2015). Thus a new area of global emission reduction and energy conservation can be created if the organic pollutants in wastewater can be recovered and reused with new treatment technologies. Here we report a new type of recyclable wastewater treatment agent with the ability to recover organic material and ions that can be used to solve the aforementioned sewage treatment problems such as high energy consumption, inefficient use of time, CO2 production, and other defects such as eutrophication (Vijayaraghavan and Yun, 2008; Kartal et al., 2010). The agent is a selected mutant bacterial cell (Genetically Engineered Bacillus subtilis, GEBS), with the yqfY and spo0A genes knocked-out. By knocking-out the yqfY gene, the acetylation reaction during cell wall peptidoglycan synthesis is eliminated, and the resulting cells become more polar and positively charged due to the increased abundance of free amines and hydroxyl groups on the cell wall. Additionally, knocking-out the spo0A gene prevents the formation of spores, which has no organic waste adsorbing capability. The resulting GEBS clone has better affinity and enhanced adsorbing capacity for organic molecules (details of the construction and characterization of GEBS clone will be published in a separate paper). Our results show that GEBS can rapidly adsorb Direct Blue dye type organic molecules and ions in waste water, and can rapidly lower the chemical oxygen demand (COD) level to the required standard, and in some cases even to zero. In addition, the cells can be regenerated and reused in a short time (3e5 min). Using GEBS biomass to rapidly and efficiently recover organic matter and ions in wastewater is an important method towards reducing carbon emissions (Xie et al., 2013). The application of this new biosorbent can overcome the defects of the traditional biochemical treatment method by improving the energy efficiency and greatly shortening the process flow and time of organic wastewater treatment. The new application can also reduce the land required for wastewater treatment, and will furthermore recycle water resources and reduce carbon emission. The novel agent and application process may serve as an important contribution for both global energy and pollution reduction. 2. Materials and methods 2.1. GEBS selection and culture A bacterial strain B. Subtilis (Accession: JN165753) (isolated from the South Lake, Wuhan, China) was irradiated by UV (power 15 W, irradiation distance 30 cm, irradiation time 70 s) to induce random mutations. A mutant ZN0871v11 was selected by the highest capacity of adsorbing Direct Blue 2B dye. Mutant ZN0871v11 was further genetically engineered by reforming the cell wall peptidoglycan synthesis pathway (to knock out the yqfY and spo0A genes). The clone is called GEBS. The GEBS clone was inoculated in LB medium (Tryptone 1.0 g, yeast extract 0.5 g, NaCl 0.5 g, H2O 100 mL), and grown at 37 C for 48 h. The resulting culture was centrifuged at 7280 g for 5 min, and the supernatant was discarded. Cell pellets were resuspended and boiled for 5 min, washed 3 times with distilled water, and then added to the samples to be treated in predetermined proportions. The ratio of wet weight (WT)
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and dry weight of the cells is 6.88:1. 2.2. Adsorbing efficiency at different temperatures, pH, and with different ions The concentration of Direct Blue 2B solution was 250 mg/L. The effect of temperature on the adsorbing ability of GEBS biomass was tested from 20 C to 42 C. The pH range used in this study was from 4.0 to 10.3 at room temperature. The following buffer systems were used: 0.1 M potassium acetate buffer solution (pH 4.0e5.5), 0.1 M potassium phosphate buffer (pH 6.0e7.0), 0.1 M TriseHCl buffer solution (pH 7.5e8.5) and CAPS (3-(cyclohexylamino) -1-propane sulphonic acid) (Amresco, Solon, OH) buffer (pH 10e13). The concentrations of Fe2þ, Cu2þ, Zn2þ, Ca2þ, Mg2þ, Kþ, Naþ tested were 20, 50, 100, 150, 200 mg/L respectively. 3. Results 3.1. Effects of temperature and pH on adsorbing efficiency The adsorbing efficiency of GEBS biomass at different pH is shown in Fig. 1a. The figure shows significant differences in adsorbing efficiency at the measured pH range between 5.0 and 10.3. The efficiency is the highest at the low pH, which is 93% at pH 5.0, and at pH 5.5 it has already decreased to 60%, with further decreased adsorbing efficiency at higher pH. The optimal pH of the adsorbing efficiency of GEBS biomass is significantly higher than other reported biosorbents. In reported literatures, when various bacterial cells were used as biosorbents, the required pH was very low (e.g. pH1.0), and the adsorption time was quite long (e.g. 12e24 h) (Baoe and Xiu, 2011; Won et al., 2009; Vijayaraghavan and Yun, 2007; Won and Yun, 2008). Fig. 1b shows that the adsorbing efficiency of GEBS biomass for Direct Blue dye is over 91% in a range of temperatures from 20 C to 42 C. The efficiency reached a maximum of 100% when measured at 37 C and 42 C. In general, the results showed that temperature has no obvious effect on the adsorbing efficiency (Fig. 1b). 