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Glycine induced culture-harvesting strategy for Botryococcus braunii
Journal of Bioscience and Bioengineering VOL. 121 No. 4, 424e430, 2016 www.elsevier.com/locate/jbiosc
Glycine induced culture-harvesting strategy for Botryococcus braunii Ying Shen,1, * Wenzhe Zhu,1 Chaozhou Chen,1 and Yilei Nie2 College of Mechanical Engineering and Automation, Fuzhou University, Fuzhou, Fujian 350116, China1 and Fujian Institute of Microbiology, Fuzhou 350007, China2 Received 28 January 2015; accepted 6 August 2015 Available online 6 November 2015
The objective of this study was to investigate the effects of culture conditions, including carbon sources and concentration, culture period, and precondition time, on the production of extracellular polymeric substances (EPS) and its influence on microalgal flocculation. EPS are natural high molecule polymer, excreted by microalgae themselves. EPS can accelerate the formation of microbial aggregates through binding cells closely. Organic carbon sources, such as glucose, glycerol, acetate and glycine were compared to select the optimal source to stimulate EPS accumulation. Subsequently, the effect of culture period, glycine dose and precondition time on EPS production and its influence on biomass growth and flocculation efficiency were investigated. As the main parts of EPS, tightly bound EPS were found positively related to suspended solids concentration. However, the loosely bound EPS may weaken the floc structure, leading to poor water-cells separation. Under the optimal condition with culture period of 16 days, glycine dose of 0.5 g lL1 and precondition time of 5 days, the biomass concentration increased from 1.49 to 2 g lL1, and the maximum suspended solids concentration of 7.06% with biomass recovery rate of 70.6% was achieved. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Flocculation; Botryococcus braunii; Extracellular polymeric substances; Suspended solids concentration; Biomass recovery rate]
Microalgae are one of the most promising renewable energy sources because of their tremendous potential in lipid production, less reliance on freshwater and arable lands compared to other oil crops (1,2). However, due to the small size (5 to 20 mm), negative surface charge (7.5 to 40 mV) and low biomass concentration (0.5 to 1 g l1), microalgal biomass harvest from growth medium is still a challenge, which accounts for 20 to 30% of the total production cost (3). Conventional harvesting processes such as centrifugation and filtration require large capital and operational cost (4,5). It was estimated that the capital and operational cost of centrifugation reached up to 12,500 dollar per hectare of harvested land and 5 to 13 dollar per liter medium (5,6). Compared to centrifugation or filtration, the capital (around 2000 dollar per hectare) and operational cost (around 0.05 to 0.4 dollar per liter) of flocculation is relatively lower (5,6). However, a major challenge is the recovery of the flocculants (usually multivalent metal salts) from medium, which will ultimately contaminate the water and final products. In addition, low suspended solids concentration and biomass recovery rate are the bottleneck of flocculation. As shown in Table 1, assuming that the cell concentration of microalgal medium is 2 g l1, there are around 2 g algal biomass dry weight, 8 g cell water (about 80% of the cell weight), and 990 g free water in 1 l medium (4). Centrifugation can remove almost all the free water to achieve about 20% suspended solids concentration and over 90% biomass recovery rate (6). However, the suspended solids concentration and biomass recovery rate achieved by flocculation were 4e6% and 50e90%, respectively (6). To increase the flocculation efficiency, it is important to achieve the
* Corresponding author. Tel.: þ86 13950301207; fax: þ86 0591 22866261. E-mail address: [email protected] (Y. Shen).
highest possible suspended solids concentration in algae harvesting to reduce energy consumption in drying while keeping biomass recovery rate in consideration. It is well known that extracellular polymeric substances (EPS) play an important role in biofilm formation of bacteria. However, EPS are not unique to bacteria since some of the most abundant EPS producers are microalgae (in particular, diatoms). The green alga Chlorella vulgaris JSC-7 and Ettlia texensis showed good autoflocculation efficiency due to the production of large amounts of EPS (predominantly polysaccharides) (7,8). Four filamentous cyanobacteria Microcoleus vaginatus, Scytonema javanicum, Phormidium tenue and Nostoc sp. and a coccoid single-cell green alga Desmococcus olivaceus were also found as the producers of EPS (9). EPS are composed of some high molecular weight compounds, including polysaccharide, protein, nucleic acids, humic substances, and ionisable functional groups like carboxylic, phosphoric amino and hydroxyl groups (10,11). It was reported that EPS present a dominant bridging mechanism between the floc components, namely cellular, bio-organic, and inorganic compounds (12e14). EPS are often divided into two major fractions: soluble EPS and bound EPS (15). The adhesion of soluble EPS to cells is weak, as a result of which soluble EPS are often dissolved in solution. The structure of bound EPS is generally depicted by a two layer model (16). The inner layer consists of tightly bound EPS, which has a certain shape and is bound tightly and stably with the cell surface. The outer layer, which consists of loosely bound EPS, is a loose and dispersible slime layer without an obvious edge. The content of the loosely bound EPS in microbial aggregates is always less than that of the tightly bound EPS (15,17). Some investigators have suggested that the composition and properties of EPS, rather than the quantity, are more important in flocculation (18,19).
