J. Cent. South Univ. (2015) 22: 95−102 DOI: 10.1007/s11771-015-2499-5
Characterization of Microcystis Aeruginosa immobilized in complex of PVA and sodium alginate and its application on phosphorous removal in wastewater LI Fei(李飞)1, 2, MAO Wen-juan(毛文娟)1, 2, LI Xue(李雪)3, WANG Xiao-yu(王晓钰)1, 2, XIAO Zhi-hua(肖智华)1, 2, ZHOU Yao-yu(周耀渝)1, 2, ZENG Guang-ming(曾光明)1, 2 1. College of Environmental Science and Engineering, Hunan University, Changsha 410082, China; 2. Key Laboratory of Environmental Biology and Pollution Control of Ministry of Education (Hunan University), Changsha 410082, China; 3. Department of Bioengineering and Environmental Science, Changsha University, Changsha 410003, China © Central South University Press and Springer-Verlag Berlin Heidelberg 2015 Abstract: Based on ecological niche theory, Microcystis Aeruginosa (MA) immobilized in the complex of polyvinyl alcohol (PVA) and sodium alginate (SA) crosslinked by CaCl2, was treated as a new kind of special species, and its properties were investigated. Chlorophyll a was used to characterize the bioactivity of the immobilized MA. Results reveal that the gel beads have mechanical strength and chemical stability even under non-sterile harsh conditions, which may be attributed to the rarely seen structure (including three different layers: dense surface, tubular-shaped divergent structure and honeycomb crystal lattice layer) of the immobilized MA determined by scanning electron microscope (SEM). SEM also displays that more quantity of MA is attached to the inwall after cultivation, which demonstrates that the MA within beads maintains high bioactivity. Removal capacities on phosphorous (P) removal in wastewater in the presence and absence of the BG-11 medium were examined, and the removal ratios are 80.3% and 76.7%, respectively, which indicates that the beads without providing ample nutrients still have high capacity of P removal. In addition, control experiment, utilizing polyvinyl alcohol and sodium alginate (PVA-SA) beads without immobilized MA, demonstrates that MA within beads plays the key role in absorbing P. Key words: eutrophication; immobilized Microcystis Aeruginosa (MA); bioactivity; phosphorous removal
1 Introduction Eutrophication has been one of the most serious environmental problems for decades. Previous researches [1−8] have demonstrated that P is a key problematic nutrient causing eutrophication. It accelerates blooms of phytoplankton bringing about eutrophication, and changes the water from a macrophysics-dominated clear state to a phytoplankton-dominated turbid state, which probably causes deleterious effects on the whole ecosystem [9−10]. It is therefore mandatory to reduce its abnormal concentration level in water body and wastewater. The treatment of P can be achieved by several physical, chemical and biological methods [11−17]. However, present researches have not been satisfied enough in the processes for the removal of P in wastewater. The issue of eutrophication has also not been solved successfully. In ecology, a niche is a term depicting the way of life of a species. Each species is deemed to have a
separate, unique niche. The ecological niche describes how an organism or population responds to the distribution of resources and competitors (e.g., by growing when resources are abundant, and when predators, parasites and pathogens are scarce) and how it responds to those opposite factors (e.g., limiting access to resources by other organisms, acting as a food source for predators and a consumer of prey). Based on ecological niche theory, we try to find such a creature to be the dominant species to replace the niche of algae which have the ability of causing eutrophication. However, the long-term natural selection has proved that the algae are the dominant species in the eutrophic water body. Thus, supposing a method, the algae are directly taken as the special creature through operation evolution. On one hand, the content of phosphorus in the water could be reduced since the treated algae have ability in taking up P, so the original algae under freely growing conditions would be inhibited. On the other hand, the easy recovery of algae can help avoid the secondary pollution.