3.2. Adsorbing efficiency of GEBS biomass in the presence of different ions As shown in Fig. 1c, we studied the effects of 7 common metal ions on the adsorbing efficiency of GEBS cells. It is important to note that in our adsorption system, the tested metal ions don't interfere with dye biosorption as it does in other systems; on the contrary, the ions enhanced the absorbing rate and increased the adsorbing efficiency. Results show that at concentrations of 20, 50, 100, 150, and 200 mg/L, the metal ions significantly promoted the adsorbing efficiency. The efficiency increases as the concentration of ions increased. Among different ions, the influence of Fe2þ, Cu2þ, and Zn2þ are the most significant (Fig. 1c). In the testing conditions, when the concentration of Fe2þ was present in the low range of 20 mg/L, the adsorbing efficiency was only 28%, but the adsorbing efficiency increases to 50% at 50 mg/L, and to 100% at 150 mg/L. The adsorbing efficiency profile of Cu2þ is similar to that of Fe2þ: at low concentration (20e100 mg/L), the efficiency is about 12e38%, but when the concentration reached 150 mg/L and 200 mg/L, the efficiency increased to the maximum of 97% and 100%, respectively. At low concentrations (20 mg/L and 50 mg/L), Zn2þ had a small positive effect of 11e37% on adsorbing efficiency, but the effect reached to 90% at a high concentration of 200 mg/L. The adsorbing efficiency was not enhanced significantly by Ca2þand Mg2þ in the tested concentrations, with a moderate adsorbing efficiency increase of 53e66% for Ca2þ and 44e55% for Mg2þ, respectively. Monovalent ions (Naþ and Kþ) have limited effects on the adsorbing efficiency of
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Fig. 1. The adsorbing efficiency at different temperature, pH and ions using Direct Blue dye solution. The reaction volume is 100 mL; the GEBS concentration is 1200 mg/L; and the concentration of Direct Blue solution was 250 mg/L. (a) The adsorbing efficiency of pH at room temperature for 30 min. (b) The adsorbing efficiency of temperature at pH5.0 for 30 min. (c) The adsorbing efficiency of metal ions on the efficiency of GEBS biomass (metal ions concentration: 20, 50, 100, 150, 200 mg/L, temperature: 25 C, pH 5.0, time: 3e5 min) to adsorb Direct Blue.
GEBS cells for Direct Blue dye with adsorbing efficiency of 15e29% for Kþ and 11e18% for Naþ. Among the common ions tested, the most efficient adsorption promoters are Fe2þ and Cu2þ, followed by Zn2þ. We then tested the adsorbing efficiency of GEBS cells at an optimal condition of pH 5.0, 37 C, and Fe2þ or Cu2þ at concentration of 150e200 mg/L. Precipitation appeared immediately after the GEBS cells and Direct Blue dye solution was mixed. The time of incubation for biosorption was shortened from about 50 min to 2e10 min when ions were added. Results showed that the adsorbing efficiency was 100% either by measuring at OD545 or COD in the supernatant. The residual Fe2þ and Cl in the supernatant decreased from 73.68 and 532.5 mg/L to 4.01 and 0.7 mg/L respectively. The adsorbing efficiency were 94.56% (Fe2þ) and 99.83% (Cl) respectively, which indicated that the GEBS cells can capture organics and ions from wastewater simultaneously and achieve co-precipitation within several minutes. The results also demonstrated the superior adsorbing efficiency and high adsorbing rate of GEBS cells compared with other biosorbents reported in the literatures (Baoe et al., 2011; Won et al., 2009, 2013; Xi et al., 2013; Vijayaraghavan and Yun, 2007; Won and Yun, 2008). 3.3. The uptake of organic molecules and adsorbing equilibration The GEBS biomass was added to five bottles containing Direct Blue dye solution according to the arithmetic progression method. After mixing evenly, the mixtures were placed at room temperature. The adsorbing phenomenon of Direct Blue dye can be observed visibly by appearance of coagulation and sedimentation, resulting in GEBS cells turning blue and the simultaneous fading of the color of the dye solution. Fig. 2a shows the equilibrium of a linear decrease of supernatant at 545 nm at increasing amount of GEBS cells. The linear equation is given as: y ¼ 0.1208x, where y (mg) is the adsorbed organics and x (mg) is the amount of the biosorbent (WT).