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.08.004
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TABLE 1. Parameters obtained with centrifuge or flocculation. Parameters Algal biomass dry weight (g) Cell water (g) Free water (g) Suspended solids concentration (%) Dewatering rate (%) Biomass recovery rate (%)
MATERIALS AND METHODS
Culture
Centrifuge
Flocculation
2 8 990 0.2 e e
>1.8 >7.2 0 <20 99 >90
1e1.8 4e7.2 20e70 <6 92e97 50e90
TABLE 2. Influence of organic carbon sources on flocculation efficiency. Organic carbon source Glucose Glycine Acetate Glycerol
Suspended solids concentration (%) 3.48 4.03 2.05 2.27
0.12 0.17 0.09 0.10
Biomass recovery rate (%) 64.34 77.21 66.44 70.07
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3.23 4.54 2.33 3.45
Nutrients, especially the carbon sources and nitrogen concentration are significant factors that influence the excretion of EPS (12,20e22). The effect of organic carbon (acetate, glucose and glycerine) on flocculation of pleurochrysis carterae was investigated (13). The results indicated that under stress, due to nutrient depletion, the microbes produced EPS that promote flocculation of P. carterae. The results also showed that the recovery efficiency is positively correlated with the precondition time. In this study, four organic carbon sources were compared to select the optimal source for the culture-harvesting strategy for freshwater alga Botryococcus braunii. The effects of culture period, glycine dose and precondition time on biomass concentration, as well as EPS production were analyzed. In addition, the influence of EPS on suspended solids concentration and biomass recovery rate obtained from flocculation of B. braunii was investigated.
Algal strain and subculture The freshwater alga B. braunii (FACHB-357) was obtained from the Freshwater Algae Culture Collection of the Institute of Hydrobiology (Wuhan, China). The alga was maintained in Modified Basal medium (23) in polyethylene bags with diameter of 7 cm and volume of 2 l (each containing 1.5 l medium). The illumination was provided by cool white fluorescent light at 100 to 110 mmol m2 s1. The aeration of 0.2 vvm (volume to volume per minute) was provided by an air pump. Constant temperature of 26 2 C was provided. Influence of organic carbon sources To make an organic carbon concentration of 0.1 g l1, a small aliquot of stock solution (glucose, acetate, glycerin or glycine with concentration of 50 g l1) was added into the microalgal medium after 16 days culture. Based on previous experience (24), B. braunii usually reached stationary growth stage after 2 to 3 weeks cultivation. The amount of EPS was increased with cultivation time and usually reached its maximum concentration at stationary growth stage, thus organic carbons were supplied in day 16 (25). The flocculation efficiencies were determined after 24 h precondition time. Based on the suspended solids concentration and biomass recovery rate, glycine was selected for further investigation. Influence of culture period, glycine dose and precondition time The effect of culture period (11, 16 and 21 days) on EPS production was carried out with 1 glycine dose of 0.5 g l and precondition time of 3 days. Furthermore, the influence of EPS on suspended solids concentration and biomass recovery rate obtained from flocculation of B. braunii was studied. The comparison of glycine dose (0.1, 0.5 and 1 g l1) was carried out with culture period of 16 days and precondition time of 3 days. In addition, the effect of precondition time (1, 3 and 5 days) was evaluated with culture period of 16 days and glycine dose of 0.5 g l1. The following procedures were followed in all experiments. First, B. braunii was cultured in polyethylene bags for 11 to 21 days, and then the algal medium was evenly transferred to 250 ml Erlenmeyer flasks (each containing 150 ml medium). Secondly, aliquot organic carbon source solution was added into each sample. The samples were cultivated in a rotary shaker (125 rpm) with light intensity of 100 to 110 mmol m2 s1 for the purpose of biomass and EPS accumulation. Thirdly, after the selected precondition time was reached, 100 ml of the sample was transferred to a 100 ml measuring cylinder. Four hours was determined as the settling time based on previous study (4). At the end of the 4 h period, the volume of the upper clear supernatant was read, and a fraction of the supernatant was carefully removed using a fixed volume pipette without disturbing the bottom concentrated algae. Biomass dry weight (in the supernatant) was measured with gravimetric method. The algae were washed with de-ionized water to remove the attached salt, and then filtered
FIG. 1. Comparison of microalgal particles with different treatment after 16 days culture. (A) Before precondition, (B) 3 days precondition with 0.1 g l1 glycine, (C) 3 days precondition with 0.5 g l1 glycine; (D) 3 days precondition with 1 g l1 glycine.
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with pre-weighted 0.45 mm GF/C filter membrane. The filter was dried at 105 C in an oven for 5 h. Algal biomass dry weight was determined by the difference of the two weights. Finally, another 50 ml sample was also washed with de-ionized water, and then applied for biomass (before flocculation) and EPS measurement. Three parts of the EPS were isolated in turn, including soluble EPS, loosely bound EPS and tightly bound EPS (26). The EPS composition in terms of polysaccharide and protein were then measured by anthrone colorimetry method (27) and coomassie brilliant blue method (28). For each treatment, two replicate samples were conducted. The replicates were analyzed twice, and the average value was presented.
but also high dewatering rate. In addition, the initial biomass concentration was also one of the significant factors on flocculation efficiency (4). As shown in Eq. 4, the suspended solids concentration was proportional to biomass concentration, biomass recovery rate and dewatering rate. Thus the suspended solids concentration was used as an indicator of flocculation efficiency in this study.
Analytical methods Biomass recovery rate, dewatering rate, concentration factor and suspended solids concentration were defined by Eqs. 1e4.
