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Higher Biomass Productivity of Microalgae in an Attached Growth System, Using Wastewater
Higher Biomass Productivity of Microalgae in an Attached Growth System, Using Wastewater
Journal of Microbiology and Biotechnology. 2014. Nov, 24(11): 1566-1573
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : June 20, 2014
  • Accepted : August 06, 2014
  • Published : November 28, 2014
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About the Authors
Seung-Hoon Lee
University of Science and Technology (UST), Daejeon 305-350, Republic of Korea
Hee-Mock Oh
University of Science and Technology (UST), Daejeon 305-350, Republic of Korea
Beom-Ho Jo
Environmental Biotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea
Sang-A Lee
Department of Biological Science, School of Biological Sciences and Biotechnology, Chungnam National University, Daejeon 305-764, Republic of Korea
Sang-Yoon Shin
University of Science and Technology (UST), Daejeon 305-350, Republic of Korea
Hee-Sik Kim
University of Science and Technology (UST), Daejeon 305-350, Republic of Korea
Sang-Hyup Lee
Graduate School of Convergence Green Technology and Policy, Korea University, Seoul 136-701, Republic of Korea
Chi-Yong Ahn
University of Science and Technology (UST), Daejeon 305-350, Republic of Korea
cyahn@kribb.re.kr

Abstract
Although most algae cultivation systems are operated in suspended culture, an attached growth system can offer several advantages over suspended systems. Algal cultivation becomes light-limited as the microalgal concentration increases in the suspended system; on the other hand, sunlight penetrates deeper and stronger in attached systems owing to the more transparent water. Such higher availability of sunlight makes it possible to operate a raceway pond deeper than usual, resulting in a higher areal productivity. The attached system achieved 2.8-times higher biomass productivity and total lipid productivity of 9.1 g m -2 day -1 and 1.9 g m -2 day -1 , respectively, than the suspended system. Biomass productivity can be further increased by optimization of the culture conditions. Moreover, algal biomass harvesting and dewatering were made simpler and cheaper in attached systems, because mesh-type substrates with attached microalgae were easily removed from the culture and the remaining treated wastewater could be discharged directly. When the algal biomass was dewatered using natural sunlight, the palmitic acid (C16:0) content increased by 16% compared with the freeze-drying method. There was no great difference in other fatty acid composition. Therefore, the attached system for algal cultivation is a promising cultivation system for mass biodiesel production.
Keywords
Introduction
Microalgae have the inherent potential of a biofuel resource, but algal biofuel production has not been commercialized owing to the high production cost. This is mainly due to the inefficient algal cultivation and harvesting technique [4 , 14] .
The main advantage of the raceway pond is its relative low building and operating cost. The open raceway pond system is less efficient when compared with a closed photobioreactor, with respect to biomass productivity. Light limitation due to top layer thickness may decrease the algal biomass productivity in raceway ponds. The depth of most raceway ponds needs to be shallow (<0.3 m) owing to this limitation [4 , 22] . The biomass concentration in the raceway pond is relatively lower, ranging from 0.1 to 1 g/l ( i.e. , 0.01-0.1%) [12] . Various attached cultivation techniques had been tried in order to increase the microalgal biomass concentration [6 , 12 , 17] .
Microalgae cultivation as planktonic cells, suspended in liquid nutrient media or wastewater, is typically performed in an open raceway pond and closed photobioreactor [2] . The disadvantage of the raceway pond system is the difficulty and associated cost of harvesting the small, suspended microalgae. Regardless of the benefits or limitations of the open and closed suspended algal cultivation methods, both involve substantial challenges of biomass harvesting that can account for up to 30% of total costs [7 , 8] . Many harvesting methods such as filtration, flotation, flocculation, sedimentation, and centrifugation have been investigated. The small size of some algal cells (typically in the range of 2-40 µm) makes the harvest of biomass difficult [16] . There is therefore an interest in using surface-attached algal cultivation systems that are naturally concentrated and more readily harvestable as compared with suspended algae [11] . The attached algal cultivation system reduces the cost related to algae harvesting and downstream processes [6 , 12 , 20] .
In this study, an attached algal system was tried using mesh-type materials to accomplish high algal productivity and cost-effective harvesting processes. The algal biomass productivity and fatty acid composition were comparatively evaluated between the attached algal system and suspended culture. A cost-effective attached system for algal harvesting was also developed.
Materials and Methods
- Wastewater Characterization
Effluent wastewater from the 2 nd lagoon was collected at Daejeon Metropolitan City Facilities Management Corporation in Korea. The biochemical oxygen demand (BOD), chemical oxygen demand (COD), total nitrogen (TN), and total phosphorus (TP) were 1.54 ± 0.45 mg/l, 8.63 ± 0.43 mg/l, 9.03 ± 0.82 mg/l, and 0.15 ± 0.02 mg/l, respectively.