Fig. 2. Adsorbing equilibrium of GEBS cells. (a) Capacity of biosorbent capturing Direct Blue dye. Y: adsorbed organics (mg); X: GEBS cells (mg, wet weight). (b) Kinetic curve using Lagergren models applied to biosorption of Direct Blue 2B on GEBS biomass. Y: Dye adsorbed (mg)/Biomass(g,wt); X: Time (s).
The equation shows that the dose of the biosorbent added is inversely proportional to the color of the supernatant. When the GEBS cells added was 77.98 mg (WT), the color of the supernatant was reduced to 0. At the ratio of GEBS cell mass (dry weight) and dye mass of 1:1.25 or more, the color and COD of the treated dye solution were reduced to “0”. The adsorbing efficiency is obviously higher than the adsorption capacity of agricultural solid wastes
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and microorganisms for the removal of dyes (Guo et al., 2013; Won et al., 2013; Xi et al., 2013; Won and Yun, 2008; Pajot et al., 2011). The kinetic curve for biosorption of Direct Blue 2B by GEBS biomass followed by the Lagergren and Ho et al. models is shown in Fig. 2b. From the curve we derived the following equation: y ¼ 2.3354Ln(x) þ 24.411, where y is the milligram dye adsorbed per gram GEBS biomass (WT), and x is the time in seconds. The figure shows that the capturing or biosorption was very quick, almost reaching the maximum in 5 s. In addition, the biosorption was irreversible; therefore the curve shown in Fig. 2b significantly differs from the curves of the reported biosorbents, though the equilibrium equation used was the same (Chu and Chen, 2002).
3.4. Recovery efficiency of the adsorbed organic molecules To test possible regeneration and reuse, GEBS cells with adsorbed Direct Blue 2B dye (Org-GEBS) were mixed with six different regeneration eluents, incubated for 2 min, and centrifuged at 7280 g for 2 min. The resulting supernatants turned blue, which were quantified by OD545 measurement. The results showed that the captured dye molecules could be eluted from the biosorbent by the tested regeneration eluents, thereby recovering the used GEBS cells. The total amount of dye eluted into the supernatant increased with increasing volume of regeneration eluents, and the dye color on GEBS cells gradually disappeared (Fig. 3a, b). When the added regeneration eluent reaches a certain volume, the captured dye can be washed away completely, and GEBS cells gradually became white and finally returned to their original state. At room temperature, the elution formula for each eluent can be expressed as the following, as shown in Fig. 3.
For For For For For For
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Eluent A: y ¼ 0.0097x. Eluent B: y ¼ 0.0205x Eluent C: y ¼ 0.0218x detergent solution (2 g/L): y ¼ 4.5141x Na2CO3 (5 mM): y ¼ 8.6897x NaHCO3 (10 mM): y ¼ 5.6145x
According to the formula, in the experimental range shown in Fig. 3, the amount of dye released is directly proportional to the volume of the eluent used. The elution efficiency of the first three eluents is eluent B > eluent C > eluent A. One mL of eluent A, B, and C can release 9.7 mg, 21.8 mg, and 20.5 mg of Direct Blue dye from GEBS cells, respectively. Also, 1 mg of Na2CO3, NaHCO3, and detergent powder could release 8.69 mg, 5.61 mg, and 4.51 mg of Direct Blue dye from the GEBS cells, respectively. Accordingly, the elution capability of Na2CO3 is about two times that of detergent powder's. For all of the six eluents tested, at a certain range, the elution capacity is also inversely proportional to the elution concentration: the higher the elution concentration, the less elution volume will be required for GEBS cell recovery. 3.5. Efficiency of recycling and reuse of GEBS biomass The regenerated GEBS biomass can be used to adsorb the organic dye again with high efficiency. Among the 6 regeneration eluents tested, Na2CO3 and NaHCO3 solution produced the best recycling results, which showed that the regenerated GEBS cells can still efficiently eliminate color of the tested dye solution (Table 1). We recycled 26.7 mg of wet weight Org-GEBS 3 times using the above 6 eluents. After regenerating with Eluent A for total of 3 times, the recovered GEBS cells still have over 85% of the original color elimination ability and 93.6e90.3% of the original COD elimination ability, after each round of regeneration respectively. The results did show that the color and COD elimination ability decrease with each round of regeneration. For eluent B, the same experiments showed over 88.65% of the original color elimination ability and 98.08e91.52% of the original COD elimination ability. For Eluent C, the data were over 95.25% for color elimination and 97.31%e92.18% for COD elimination, respectively. The reasons of decreased color and COD elimination efficiency could be that some GEBS cells were lost due to cohering on the vessel wall following each elution, or/and the alkaline eluents may cause permanent structural damage to the cells. The adsorbing efficiency of the GEBS cells recycled with 2 g/L detergent powder solution for 3 times were 90.1 e 87% for color elimination and 93.4e86.9% for COD elimination, respectively. The data for 5 mM Na2CO3 solution was 100 e 95.2% for color elimination, and 96.7e92.1% for COD elimination; while for 10 mM NaHCO3 solution was 100% for color and 97.4e95.6% for COD. 3.6. Industrial wastewater treatment with GEBS cells