As shown in Table 2, the suspended solids concentration and biomass recovery rate achieved from the flocculation experiments were significantly affected by the supplied organic carbon sources. The maximum suspended solids concentration of 4.03 0.17% with biomass recovery rate of 77.21 4.54% was achieved by using glycine, followed by glucose, glycerol and acetate. The differences in flocculation efficiency were probably due to the effect of EPS on flocculation. The influence of glycine dose was further investigated. As shown in Fig. 1, the algae formed large flocs in the flask after 3 days precondition time, when applying 1 g l1 of glycine (Fig. 1D). Increasing glycine dose increased the suspended solids concentration (Fig. 2A). The maximum suspended solids concentration of 5.34 0.35% with biomass recovery rate of 57.1 3.3% was obtained from 1 g l1 glycine induced flocculation. As shown in Fig. 2A and B, a positive tendency was found between suspended solids concentration and tightly bound EPS. Fig. 2C shows that by supplying glycine with concentration of 0.1, 0.5 and 1 g l1, the biomass dry weight increased from 1.49 0.01 to 2.02 0.02 g l1, 1.49 0.01 to 1.99 0.03 g l1, 1.49 0.01 to 2.34 0.02 g l1, respectively. The results indicated that glycine was a preferred
BC1 0:1 BC2 V 100% BC1 0:1
(1)
V 100% 0:1
(2)
0:1 1 BRR ¼ BRR V DR
(3)
BC1 0:1 BC2 V BC1 BRR 100% ¼ 100% ð0:1 VÞ 1000 1 DR
(4)
BRR ¼
DR ¼
CF ¼
SSC ¼
where BRR is biomass recovery rate (%), DR is dewatering rate (%); CF is concentration factor; SSC is suspended solids concentration (%), BC1 is the initial algal biomass concentration before flocculation (g l1); BC2 and V are the biomass concentration (g l1) and volume (l) of the supernatant. Both concentration factor and suspended solids concentration were commonly used to evaluate the flocculation efficiency (4,29,30). As shown in Eq. 3, the concentration factor is proportional to biomass recovery rate, and inversely proportional to dewatering rate. It’s suggested that either increasing the biomass recovery rate or decreasing the dewatering rate, increased the concentration factor. As a result, to reduce the cost of harvest and downstream processes, it is critical to achieve not only high biomass recovery rate
RESULTS
FIG. 2. Influence of glycine dose on suspended solids concentration and biomass recovery rate (A), tightly and loosely bound EPS (B), biomass dry weight (C).
VOL. 121, 2016 nitrogen source to B. braunii. The addition of glycine increased biomass as well as EPS growth which was in consistent with our previous research (31). In the comparison of culture period, both suspended solids concentration and biomass recovery rate were decreased first and then increased when increasing culture period from 11 to 21 days (Fig. 3A). The maximum suspended solids concentration of 5.77 0.17 % with biomass recovery rate of 70.8 2.9% was achieved in the culture period of 21 days. As shown in Fig. 3A and B, the tightly bound EPS showed a positive tendency with suspended solids concentration, but loosely bound EPS showed an opposite tendency with suspended solids concentration/biomass recovery rate. Meanwhile, in culture period of 11, 16 and 21 days, the addition of glycine with concentration of 0.5 g l1 increased biomass concentration from 0.87 0.05 to 1.4 0.02 g l1, 1.49 0.01 to 1.99 0.03 g l1, and 1.75 0.01 to 2.16 0.02 g l1, respectively. As shown in Fig. 4A, increasing the precondition time increased the suspended solids concentration. However, the biomass recovery rate was decreased firstly, and then increased with the rising of precondition time. The maximum suspended solids concentration of 7.06 0.50% with biomass recovery rate of 70.6 0.5% was achieved with precondition time of 5 days. As shown in Fig. 4A and B, a positive tendency was found between suspended solids concentration and tightly bound EPS, while an opposite tendency was found between biomass recovery rate and loosely bound EPS. Meanwhile, with precondition time of 1, 3 and 5 days, the biomass concentration increased from 1.49 0.01 to 1.87 0.09 g l1, 1.49 0.01 to 1.99 0.03 g l1, and 1.49 0.01 to 2.00 0.14 g l1, respectively.
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DISCUSSION Influence of organic carbon sources EPS are high molecular weight compounds secreted by microorganisms with their growth, and accumulated on cell surfaces (27). EPS provide threedimensional polymer network that interconnects and transiently immobilizes cells, and possibly promote microalgal flocculation. Carbon sources are one of the most important factors that influence the production and components of EPS. As shown in Table 2, glycine achieved higher suspended solids concentration than other carbon sources, which was probably due to the specific metabolic pathways of the different carbon sources. The citric acid cycle plays a major role in the metabolism of organic compounds and the biosynthesis of microbial materials. Acetate can enter the citric cycle directly, but other organic sources have to be first degraded to pyruvate and then oxidised to form acetylCoA before it can enter the cycle (15,32). In comparison to acetate degradation, the metabolism of other organic carbons may be more complex that likely involves more enzymes including extracellular enzymes. Therefore, a higher EPS abundance is expected in the biomass fed on glucose or glycine than grown on acetate. In addition, the carbon to nitrogen ratio was also significant on the components of EPS (17). Researchers have found that activated sludge growing in wastewater with a low carbon to nitrogen ratio tended to produce EPS with a high proteins/carbohydrates ratio (17,33). And the EPS components, namely proteins and carbohydrates had a more profound effect on bridging the particles, with proteins being more significant than carbohydrates (12). Since the carbon to nitrogen ratio of
FIG. 3. Influence of culture period on suspended solids concentration and biomass recovery rate (A), tightly and loosely bound EPS (B), biomass dry weight (C).