Foundation item: Projects(51178172, 51308076) supported by the National Natural Science Foundation of China; Project(13JJ4107) supported by Hunan Provincial Natural Science Foundation, China; Project(K1207026-31) supported by Changsha Planning Project of Science and Technology, China Received date: 2013−09−17; Accepted date: 2013−12−26 Corresponding author: LI Xue, PhD; Tel: +86−18684940100; E-mail:
[email protected]
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Immobilization techniques provide an alternative solution. They have been widely applied to wastewater treatment, such as phenol degradation [18], nitrogen removal [19−20], sulfate reduction and copper removal [21], and pineapple wastewater treatment [22]. However, few systematic studies have been carried out to investigate the application of immobilized microorganism for P removal. The application of immobilized microorganism not only helps maintain high bacterial cell concentrations, but also increases the biological activity of the microorganisms, makes control separation easy, and protects cells against toxic substances and changes in environment factors [10, 20, 22−27]. Although many natural and synthetic polymers have been used, each has its own drawbacks [24, 26, 28−30]. In this work, a specially designed complex of PVA solution and sodium alginate solution crosslinked by CaCl2 was prepared for immobilization of Microcystis Aeruginosa (MA). MA is a kind of common algae causing eutrophication. This method, which has not been previously reported, might not only avoid the agglomeration of PVA gel beads and improve PVA network structure, but also make MA remain proliferation and bioactivity in gel beads without leakage. The objectives of this work were to determine the feasibility of using PVA as a gel matrix for immobilization of MA, and to establish a simple and fast procedure for MA immobilization on a laboratory scale, which might be further adapted to the industry. Further, the properties of the immobilized beads and the efficiency of P removal by cultivation immobilized beads as a new kind of special species were investigated. PVA-SA without immobilized MA was included as control to determine which was the key role (MA within beads, immobilization matrix or both) during the process of P removal.
2 Materials and methods 2.1 Microorganism and medium A strain of Microcystis Aeruginosa (MA) in this work was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences. The algae and the immobilized beads were cultivated in the BG-11 medium containing 1.5 g/L NaNO3, 0.04 g/L K2HPO4·3H2O, 0.075 g/L MgSO4·7H2O, 0.036 g/L CaCl2·2H2O, 0.006 g/L citric acid, 0.006 g/L ferric ammonium citric acid, 0.001 g/L EDTA, 0.02 g/L Na2CO3 and 1 mL A5+Co solution. All the chemicals used in the experiments were of analytical grade. Before inoculation and cultivation, the BG-11 medium and apparatus were autoclaved at 110 °C for 30 min. The initial pH of the medium was adjusted to 7.0−7.3 by using 0.1 mol/L HCl
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or 0.1 mol/L NaOH solutions. 2.2 Preparation of MA suspension MA was inoculated into 400 mL BG-11 medium in a 1000 mL conical flask. Cultivation was carried out at 25 °C under the illumination intensity of 2000 lx with 6−7 h per day for 20 days until the media changed in color from light green to dark green. After cultivation and following centrifugation, 2300 r/min was processed for 15 min to remove supernatant. The harvested microorganisms were washed aseptically with distilled water. Finally, the total volume of concentrated suspension was about 100 mL. 2.3 Immobilization procedure PVA (polyvinyl alcohol) is a promising type of synthetic polymer for immobilization due to its attractive characteristics such as high biological compatibility, durability, high chemical stability, low material cost, nontoxity to microorganisms and highly porous structure [21−22, 27−29, 31−36]. Therefore, PVA has gained increasing attentions on microbial immobilization. This procedure was performed in sterile condition. PVA, 20 g, with 2000 degree of polymerization and 2 g sodium alginate were diluted with distilled water to 100 mL and heated until being dissolved. After the mixture cooled to room temperature, the 100 mL concentrated suspension was added to the mixed solution. The final concentrations of PVA and SA were 10% and 1%, respectively. Then the solution was extruded with the form of droplets into 4% (w/v) CaCl2 solution through a syringe and gently