- Attached Algal Culture
Two raceway ponds were constructed in a wastewater treatment plant: one was operated for attached algal cultivation, and the other for suspended cultivation. Effluent wastewater from the 2 nd lagoon was used as cultivation media for microalgae. Microalgae existing in the effluent from the 2 nd lagoon, such as Scenedesmus, Chlorella, Pediastrum, Nitzschia, Cosmarium , filamentous microalgae, and others, were cultivated in an 8,000 L raceway pond (20.6 m 2 ) for 18 days. Each raceway pond contained a paddlewheel and operated at a water depth of 0.4 m.
Different materials tested as attaching materials for microalgal cultivation included polycarbonate plate, polyethylene plate, nylon mesh, and stainless mesh. The selection of the materials was based on the criteria of suitability for microalgal attachment and low cost. Microalgal attachment performance was determined by measuring the optical density after harvest of attached microalgal biomass, at a wavelength of 680 nm, using a UV-Vis spectrophotometer (Model UV-2450, Shimadzu, Japan).
Fig. 1 shows the schematic of the substrate of the attached algal system. The attaching substrates consisted of 126 nylon meshes 0.3 m × 0.4 m (W × H) in size, and 45 nylon meshes of 1.0 m × 0.4 m (W × H) in size. The total area of attached substrates was 33.1 m 2 .
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Schematic of the substrate of the attached algae culture system. (A) The schematic of attaching materials. (B) Actual photograph of the attached system.
- Suspended Algal Culture
The other raceway pond was operated for comparative performance as a suspended system. The microalgal cells were grown under the same conditions, except that no attaching materials were installed in the raceway pond. The wastewater used as culture medium, water depth, working volume, and sunlight intensity were the same as those used in the attached system.
- Sunlight Penetration Rate Measurement
Sunlight intensity was measured by using a DataLogger (LI-1000, LI-COR) equipped with a quantum sensor. The sunlight intensity in the air was determined at 2 m above both raceway ponds. The sunlight intensity to the surface and bottom was determined right under the surface and on the bottom of the raceway pond, respectively. The sunlight penetration rate to the surface and on the bottom were calculated as the sunlight intensity to the surface divided by the sunlight intensity in the air, and the sunlight intensity on the bottom divided by the sunlight intensity in the air, respectively.
- Algal Biomass Determination
The dry cell weight (DCW) of the microalgal biomass was estimated by filtering a 15 ml aliquot of the culture through glass microfiber filter (GF/C, Whatmann) in order to calculate the algal biomass in suspension, of both systems. The retained cells were dried at 105℃ for 24 h, and the DCW was measured.
In order to calculate the attached algal biomass, three of the microalgae-attached materials were taken out from the raceway pond at every 3 days, and the algal biomass was harvested by scraping the “mat” from the attached material using a sharpedged tool. After harvesting, the microalgal biomass was properly diluted with deionized water. The DCW was then estimated by filtering a 5 ml aliquot of the diluents through glass microfiber filter (GF/C, Whatmann). The retained cells were dried at 105℃ for 24 h, and the DCW was measured.
- Lipid Extraction
Total lipids were extracted by mixing chloroform–methanol with the samples in a proportion of 1:1 (v/v), using a slightly modified Bligh and Dyer method [1] . The mixtures were transferred to a separatory funnel and shaken for 5 min. The lipid fraction was then separated from the separatory funnel and the solvent evaporated using a rotary evaporator. The weight of the crude lipid obtained from each sample was measured using an electronic scale
- Fatty Acid Analysis
The fatty acids were analyzed using the modified method of Lepage and Roy [15] . The crude lipid (~10 mg) was dissolved using 2 ml of a freshly prepared chloroform-methanol mixture [2:1 (v/v)] and transferred into a capped test tube. One milliliter of chloroform containing nonadecanoic acid (500 mg/l) as internal standard, 1 ml of methanol, and 300 ml of sulfuric acid as transmethylation reagents were mixed for 5 min and then incubated at 100℃ for 10 min. The fatty-acid-containing phase was separated by adding 1 ml of distilled water and then recovered. The organic phase was filtered using a hypodermic 0.22 µm PVDF syringe filter (Millex-GV, Millipore, USA). Fatty acid methyl esters (FAMEs) were analyzed using a gas chromatograph (GC-7890, Agilent, USA) equipped with a flame ionization detector and HP-INNO wax capillary column (Agilent Technologies, USA). The temperatures of the injector and detector were set at 250℃ and 275℃, respectively. Oven temperature conditions were maintained at 50℃ for 1 min, 200℃ for 12 min, and 250℃ for 2 min. Mix RM3, Mix RM5, GLC50, GLC70 (Supelco Co., USA), and α-linolenic acid (Sigma Chemical Co., USA) were used as standard materials. All reagents were of analytical grade. The components were identified by comparing their retention times and fragmentation patterns with those of the standards [27] .