Fig. 3. Recovery efficiency of six different eluents. GEBS cells with adsorbed Direct Blue dye were mixed with six different regeneration eluents, incubated for 2 min, and centrifuged at 7280 g for 2 min. The resulting supernatants turned blue, which were quantified at 545 nm after diluting 80 times. (a) The recovery efficiency of eluent A, B and C to Direct Blue adsorbed by GEBS cells. (b) The recovery efficiency of Na2CO3, NaHCO3, and detergent powder to Direct Blue adsorbed by GEBS cells.
Raw industrial wastewater samples (1 sample each from a textile and paper mill industry, and 2 samples from a petrochemical enterprise) were treated with GEBS cells at pH 5.0, with an addition of 200 mg/L of Fe2þ. The mixtures were incubated for 3e5 min at room temperature after 2 g of wet weight GEBS cells were added to 1 L wastewater. The resulting GEBS cells were then regenerated three times with 10 mM NaHCO3 eluent and their adsorbing ability tested each time after regeneration (Table 1). The color (OD545) elimination rate of all samples was 100% for the regenerated GEBS cells. The COD elimination rate of the sample from textile enterprise were between 99.39 and 86%, while data for sample from the paper mill were 97.55%e89.07%. The data for the 2 samples from
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petrochemical enterprise were 95.88 e 90.2%, and 97.57e91.65%, respectively. 4. Discussion This study shows that at a fixed ratio, our adsorbing agent can adsorb organic and ions pollutants in wastewater, and can essentially eliminate dye color and COD in less than a minute. The ratio of the biosorbent to the Direct Blue 2B organic dyes can be amounted to 1:1.25, demonstrating a very high adsorbing capability. Importantly, this biosorbent can be regenerated within 3e5 min, and therefore has high regeneration efficiency and presents low cost for future industrial application. The pH has a significant effect on the capturing capacity. One of the reasons may be that at acidic conditions (pH 5.0e5.5), the polarity of the organic molecules undergoes changes leading to different ionic states and binding profiles. Results showed that metal ions, especially Fe2þ and Cu2þ can consolidate the polarity and ionic state of organic molecules to promote the capturing capacity and rate of GEBS biomass. The residue of cations, anions and organics in the supernatant decreased to very low concentration indicates that the cations and anions not only may affect the dye molecules and the surface of GEBS biomass, but also achieve coprecipitation with the biosorbent and organics in a very short time. Both the cations and anions take part in the interaction between the surface of GEBS cells and dye molecules during the biosorption. Compared with many reported biosorbents (Xiong et al., 2010), the GEBS cells differentiate themselves with their ability to capture both organics and ions from wastewater, which is very important in wastewater treatment. As described above, our biosorbent has significant practical advantages. Many reported adsorbents have relatively low adsorbing capability and some need lengthy times (e.g. 5 d) to reach a high adsorption ratio, in addition to difficulties in their recycling ra and Aicha, 2006; Sivasamy and reuse (Pathak et al., 2014; Nace and Sundarabal, 2011; Xiong et al., 2010; Inoue et al., 2013; Kalpana et al., 2011; Umpuch et al., 2015) (Table 2). Among the general applied absorbents, active carbon is used the most because it can be regenerated and reused. However, the used active carbon needs to be regenerated in a factory, demanding sophisticated equipment, consuming high energy, and resulting in costly recycling (Chen et al., 2012). One of the unique aspects of the GEBS cells is that it does not obey the complex Lagergren equation (Won and Yun, 2008; Pajot et al., 2011; Chen et al., 2012), but rather a simple linear proportional relationship. The biosorbent can complete adsorption reaction in a short time, and has overwhelming capturing capabilities for organic molecules. Another feature is that GEBS cells can overcome many weakness and difficulties