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FIG. 4. Influence of precondition time on suspended solids concentration and biomass recovery rate (A), tightly and loosely bound EPS (B), biomass dry weight (C).
glycine was lower than other organic carbon sources, it is reasonable that higher proteins/carbohydrates ratio was obtained. As a result, higher flocculation efficiency was achieved. It was proved that more than 83% of phytoplankton species from 9 algal classes can utilize glycine as nitrogen source for growth (34). As a nitrogen and carbon source, it is possible that the glycine may be applied for both microalgal biomass and EPS accumulation. Influence of glycine dose Nitrogen concentration is not only important to microalgal biomass growth but also significant to EPS production (12,31). Pal and Paul (35) optimized the culture conditions for the production of EPS by Serpentine Rhizobacterium Cupriavidus pauculus KPS 201. The results indicated that the biomass growth and EPS production were affected by many factors, such as pH, temperature, organic carbon sources, nitrogen and phosphorus concentration, and culture period (35). When increasing NH4Cl from 0 to 1.75 g l1, the productivity of EPS was enhanced to 129.2 mg l1 (35). The positive effect of nitrogen on EPS production has been widely reported, which was in consistent with this study (36,37). As shown in Fig. 2A, the tightly bound EPS were significantly enhanced when increasing glycine dose from 0.1 to 1 g l1. Due to the active participation in the formation of stabilized flocs, tightly bound EPS have been extensively reported to have positive impact on the dewaterability (38e40). Therefore, increasing the production of tightly bound EPS was supposed to increase the compactness of the particles, and achieve higher suspended solids concentration. As the outer layer of EPS, the loosely bound EPS only take up a small part of bound EPS. As a result, the variance of loosely
bound EPS was not significant when increasing glycine dose from 0.1 to 1 g l1. Poxon and Darby (41) found out that the loosely bound EPS contained an abundant amount of bound water. Increasing the amount of loosely bound EPS brought a greater amount of bound water into the aggregates. Thus, highly porous sludge flocs with a lower density were achieved (41). The results were in consistent with this study. Furthermore, based on Eq. 3, biomass recovery rate was positively related to suspended solids concentration, but negatively related to dewatering rate and biomass concentration. Generally, decreasing either dewatering rate or initial biomass concentration will enhance biomass recovery rate, but also increase the downstream operation cost. Therefore, a cost-optimal balance between biomass recovery rate and suspended solids concentration, rather than only biomass recovery rate, should be considered. Influence of culture period A number of researchers have pointed out that growth stage was one of the most significant factors that influenced the production of EPS (20,35,42). For instance, it was reported that the production of EPS was closely related to the bacterial growth stage, during the exponential growth period, EPS grew with cultivation time (42). However, the EPS productivity (presented as the gram of EPS per unit gram of biomass) was also affected by the biomass growth rate. It is reasonable that increasing biomass growth rate from day 11 to 16 decreased the productivity of tightly bound EPS (Fig. 3B and C). Continuously prolonging the culture period from day 16 to 21 did not substantially increase the biomass dry weight, but significantly enhanced the productivity of tightly bound EPS. As
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previously explained, the suspended solids concentration was positively affected by tightly bound EPS. As a result, the suspended solids concentration decreased first and then increased when prolonging culture period from 11 to 21 days. Furthermore, it has to be pointed out that the productivity of loosely bound EPS decreased when prolonging the culture period to 21 days. One possible explanation was the biodegradation due to the nutrient deficiency (40). Since loosely bound EPS are a loose and dispersible slime layer on the outside. It is possible to believe that loosely bound EPS may be biodegraded preferentially in nutrient deficient situation. As previously explained, high loosely bound EPS resulted in poor performance of sludge sedimentation and compression. The theory explained the opposite tendency between loosely bound EPS and suspended solids concentration/ biomass recovery rate. Influence of precondition time Nutrient and cultivation time are both critical to the productivity and components of EPS (22,39). Generally, the EPS productivity increased with cultivation time in nutrient sufficient situation (41). However, the small molecular substances that are produced as a result of EPS degradation can be used as carbon and energy sources for cell growth in conditions of nutrient shortage (32). Since the comparison of different levels of precondition time was conducted with the addition of 0.5 g l1 glycine. The supplied glycine served as nutrient for not only biomass, but also EPS growth. Therefore, the tightly bound EPS, which were the major parts of EPS, increased with the precondition time, hence enhanced the suspended solids concentration. However, when the precondition time was increased to 5 days, part of loosely bound EPS might be biodegraded due to the nutrient shortage (Fig. 4B). As previously explained, excessive loosely bound EPS had a negative effect on flocculation. Decreasing loosely bound EPS was however, increased the biomass recovery rate. In conclusion, glycine was found an effective carbon and nitrogen source for both biomass and EPS accumulation of B. braunii. Under the optimal condition with culture period of 16 days, glycine dose of 0.5 g l1 and precondition time of 5 days, the biomass concentration increased 34%. Meanwhile, after settling, 98% of the medium was removed. The maximum suspended solids concentration of 7.06% (concentration factor of 34.6) with biomass recovery rate of 70.6% was achieved. Compared to the average level (suspended solids concentration of 4e6%, concentration factor of 4e35), the suspended solids concentration achieved was enhanced (6,29). In addition, the supernatant medium was successfully recycled as inoculum to perform the continuously culture-harvest strategy. This strategy may also be effective on microalgal species which can take glycine as a nitrogen source. For instance, we proved that the addition of glycine with concentration of 0.1 g l1 was effective on flocculation of Scenedesmus dimorphus, Chlorococcum humicola, and Chlorella sp. (data not shown). However, the extra cost of glycine must be considered when applying this strategy into industry. To reduce the precondition time and the chemical cost, the application of isolated EPS needs further investigation.