Results and Discussion
- Microalgal Attachment on Different Attaching Materials
The algal attachment experiment was conducted by culturing mixed microalgae in the 2 nd effluent wastewater with four attaching materials: polycarbonate plate, polyethylene plate, nylon mesh, and stainless mesh. Fig. 2 shows the attachment performance of microalgae grown on the surface of different materials. The results showed that the attachment material was a very important parameter in biofilm development. Microalgal attachment in nylon and stainless mesh was much higher than that on polycarbonate and polyethylene plate. Nylon was chosen as the attaching material since it was cheaper than stainless mesh. Johnson and Wen [12] reported greater Chlorella attachment to polystyrene foam than to cardboard, polyethylene fabric, or loofah sponge. Greater attachment to cotton rope than nylon, polypropylene, cotton, acrylic, and jute was observed for the mixed microalgal cultivation [6] . The two studies used different microalgal species, and hence different materials for microalgal cultivation were selected. Liu et al . [17] reported that among four different microalgal species, Scenedesmus obliquus had the highest biomass productivity using glass plate and filter paper as attached material. Thus, study-dependent differences in selections of attaching material are likely due to differences in microalgal species.
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Comparison of the attachment performance of microalgae grown on the surface of different attaching materials.
- Light Penetration of Attached and Suspended System
Fig. 3 A shows sunlight penetration to the surface and bottom of the raceway pond, in both attached and suspended systems. The sunlight penetration to the surface in the attached system was approximately 20-30%, which was almost equal to that of the suspended system for all cultivation times. The sunlight intensity to the surface in both cultures was 272-520 µmol photon m -2 s -1 , except for rainy days: 3, 6, 11 days ( Fig. 3 B). Since the biomass productivity of Scenedesmus obliquus tended to increase significantly along with the increase in light intensity up to 540 µmol photon m -2 s -1 [10] , this light intensity to the surface in both systems was sufficient for photosynthesis and growth in microalgae.
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Sunlight penetration (A) and intensity (B) at the surface and bottom of the raceway pond of both attached and suspended systems (●, surface of the attached system; ○ , bottom of the attached system; ▼ , surface of the suspended system; △ , bottom of the suspended system).
There was a big difference in sunlight penetration at the bottom of the attached and suspended systems. The main disadvantage of the suspended system using the raceway pond seems to be the light limitation in the high layer thickness [22] . After 4-day cultivation in the suspended system, the sunlight penetration to the bottom was less than 5%. Moreover, the sunlight intensity at the bottom of the suspended system decreased to 0% after 10 days of cultivation ( Fig. 3 A). The sunlight intensity at the bottom of the suspended system was 2-24 µmol photon m -2 s -1 ( Fig. 3 B). According to Suh and Lee [23] , it is generally accepted that a culture can be considered under dark condition when the light intensity is below a certain limit ranging from 0 to 15 µmol photon m -2 s -1 . The sunlight intensity of the suspended system was too low for microalgae at the bottom to grow. Therefore, microalgae cultured deeper in the suspended system in this study were light-limited. The algal concentration absorbs almost all available light within the top 15 cm of the raceway pond, leaving the rest of the pond depth in a light-limited condition [21] .
After 4 days of cultivation of the attached system, the sunlight penetration to the bottom was 11–21%, which corresponded to 191–354 µmol photon m -2 s -1 of light intensity. This was sufficient for microalgae to grow. Because little suspended microalgal growth occurred in the attached system ( Fig. 4 ), the light penetration to the bottom was much higher than that of the suspended system. Whereas the suspended system created light-limited conditions due to top layer thickness, sunlight penetrated deeper and stronger in the attached system.
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Microalgal biomass in both the attached and suspended systems of algal cultivation (●, total biomass of the attached system; ○ , total biomass of the suspended system; ▼ , suspended biomass of the attached system).
The depth of most raceway ponds remains shallow (approximately 15–30 cm) because of light-limited conditions during algal cultivation [4 , 22] . Both raceway ponds used in this study operated at a deeper water depth of 40 cm. Nevertheless, the attached system maintained transparency of the culture medium and allowed sufficient light intensity for microalgal cultivation. The raceway pond could be operated deeper than usual, using an attached system to increase microalgal biomass productivity per unit area.