stirred, and later submerged for 8 h at 4 °C to form spherical and complete solidification. After gelling, the beads were removed and washed with normal saline for two times, and then stored at 4 °C for further use. 2.4 Acclimation of immobilized beads The harvested beads were transferred into fresh BG-11 medium at the ratio of 1:3 in volume. In order to investigate the bioactivity of the immobilized gels, Chlorophyll a concentrations were analyzed by the method [37−38] based on acetone extraction and finally determined by using ultraviolet spectrophotometer (UV754N, Shanghai, China). In addition, the Chlorophyll a value in medium was also measured as the symbol whether there was leakage of the algae or not. 2.5 Characterization 2.5.1 Morphological observation Samples of the MA immobilization beads were rinsed with distilled water two times, and then fixed with 2.0% (w/v) glutaraldehyde for 20 min. The fixed gel beads were then dehydrated by sequential immersion in
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increasing concentrations of acetone (50%, 60%, 70%, 80%, 90%) (w/v), then twice in anhydrous acetone to remove the final traces of water. The dehydrated beads were then freeze-dried (FD-1, Beijing, China), cut in half with a sterile scalpel, and coated with gold. The surface and cross-sectional morphologies of the immobilized beads before and after culture were examined by scanning electron microscopy (FEI QUANTA200, USA, operated at 10 kV). 2.5.2 Dilatability test A method described by WANG et al [29] was utilized in this work for dilatability test. Fifty MA-PVA beads were immersed in distilled water at 25 °C for 15 d. Diameters of these beads were measured by a vernier caliper. The dilatability (I) was calculated from the ratio: I=D2/D1
(1)
where D1 is the average diameter before immersion; D2 is the average diameter after immersion. The experiment was performed in triplicate and the average data were used in data analysis. 2.5.3 Mechanical stability test Based on KHOO and TING’s research [39], there were some changes in this work. A beaker with a diameter of 7.7 cm and height of 12 cm was divided into four equal regions by the baffles (1.1 cm wide each). Fifty beads were added to the beaker, followed by distilled water with the height of 7 cm. The agitation speed was controlled at 2000, 3000 and 4000 r/min by a tachometer (eGL2-DT2236). The beads were agitated in the beaker for 7 d and the surviving beads were counted. 2.5.4 Chemical stability test According to the method reported previously [28, 40−41], acid solutions with pH 4−6 using hydrochloric acid, and alkali solutions with pH 8−10 were prepared using sodium hydroxide. One hundred beads were immersed in each solution, and 5 h later, the remaining beads were counted. pH meter (FE20 Mettler Toledo, Shanghai, China) was used for pH determination. 2.6 P removal estimation The beads, which had been cultured, were put into a synthetic wastewater containing 30 μg/L K2HPO4. 25 mL samples were taken once every other day throughout the experiment for the measurement of P and Chlorophyll a at 25 °C, respectively. In addition, the PVA-SA beads without immobilizing MA were also carried out to investigate the efficiency for P removal. Molybdenum Blue method [42] was used for phosphate analysis.
3 Results and discussion 3.1 Cultivation of MA-PVA beads Chlorophyll is the essential component of biological photosynthesis. Microcystis Aeruginosa (MA) is a kind
of photoautotrophic algae which can use Chlorophyll to carry out photosynthesis under the sunlight, and provide the energy for its own metabolism, growth and reproduction. Therefore, the increase of Chlorophyll indicates that the number of algae and its bioactivity are enhanced at the same time [43−44]. As depicted in Fig. 1, within the planned period of time, the concentration of Chlorophyll a increases to 186.5 mg/L, which indicates that the immobilized MA keeps bioactivity in beads and can be able to carry on schizogamy. Meanwhile, the color changes of the beads are observed during cultivation. Light green turns into dark green with time during cultivation. On the contrary, in Fig. 1, the amount of Chlorophyll a is almost zero in Fig. 1(b), which demonstrates that there are nearly no residual algae in nutrient solution, so it is conceivable that the increase of Chlorophyll a is mainly due to the algae immobilized in matrix. As a result, it is well proved that the presented method is effective in immobilizing algae, without leakage.