inherent in the existing water treatment systems because they can adsorb organic pollutants and ions in short time (5e10 min) and do not require special equipment and conditions. Moreover, the biosorbent can be recycled and reused many times, requiring only small volumes of eluent to achieve regeneration, and thus greatly reduces the cost for treating wastewater and polluted water. The adsorbed organic pollutants during the regeneration process can also be recovered and used, making complete reservation of energy and environment possible. In this study, we also compared the regeneration efficiency of 6 different eluents (Table 1). Results showed that the biosorbent can be regenerated with high efficiency by all 6 of the eluents. The principle of each of the above 6 eluents on the regeneration of the biosorbent, however, is different. For eluents A, B, and C, a strong electrostatic force and affinity to the adsorbed organic molecules may be the main regeneration forces. Eluent A and C also contain
SDS, which is a strong surfactant and may add to the regeneration. The regeneration ability of Na2CO3 and NaHCO3 could be due to ion exchange interactions. The biosorbent captures organic molecules at acidic conditions, and when the pH shifts to strongly alkaline in Na2CO3 and NaHCO3 solutions, the captured molecules could be released by the changes in charge. The elution principle of the detergent used in this study may be the same as it is in laundry: the detergent helps to “wash out” the adsorbed pollutants and regenerates the biosorbent. For industrial applications in sewage treatment, optimal eluent selection may depend on the type of pollutant. Compared with the existing organic wastewater processing techniques (Table 2), treating water pollution with the biosorbent described in this study is faster, more efficient, and overcomes many practical problems facing the classical sewage processing ra and Aicha, 2006; Xiong et al., 2010). In addition, the GEBS (Nace cells used in this study are easy to produce and regenerate, and the released organic pollutants from the regeneration process can be used in energy generation such as methane fermentation, further reducing the environmental impact (Kalpana et al., 2011). In summary, the method described in this study is a new technology based upon a highly efficient wastewater treatment agent, which has high adsorption rate, low producing cost, low energy consumption, reduced land use, low demand for equipment and environment, no secondary pollution, and is both recyclable and reusable. Although not tested, theoretically, the biosorbent may even be used to enrich and clean nuclear pollutants released into the environment in emergent situations. 5. Conclusions In this study, we used direct blue dye solution as a model for polluted water treatment with the biosorbent GEBS cells because the dye is easy to monitor by visible color change, and easy to quantify by spectrophotometric methods. The results show that, compared with the existing flocculent and adsorbents, the GEBS biomass offers significant advantages in several aspects. First, because of the genetic selection and engineering, the capturing capacity is a perfect combination of electrostatic, hydrophobic, and affinity interactions, and therefore has a much higher capturing capacity than the existing flocculent and adsorbents do. Second, the GEBS cells can adsorb organics and cations and anions simultaneously from wastewater and achieve co-precipitation within several minutes. Third, the capturing and regeneration rate is much faster, in the range of 3e5 min; this property may enable more efficient and practical industrial applications. In summary, from GEBS cell production to recycling, the whole process is simple to use, with low energy consumption, and without toxic secondary pollution. Fourth, the results of raw industrial wastewater samples show that the GEBS cells can capture many kinds of complex organic molecules in raw industrial pollution waters and can be easily and rapidly regenerated in several minutes and reused in a simulated “real world” situation. These results demonstrate that GEBS cells can have broad application in water pollution treatment. This is therefore a new environment-friendly technology for treating water pollution in general, and for treating difficult to treat organic water pollution in particular. Acknowledgments We thank Professors Buhai Li and Xiaomei Sun for reagents. This study is supported by the Fundamental Research Funds for the Central Universities of China (Grant No. CZY12023; ZZY10009), the Natural Science Key Foundation of South-Central University for Nationalities (Grant No. YZZ07014).
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