- Microalgal Growth in Attached and Suspended Systems
Microalgal biomass productivity of the attached system was compared with that of suspended system to evaluate the advantage for microalgae cultivation. When algal cells were cultured in the raceway ponds with the attached system, it took 3–4 days for almost all the cells to settle on the attaching materials. After 6 days of algal cultivation using wastewater, the suspended biomass in the attached system decreased to approximately 0 g/m 2 ( Fig. 4 ), which was similar to that reported by Johnson and Wen [12] . They concluded that almost all the microalgal cells settled on the surface of the supporting materials after 2-3 days of incubation. Once the microalgal cells attached to the nylon mesh, continual growth led to the formation of a thick “mat”, which made the microalgal cells in suspension negligible. However, in the suspended system, microalgal cells in suspension continuously increased up to a biomass of 58.4 ± 1.8 g/m 2 for 18 days ( Fig. 4 ).
The microalgal biomass in suspension was equal to the total biomass in the suspended system. Fig. 4 compared the growth curves of the microalgal biomass of the attached and suspended systems. The total biomass productivity of the suspended system was 3.2 g m -2 day -1 . The total microalgal biomass in the attached system was 136.9 ± 11.2 g/m 2 after 18 days, corresponding to the average biomass productivity of 9.1 g m -2 day -1 ( Table 1 ). The maximum biomass productivity of attached and suspended systems was 13.5 g m -2 day -1 and 6.1 g m -2 day -1 , respectively. The average biomass productivity of the attached system was approximately 2.8 times higher than the suspended system ( Table 1 ). Ho et al . [10] and Liu et al . [17] reported that the biomass productivity of attached microalgae of S. obliquus increased with increasing light intensity. The attached system for algal cultivation allowed the microalgal cells attached on the surface of the supporting material to receive light and nutrients, whereas the suspended system had a severe problem of light limitation [12] . Since the microalgae in the photoautotrophic culture obtain energy for their growth from light, an insufficient supply of light does not sustain optimal growth [3] . Therefore, this result indicated that the attached system allowed a better light availability than the suspended system, which means that the attached system was more efficient in microalgal biomass productivity.
Comparison of performance between attached and suspended algal culture systems.
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Comparison of performance between attached and suspended algal culture systems.
As shown in Table 2 , the biomass productivity of the attached system in this study was relatively high, as compared with other biofilm-based production methods, except for two recent studies. Liu et al . [17] developed an attached photobioreactor using filter papers as attaching material and cultured S. obliquus at a biomass productivity of 50-80 g m -2 day -1 under outdoor conditions. However, they used a synthetic medium (BG-11) for cultivation and operated on a very small scale of 10 cm 2 supporting material. Christenson and Sims [6] reported the biomass productivity of 20–31 g m -2 day -1 for biofilm algae cultivation in municipal wastewater in a small biofilm reactor (4.26 m 2 ). The supporting material area and working volume of 13.5 m 2 and 8,000 L, respectively, in the attached system of this study was on a bigger scale than other biofilm-based studies. Considering that microalgal growth was not optimized in this study, biomass productivity could be further improved by optimizing the culture conditions of the attached system.
Comparison of the attaching materials, areas, working volumes, culture media, illumination, microalgal species, biomass productivity, and harvesting method of attached growth systems.
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Comparison of the attaching materials, areas, working volumes, culture media, illumination, microalgal species, biomass productivity, and harvesting method of attached growth systems.
- Lipid Content and Productivity of Attached and Suspended Systems
The total lipid content of the attached system was 21.3 ± 5.2% of the DCW, which was comparable to the suspended system of 20.4 ± 5.3% ( Table 1 ). Lv et al . [18] reported a biomass concentration and lipid content change at a light intensity of 60 µmol photon m -2 s -1 . However, there were no significant differences in lipid content of both systems ( Table 1 ), considering the differences in light penetration ( Fig. 3 ). This result was also observed by Chrismadha and Borowitzka [5] . They demonstrated that the correlation of light intensity and lipid content is usually species-specific. Both systems in this study contained a wide variety of microalgal species.
Since the lipid contents of both systems were approximately equal, the lipid productivity was highly related to biomass productivity. The maximum lipid productivity of the attached system (3.7 g m -2 day -1 ) was 3.3-times higher than that of the suspended system (1.1 g m -2 day -1 ). The average lipid productivity of the attached system, 1.9 g m -2 day -1 , was 2.7-times higher than that of the suspended system. Christenson and Sims [6] reported a higher fatty acid productivity of 2.2–2.5 g m -2 day -1 , which was about 2-times higher than that of the suspended system of 1.0–1.2 g m -2 day -1 . Considering that microalgal culture condition and lipid accumulation were not optimized in this study, the lipid productivity can be improved under more favorable conditions. Among the attached systems studied so far, the scale in this study was the largest ( Table 2 ). Moreover, the increase in the rate of lipid productivity by the attached system in this study was higher than other attached systems [6 , 12] .