Fig. 1 Variation of chlorophyll a in beads and medium during cultivation
3.2 Morphological observation As shown in Fig. 2, the surface and interior structure of the gel beads and the microbial population development and distribution within them are characterized by SEM. It can be seen in Fig. 2(a) that the microstructure of single spherical bead has many severe wrinkles on its surface, which is caused by the partial collapsing of polymer network during dehydration and drying. There is a sunken trough width of 10 µm between every two parts of heliciform embossment (Fig. 2(b)). After cultivation, a large quantity of spore-shaped matter growing on the original sunken trough is observed in Fig. 2(c), which might be contributed to blocking the leakage of algae. Figure 2(d) shows a transverse section of the gel bead. It is obvious that there are three layers of different structures (the wrinkled peripheral surface, tubular-shaped divergent structure and honeycomb
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Fig. 2 SEM images of immobilized beads: (a) Entire exterior of gel bead; (b) Peripheral surface of gel bead before culture; (c) Peripheral surface of gel bead after culture; (d) Cross-sectional image of gel bead; (e) Middle layer; (f) Biological attachments after culture ((f1) Before culture; (f2) Interior layer)
support layer) from surface to interior. Through magnifying the middle layer (Fig. 2(e)), a huge specific porosity and surface area can be seen. Remarkably, the diameters of the pores are larger than the thickness of the
wall, resulting in high surface to volume ratios, which provides sufficient space for the algae growth and facilitates substrate diffusion and gas transfer. Figure 2(f) (after cultivation) displays more cells attached to the
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inwall than in Fig. 2(f1) (before cultivation). The results seem to correspond to the Chlorophyll a test that the immobilized algae within beads maintains bioactivity, and can grow and propagate. Figure 2(g) presents the interior structure of the beads. As we know, the honeycomb structure is one of the most fastness constructions in organic sphere, which implies that the gel beads own hard mechanical capacity. This special structure of the gel beads has never been reported and the reasons for formation are comprehensive. It was reported that PVA had a hydrophilic nature due to the hydroxyl groups in its molecular structure. When PVA was used as carriers of microorganisms, the hydrogen bonding ability could be maintained, which could create a hydrophilic microenvironment for the metabolism of the immobilized microorganisms and protection against detrimental conditions [21, 23, 36]. On the other hand, the addition of sodium alginate gave PVA a beneficial influence on the physical properties, such as reducing the tendency of the beads to agglomerate and the solubility of the PVA gels, as well as improving the porosity of the PVA [29, 34]. Besides, SA could form a reticulated structure when it contacted with Ca2+, which facilitated the formation of hydrogen bonds between PVA molecules and promoted the formation of the interpenetrating gel network, therefore contributing to improvement of the strength and durability of the beads [45−47]. 3.3 Stability of beads Stability is the key parameter for the potential application of immobilized MA in industries. Dilatability test and mechanical strength test were performed to examine the stability of gels. All of the beads are about 4.1 mm in diameter before immersion. After immersion for 15 d, most of the beads do not change notably. The diameter is around 5.3 mm. The distention index (I) is given in Table 1, while the beads are somewhat swollen, but the effect is almost negligible. It is deduced that the PVA-alginate beads fabricated may be characterized as highly rubbery and elastic probably due to the right amounts and the ratio of the forming agents used [22, 28, 48]. Table 1 Physical stability of MA-immobilized bead Starting diameter/mm
Expanded diameter/mm
Distention index
Mechanical strength
4.1
5.3
1.29
High
Continuously stirring with constant speed was conducted for each group in the mechanical stability test and the results are shown in Fig. 3. As shown in Fig. 3, the percentage of entire bead remained varies from 92%,
87% and 45% for group 1, 2, 3, respectively. The Group 1 is nearly almost not broken at agitation speed of 2000 r/min. The beads have a very large mechanical strength and are very difficult to break. At the speed of 3000 r/min, the beads of Group 2 crack partially, but the value is still high. The percentage of Group 3 decreases sharply at the speed of 4000 r/min. More than half of beads are broken. While in reality, such high agitation speed of 2000 r/min does not exist in eutrophication environment, not to mention 4000 r/min. It is indicated that the beads can be applied in actual wastewater treatment systems due to its high elasticity and strong stability. This finding is in accordance with previous report [40].
Fig. 3 Mechanical stability of MA-immobilized bead
3.4 Effect of pH The pH effect on the immobilized gels was studied at 25 °C. The reactions were carried out and the results are depicted in Fig. 4. The maximum beads remaining ratio is 95% at pH 7. There is no significant difference of the ratio as pH ranges from 6 to 9. While the pH value further increases to 11, the beads are swollen and broken obviously. There is a sharp decline in the ratio. The same situation happens as the pH decreases from 5 to 3. Previous reports stated that the physical crosslinking PVA cannot withstand high acidity or alkalinity [49]. It is probably because the strong alkaline or acidic solution can weaken the intermolecular hydrogen bonds between PVA and other ions. As the acidity or alkalinity increases, the excess H+ and OH− ions are present in the solution which might act as a chelating agent, leading to the decrosslinking of the beads [41]. Moreover, the —COO− groups of SA molecules tend to transform into —COOH groups under acid condition. Conversely, in alkaline environment, the —COOH groups in SA are easy to ionize and produce — COO− ions [49], which can corrode the crosslinked network. Besides, different solution pH values may also affect the behavior of the surface electric changes on microorganisms, which can give rise to the phenomenon descried above to some extent [45, 50].