ACKNOWLEDGMENTS This research was financially supported by Education Department of Fujian Province (No. JA12018) and Fuzhou Administration of Science and Technology (No. 2014-G-60).
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Glycine induced culture-harvesting strategy for Botryococcus braunii Ying Shen,1, * Wenzhe Zhu,1 Chaozhou Chen,1 and Yilei Nie2 College of Mechanical Engineering and Automation, Fuzhou University, Fuzhou, Fujian 350116, China1 and Fujian Institute of Microbiology, Fuzhou 350007, China2 Received 28 January 2015; accepted 6 August 2015 Available online 6 November 2015
The objective of this study was to investigate the effects of culture conditions, including carbon sources and concentration, culture period, and precondition time, on the production of extracellular polymeric substances (EPS) and its influence on microalgal flocculation. EPS are natural high molecule polymer, excreted by microalgae themselves. EPS can accelerate the formation of microbial aggregates through binding cells closely. Organic carbon sources, such as glucose, glycerol, acetate and glycine were compared to select the optimal source to stimulate EPS accumulation. Subsequently, the effect of culture period, glycine dose and precondition time on EPS production and its influence on biomass growth and flocculation efficiency were investigated. As the main parts of EPS, tightly bound EPS were found positively related to suspended solids concentration. However, the loosely bound EPS may weaken the floc structure, leading to poor water-cells separation. Under the optimal condition with culture period of 16 days, glycine dose of 0.5 g lL1 and precondition time of 5 days, the biomass concentration increased from 1.49 to 2 g lL1, and the maximum suspended solids concentration of 7.06% with biomass recovery rate of 70.6% was achieved. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Flocculation; Botryococcus braunii; Extracellular polymeric substances; Suspended solids concentration; Biomass recovery rate]
Microalgae are one of the most promising renewable energy sources because of their tremendous potential in lipid production, less reliance on freshwater and arable lands compared to other oil crops (1,2). However, due to the small size (5 to 20 mm), negative surface charge (7.5 to 40 mV) and low biomass concentration (0.5 to 1 g l1), microalgal biomass harvest from growth medium is still a challenge, which accounts for 20 to 30% of the total production cost (3). Conventional harvesting processes such as centrifugation and filtration require large capital and operational cost (4,5). It was estimated that the capital and operational cost of centrifugation reached up to 12,500 dollar per hectare of harvested land and 5 to 13 dollar per liter medium (5,6). Compared to centrifugation or filtration, the capital (around 2000 dollar per hectare) and operational cost (around 0.05 to 0.4 dollar per liter) of flocculation is relatively lower (5,6). However, a major challenge is the recovery of the flocculants (usually multivalent metal salts) from medium, which will ultimately contaminate the water and final products. In addition, low suspended solids concentration and biomass recovery rate are the bottleneck of flocculation. As shown in Table 1, assuming that the cell concentration of microalgal medium is 2 g l1, there are around 2 g algal biomass dry weight, 8 g cell water (about 80% of the cell weight), and 990 g free water in 1 l medium (4). Centrifugation can remove almost all the free water to achieve about 20% suspended solids concentration and over 90% biomass recovery rate (6). However, the suspended solids concentration and biomass recovery rate achieved by flocculation were 4e6% and 50e90%, respectively (6). To increase the flocculation efficiency, it is important to achieve the
* Corresponding author. Tel.: þ86 13950301207; fax: þ86 0591 22866261. E-mail address: [email protected] (Y. Shen).
highest possible suspended solids concentration in algae harvesting to reduce energy consumption in drying while keeping biomass recovery rate in consideration. It is well known that extracellular polymeric substances (EPS) play an important role in biofilm formation of bacteria. However, EPS are not unique to bacteria since some of the most abundant EPS producers are microalgae (in particular, diatoms). The green alga Chlorella vulgaris JSC-7 and Ettlia texensis showed good autoflocculation efficiency due to the production of large amounts of EPS (predominantly polysaccharides) (7,8). Four filamentous cyanobacteria Microcoleus vaginatus, Scytonema javanicum, Phormidium tenue and Nostoc sp. and a coccoid single-cell green alga Desmococcus olivaceus were also found as the producers of EPS (9). EPS are composed of some high molecular weight compounds, including polysaccharide, protein, nucleic acids, humic substances, and ionisable functional groups like carboxylic, phosphoric amino and hydroxyl groups (10,11). It was reported that EPS present a dominant bridging mechanism between the floc components, namely cellular, bio-organic, and inorganic compounds (12e14). EPS are often divided into two major fractions: soluble EPS and bound EPS (15). The adhesion of soluble EPS to cells is weak, as a result of which soluble EPS are often dissolved in solution. The structure of bound EPS is generally depicted by a two layer model (16). The inner layer consists of tightly bound EPS, which has a certain shape and is bound tightly and stably with the cell surface. The outer layer, which consists of loosely bound EPS, is a loose and dispersible slime layer without an obvious edge. The content of the loosely bound EPS in microbial aggregates is always less than that of the tightly bound EPS (15,17). Some investigators have suggested that the composition and properties of EPS, rather than the quantity, are more important in flocculation (18,19).