- Fatty Acid Profiles of Attached and Suspended Systems
The fatty acid composition of the attached system was similar to that of the suspended system ( Fig. 5 ). In general, the lipid produced from microalgae usually contains a lipid profile of mainly C16 and C18 fatty acids and thus is suitable for biodiesel production [19] . The major fatty acids of both systems were palmitic acid (C16:0) and palmitoleic acid (C16:1) comprising 15%-20% and 7%-12% of total fatty acids, respectively. Myristic acid (C14:0), stearic acid (C18:0), oleic acid (C18:1n9c), and linolenic acid (C18:3n3) were measured as minor fatty acids. The proportion of palmitic acid (C16:0) decreased from 27% to 17% with cultivation time in the attached system, but palmitoleic acid (C16:1) and oleic acid (C18:1n9c) maintained at 10% and 5%, respectively. The suspended system showed a similar tendency with the attached system. The proportion of palmitic acid (C16:0) also decreased from 21% to 15% with cultivation time, but palmitoleic acid was maintained at 11%. Biodiesel fuels enriched in oleic acid are desirable, because a high oleic acid content increases the oxidative stability, enabling longer storage [13] . The oleic acid (C18:1n9c) proportion in the attached system was higher than that of the suspended system, 10% vs 1% ( Fig. 5 ). Thus, the lipid from the attached system is more suitable for producing biodiesel than that from the suspended system.
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Fatty acid methyl ester profile of the attached (A) and suspended (B) systems.
Additionally, the attached system has the merit of easy and cheap harvesting ( Table 1 ). The biomass in the attached system was harvested through scraping and spooling methods [6 , 12 , 20 , 26] , whereas an expensive centrifugation operation was commonly used in the harvesting of the suspended cells. The harvested biomass slurry (typically 5-15% dry solid content) is perishable and must be processed after harvest; dehydration or drying is commonly used to extend the viability depending on the final product required. Methods employed include sun drying, spray drying, drum drying, and freeze drying. Sun drying is the cheapest dehydration method, but the main disadvantages include a long drying time and the requirement for large drying surfaces. Spray drying is commonly used for extraction of high value products, but it is relatively expensive. Freeze drying is equally expensive, especially for large-scale operations. Lipids are, however, extracted more easily from freeze-dried biomass. The attached system in this study would allow for microalgal dewatering without any drying method such as freeze drying and for microalgal biomass harvesting without centrifugation, thereby decreasing the related cost. This implies a great advantage of the attached system, in terms of ease of biomass dewatering and harvesting.
The freeze-dried and sunlight-dried algal biomasses were analyzed for their fatty acid profile. Thompson [25] reported that various environmental factors (such as temperature, nutrients, and light) affect the lipid profile of microalgae, because there was a close relationship between lipids and photosystem subcomplexes. Fig. 6 shows the fatty acid profiles of the freeze-dried and sunlight-dried microalgal biomasses. When the microalgal biomass was dewatered using sunlight, the palmitic acid (C16:0) content increased significantly from 17% to 33% compared with the freeze-dried method. The sunlight-dried method makes fatty acid profiles suitable for biodiesel production by increasing the palmitic acid (C16:0) content. This phenomenon was also observed by Guihéneuf et al . [9] , who reported that the palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1) contents of microalgae were significantly enhanced under a high light intensity. Sukenik [24] reported almost the same results. Under high light intensity, Nannochloropsis sp. was associated with an increase in palmitic acid (C16:0) and oleic acid (C18:1) contents. However, the palmitoleic acid (C16:1) content decreased from 9% to 1% by the sunlight-drying method compared with the freeze-drying method. Other fatty acid contents had no great differences. This result indicated that sunlight drying was better for biodiesel production than the freeze-drying method, in terms of fatty acid profile and cost.
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Fatty acid methyl ester profile of the attached system of freeze-dried and sunlight-dried methods on day 18.
Acknowledgements
This research was supported by a grant from the Water Industry Development Program funded by K-water of Korea (Project No. WI11STU02) and the Energy Efficiency & Resources of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grant funded by the Korea Government Ministry of Trade, Industry and Energy (No. 2012T100201665), and the KIST Institutional Program (Project No. 2E24280) and the KRIBB Research Initiative.
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