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According to Fig. 5, the immobilized MA within beads plays a key role in P removal, and the mechanism is related to P adsorption and takes P as a kind of nutrient to absorb for supporting energy for growth and propagation. Therefore, the P removal by immobilized MA is permanent and the problem of desorption can be almost neglected. According to the previous report [51], MA can absorb more phosphorus even phosphorus concentration in its body has reached saturation.
Fig. 4 Chemical stability for MA-immobilized beads
On the other hand, results indicate that the immobilized MA can tolerate wider ranges of pH. This is probably due to the right amount of CaCl2 used to crosslink PVA and SA resulting in a good inner network structures and a well-protected microenvironment which can provide chemical stabilization for maintaining cell’s longevity under harsh conditions. In this sense, results from this experiment indicate that the system is more applicable for practical application [34, 50]. 3.5 P removal analysis The concentration of P was obtained with spectrophotometer by measuring the absorbance of filtrate at 700 nm, and then calculated from calibration curve. The variations of phosphorus concentrations with time of immobilized beads are depicted in Fig. 5. The P concentrations treated by immobilized MA gel beads with and without providing medium both show decreasing trend throughout the experiment. Initially, the curves fall slowly and no obvious difference among values is observed. There is a sharp decline on the curves after 5 d. The total decreased concentration of phosphorus after 13 d operation is observed in concentration without providing medium as compared to that with providing medium, whereas the value is slightly low. The removal rates of P are 80.3% and 76.7%, respectively. As it is known, ample nutrient element that microorganisms need can not be guaranteed under natural environment whenever and wherever possible. For this reason, the results indicate that the immobilized MA may have the ability to adapt to both nutrient-rich and nutrient-poor environment. The P concentration for the PVA-SA beads without immobilizing MA declines firstly, followed by ascending. The results indicate that the immobilized matrix is in possession of a certain extent of phosphorus adsorption, however, the original adsorption of P can release into the water again.
Fig. 5 Variation of P concentration with time
Figure 6 shows the concentration variations of Chlorophyll a within immobilized MA, accompanied with collected samples for P test. Initially, the concentration of Chlorophyll a decreases notably, which deduces that both bioactivity and amount of MA within beads reduce. From the 5th day on, the concentrations under two conditions both increase slowly, and the terminal values of Chlorophyll a are higher than the beginning. It is probably due to the fact that immobilization provides a shelter for cells against the adverse effects, which contributes to maintaining a certain extent of biomass. Besides, after a period of acclimation, immobilized MA can absorb phosphorus effectively, which demonstrates that MA in beads still own biological activity during P removal.
Fig. 6 Variation of chlorophyll a in MA-immobilized beads
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4 Conclusions For immobilization of Microcystis Aeruginosa (MA), the gel complex of PVA (10%, w/v) and sodium alginate (1%, w/v) using CaCl2 (4%, w/v) as crosslinking agent are deployed. The beads are excellent in fixing MA without leakage and maintaining biological activity. SEM reveals that MA can grow and propagate for its higher cell density and bioactivity. Further, it is demonstrated that the immobilized MA as a special kind of species owns relatively good mechanical, chemical stability and high bioactivity, which is attributed to the specific structure of the gel bead. Immobilized MA shows a good performance on phosphorus removal, and the removal ratio is almost 80.3% within 13 d with an initial concentration of 30 μg/L. There is a slight difference between providing medium or not, and the corresponding removal ratios are 80.3% and 76.7%, respectively. In addition, control experiment indicates that MA within beads plays a key role in P removal. This technique is demonstrated to be simple, inexpensive, lowly toxic and easily performed in present laboratory scale. To explore this kind of immobilized MA put into real eutrophic wastewater treatment, further studies (absorption mechanism) need to be done to evaluate and optimize the beads for application at industrial scales.
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