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.08.004
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TABLE 1. Parameters obtained with centrifuge or flocculation. Parameters Algal biomass dry weight (g) Cell water (g) Free water (g) Suspended solids concentration (%) Dewatering rate (%) Biomass recovery rate (%)
MATERIALS AND METHODS
Culture
Centrifuge
Flocculation
2 8 990 0.2 e e
>1.8 >7.2 0 <20 99 >90
1e1.8 4e7.2 20e70 <6 92e97 50e90
TABLE 2. Influence of organic carbon sources on flocculation efficiency. Organic carbon source Glucose Glycine Acetate Glycerol
Suspended solids concentration (%) 3.48 4.03 2.05 2.27
0.12 0.17 0.09 0.10
Biomass recovery rate (%) 64.34 77.21 66.44 70.07
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3.23 4.54 2.33 3.45
Nutrients, especially the carbon sources and nitrogen concentration are significant factors that influence the excretion of EPS (12,20e22). The effect of organic carbon (acetate, glucose and glycerine) on flocculation of pleurochrysis carterae was investigated (13). The results indicated that under stress, due to nutrient depletion, the microbes produced EPS that promote flocculation of P. carterae. The results also showed that the recovery efficiency is positively correlated with the precondition time. In this study, four organic carbon sources were compared to select the optimal source for the culture-harvesting strategy for freshwater alga Botryococcus braunii. The effects of culture period, glycine dose and precondition time on biomass concentration, as well as EPS production were analyzed. In addition, the influence of EPS on suspended solids concentration and biomass recovery rate obtained from flocculation of B. braunii was investigated.
Algal strain and subculture The freshwater alga B. braunii (FACHB-357) was obtained from the Freshwater Algae Culture Collection of the Institute of Hydrobiology (Wuhan, China). The alga was maintained in Modified Basal medium (23) in polyethylene bags with diameter of 7 cm and volume of 2 l (each containing 1.5 l medium). The illumination was provided by cool white fluorescent light at 100 to 110 mmol m2 s1. The aeration of 0.2 vvm (volume to volume per minute) was provided by an air pump. Constant temperature of 26 2 C was provided. Influence of organic carbon sources To make an organic carbon concentration of 0.1 g l1, a small aliquot of stock solution (glucose, acetate, glycerin or glycine with concentration of 50 g l1) was added into the microalgal medium after 16 days culture. Based on previous experience (24), B. braunii usually reached stationary growth stage after 2 to 3 weeks cultivation. The amount of EPS was increased with cultivation time and usually reached its maximum concentration at stationary growth stage, thus organic carbons were supplied in day 16 (25). The flocculation efficiencies were determined after 24 h precondition time. Based on the suspended solids concentration and biomass recovery rate, glycine was selected for further investigation. Influence of culture period, glycine dose and precondition time The effect of culture period (11, 16 and 21 days) on EPS production was carried out with 1 glycine dose of 0.5 g l and precondition time of 3 days. Furthermore, the influence of EPS on suspended solids concentration and biomass recovery rate obtained from flocculation of B. braunii was studied. The comparison of glycine dose (0.1, 0.5 and 1 g l1) was carried out with culture period of 16 days and precondition time of 3 days. In addition, the effect of precondition time (1, 3 and 5 days) was evaluated with culture period of 16 days and glycine dose of 0.5 g l1. The following procedures were followed in all experiments. First, B. braunii was cultured in polyethylene bags for 11 to 21 days, and then the algal medium was evenly transferred to 250 ml Erlenmeyer flasks (each containing 150 ml medium). Secondly, aliquot organic carbon source solution was added into each sample. The samples were cultivated in a rotary shaker (125 rpm) with light intensity of 100 to 110 mmol m2 s1 for the purpose of biomass and EPS accumulation. Thirdly, after the selected precondition time was reached, 100 ml of the sample was transferred to a 100 ml measuring cylinder. Four hours was determined as the settling time based on previous study (4). At the end of the 4 h period, the volume of the upper clear supernatant was read, and a fraction of the supernatant was carefully removed using a fixed volume pipette without disturbing the bottom concentrated algae. Biomass dry weight (in the supernatant) was measured with gravimetric method. The algae were washed with de-ionized water to remove the attached salt, and then filtered
FIG. 1. Comparison of microalgal particles with different treatment after 16 days culture. (A) Before precondition, (B) 3 days precondition with 0.1 g l1 glycine, (C) 3 days precondition with 0.5 g l1 glycine; (D) 3 days precondition with 1 g l1 glycine.
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with pre-weighted 0.45 mm GF/C filter membrane. The filter was dried at 105 C in an oven for 5 h. Algal biomass dry weight was determined by the difference of the two weights. Finally, another 50 ml sample was also washed with de-ionized water, and then applied for biomass (before flocculation) and EPS measurement. Three parts of the EPS were isolated in turn, including soluble EPS, loosely bound EPS and tightly bound EPS (26). The EPS composition in terms of polysaccharide and protein were then measured by anthrone colorimetry method (27) and coomassie brilliant blue method (28). For each treatment, two replicate samples were conducted. The replicates were analyzed twice, and the average value was presented.
but also high dewatering rate. In addition, the initial biomass concentration was also one of the significant factors on flocculation efficiency (4). As shown in Eq. 4, the suspended solids concentration was proportional to biomass concentration, biomass recovery rate and dewatering rate. Thus the suspended solids concentration was used as an indicator of flocculation efficiency in this study.
Analytical methods Biomass recovery rate, dewatering rate, concentration factor and suspended solids concentration were defined by Eqs. 1e4.
As shown in Table 2, the suspended solids concentration and biomass recovery rate achieved from the flocculation experiments were significantly affected by the supplied organic carbon sources. The maximum suspended solids concentration of 4.03 0.17% with biomass recovery rate of 77.21 4.54% was achieved by using glycine, followed by glucose, glycerol and acetate. The differences in flocculation efficiency were probably due to the effect of EPS on flocculation. The influence of glycine dose was further investigated. As shown in Fig. 1, the algae formed large flocs in the flask after 3 days precondition time, when applying 1 g l1 of glycine (Fig. 1D). Increasing glycine dose increased the suspended solids concentration (Fig. 2A). The maximum suspended solids concentration of 5.34 0.35% with biomass recovery rate of 57.1 3.3% was obtained from 1 g l1 glycine induced flocculation. As shown in Fig. 2A and B, a positive tendency was found between suspended solids concentration and tightly bound EPS. Fig. 2C shows that by supplying glycine with concentration of 0.1, 0.5 and 1 g l1, the biomass dry weight increased from 1.49 0.01 to 2.02 0.02 g l1, 1.49 0.01 to 1.99 0.03 g l1, 1.49 0.01 to 2.34 0.02 g l1, respectively. The results indicated that glycine was a preferred
BC1 0:1 BC2 V 100% BC1 0:1
(1)
V 100% 0:1
(2)
0:1 1 BRR ¼ BRR V DR
(3)
BC1 0:1 BC2 V BC1 BRR 100% ¼ 100% ð0:1 VÞ 1000 1 DR
(4)
BRR ¼
DR ¼
CF ¼
SSC ¼
where BRR is biomass recovery rate (%), DR is dewatering rate (%); CF is concentration factor; SSC is suspended solids concentration (%), BC1 is the initial algal biomass concentration before flocculation (g l1); BC2 and V are the biomass concentration (g l1) and volume (l) of the supernatant. Both concentration factor and suspended solids concentration were commonly used to evaluate the flocculation efficiency (4,29,30). As shown in Eq. 3, the concentration factor is proportional to biomass recovery rate, and inversely proportional to dewatering rate. It’s suggested that either increasing the biomass recovery rate or decreasing the dewatering rate, increased the concentration factor. As a result, to reduce the cost of harvest and downstream processes, it is critical to achieve not only high biomass recovery rate
RESULTS
FIG. 2. Influence of glycine dose on suspended solids concentration and biomass recovery rate (A), tightly and loosely bound EPS (B), biomass dry weight (C).
VOL. 121, 2016 nitrogen source to B. braunii. The addition of glycine increased biomass as well as EPS growth which was in consistent with our previous research (31). In the comparison of culture period, both suspended solids concentration and biomass recovery rate were decreased first and then increased when increasing culture period from 11 to 21 days (Fig. 3A). The maximum suspended solids concentration of 5.77 0.17 % with biomass recovery rate of 70.8 2.9% was achieved in the culture period of 21 days. As shown in Fig. 3A and B, the tightly bound EPS showed a positive tendency with suspended solids concentration, but loosely bound EPS showed an opposite tendency with suspended solids concentration/biomass recovery rate. Meanwhile, in culture period of 11, 16 and 21 days, the addition of glycine with concentration of 0.5 g l1 increased biomass concentration from 0.87 0.05 to 1.4 0.02 g l1, 1.49 0.01 to 1.99 0.03 g l1, and 1.75 0.01 to 2.16 0.02 g l1, respectively. As shown in Fig. 4A, increasing the precondition time increased the suspended solids concentration. However, the biomass recovery rate was decreased firstly, and then increased with the rising of precondition time. The maximum suspended solids concentration of 7.06 0.50% with biomass recovery rate of 70.6 0.5% was achieved with precondition time of 5 days. As shown in Fig. 4A and B, a positive tendency was found between suspended solids concentration and tightly bound EPS, while an opposite tendency was found between biomass recovery rate and loosely bound EPS. Meanwhile, with precondition time of 1, 3 and 5 days, the biomass concentration increased from 1.49 0.01 to 1.87 0.09 g l1, 1.49 0.01 to 1.99 0.03 g l1, and 1.49 0.01 to 2.00 0.14 g l1, respectively.
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DISCUSSION Influence of organic carbon sources EPS are high molecular weight compounds secreted by microorganisms with their growth, and accumulated on cell surfaces (27). EPS provide threedimensional polymer network that interconnects and transiently immobilizes cells, and possibly promote microalgal flocculation. Carbon sources are one of the most important factors that influence the production and components of EPS. As shown in Table 2, glycine achieved higher suspended solids concentration than other carbon sources, which was probably due to the specific metabolic pathways of the different carbon sources. The citric acid cycle plays a major role in the metabolism of organic compounds and the biosynthesis of microbial materials. Acetate can enter the citric cycle directly, but other organic sources have to be first degraded to pyruvate and then oxidised to form acetylCoA before it can enter the cycle (15,32). In comparison to acetate degradation, the metabolism of other organic carbons may be more complex that likely involves more enzymes including extracellular enzymes. Therefore, a higher EPS abundance is expected in the biomass fed on glucose or glycine than grown on acetate. In addition, the carbon to nitrogen ratio was also significant on the components of EPS (17). Researchers have found that activated sludge growing in wastewater with a low carbon to nitrogen ratio tended to produce EPS with a high proteins/carbohydrates ratio (17,33). And the EPS components, namely proteins and carbohydrates had a more profound effect on bridging the particles, with proteins being more significant than carbohydrates (12). Since the carbon to nitrogen ratio of
FIG. 3. Influence of culture period on suspended solids concentration and biomass recovery rate (A), tightly and loosely bound EPS (B), biomass dry weight (C).
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FIG. 4. Influence of precondition time on suspended solids concentration and biomass recovery rate (A), tightly and loosely bound EPS (B), biomass dry weight (C).
glycine was lower than other organic carbon sources, it is reasonable that higher proteins/carbohydrates ratio was obtained. As a result, higher flocculation efficiency was achieved. It was proved that more than 83% of phytoplankton species from 9 algal classes can utilize glycine as nitrogen source for growth (34). As a nitrogen and carbon source, it is possible that the glycine may be applied for both microalgal biomass and EPS accumulation. Influence of glycine dose Nitrogen concentration is not only important to microalgal biomass growth but also significant to EPS production (12,31). Pal and Paul (35) optimized the culture conditions for the production of EPS by Serpentine Rhizobacterium Cupriavidus pauculus KPS 201. The results indicated that the biomass growth and EPS production were affected by many factors, such as pH, temperature, organic carbon sources, nitrogen and phosphorus concentration, and culture period (35). When increasing NH4Cl from 0 to 1.75 g l1, the productivity of EPS was enhanced to 129.2 mg l1 (35). The positive effect of nitrogen on EPS production has been widely reported, which was in consistent with this study (36,37). As shown in Fig. 2A, the tightly bound EPS were significantly enhanced when increasing glycine dose from 0.1 to 1 g l1. Due to the active participation in the formation of stabilized flocs, tightly bound EPS have been extensively reported to have positive impact on the dewaterability (38e40). Therefore, increasing the production of tightly bound EPS was supposed to increase the compactness of the particles, and achieve higher suspended solids concentration. As the outer layer of EPS, the loosely bound EPS only take up a small part of bound EPS. As a result, the variance of loosely
bound EPS was not significant when increasing glycine dose from 0.1 to 1 g l1. Poxon and Darby (41) found out that the loosely bound EPS contained an abundant amount of bound water. Increasing the amount of loosely bound EPS brought a greater amount of bound water into the aggregates. Thus, highly porous sludge flocs with a lower density were achieved (41). The results were in consistent with this study. Furthermore, based on Eq. 3, biomass recovery rate was positively related to suspended solids concentration, but negatively related to dewatering rate and biomass concentration. Generally, decreasing either dewatering rate or initial biomass concentration will enhance biomass recovery rate, but also increase the downstream operation cost. Therefore, a cost-optimal balance between biomass recovery rate and suspended solids concentration, rather than only biomass recovery rate, should be considered. Influence of culture period A number of researchers have pointed out that growth stage was one of the most significant factors that influenced the production of EPS (20,35,42). For instance, it was reported that the production of EPS was closely related to the bacterial growth stage, during the exponential growth period, EPS grew with cultivation time (42). However, the EPS productivity (presented as the gram of EPS per unit gram of biomass) was also affected by the biomass growth rate. It is reasonable that increasing biomass growth rate from day 11 to 16 decreased the productivity of tightly bound EPS (Fig. 3B and C). Continuously prolonging the culture period from day 16 to 21 did not substantially increase the biomass dry weight, but significantly enhanced the productivity of tightly bound EPS. As
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previously explained, the suspended solids concentration was positively affected by tightly bound EPS. As a result, the suspended solids concentration decreased first and then increased when prolonging culture period from 11 to 21 days. Furthermore, it has to be pointed out that the productivity of loosely bound EPS decreased when prolonging the culture period to 21 days. One possible explanation was the biodegradation due to the nutrient deficiency (40). Since loosely bound EPS are a loose and dispersible slime layer on the outside. It is possible to believe that loosely bound EPS may be biodegraded preferentially in nutrient deficient situation. As previously explained, high loosely bound EPS resulted in poor performance of sludge sedimentation and compression. The theory explained the opposite tendency between loosely bound EPS and suspended solids concentration/ biomass recovery rate. Influence of precondition time Nutrient and cultivation time are both critical to the productivity and components of EPS (22,39). Generally, the EPS productivity increased with cultivation time in nutrient sufficient situation (41). However, the small molecular substances that are produced as a result of EPS degradation can be used as carbon and energy sources for cell growth in conditions of nutrient shortage (32). Since the comparison of different levels of precondition time was conducted with the addition of 0.5 g l1 glycine. The supplied glycine served as nutrient for not only biomass, but also EPS growth. Therefore, the tightly bound EPS, which were the major parts of EPS, increased with the precondition time, hence enhanced the suspended solids concentration. However, when the precondition time was increased to 5 days, part of loosely bound EPS might be biodegraded due to the nutrient shortage (Fig. 4B). As previously explained, excessive loosely bound EPS had a negative effect on flocculation. Decreasing loosely bound EPS was however, increased the biomass recovery rate. In conclusion, glycine was found an effective carbon and nitrogen source for both biomass and EPS accumulation of B. braunii. Under the optimal condition with culture period of 16 days, glycine dose of 0.5 g l1 and precondition time of 5 days, the biomass concentration increased 34%. Meanwhile, after settling, 98% of the medium was removed. The maximum suspended solids concentration of 7.06% (concentration factor of 34.6) with biomass recovery rate of 70.6% was achieved. Compared to the average level (suspended solids concentration of 4e6%, concentration factor of 4e35), the suspended solids concentration achieved was enhanced (6,29). In addition, the supernatant medium was successfully recycled as inoculum to perform the continuously culture-harvest strategy. This strategy may also be effective on microalgal species which can take glycine as a nitrogen source. For instance, we proved that the addition of glycine with concentration of 0.1 g l1 was effective on flocculation of Scenedesmus dimorphus, Chlorococcum humicola, and Chlorella sp. (data not shown). However, the extra cost of glycine must be considered when applying this strategy into industry. To reduce the precondition time and the chemical cost, the application of isolated EPS needs further investigation.
ACKNOWLEDGMENTS This research was financially supported by Education Department of Fujian Province (No. JA12018) and Fuzhou Administration of Science and Technology (No. 2014-G-60).
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