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Polypropylene Bundle Attached Multilayered Stigeoclonium Biofilms Cultivated in Untreated Sewage Generate High Biomass and Lipid Productivity S
Polypropylene Bundle Attached Multilayered Stigeoclonium Biofilms Cultivated in Untreated Sewage Generate High Biomass and Lipid Productivity S
Journal of Microbiology and Biotechnology. 2015. Sep, 25(9): 1547-1554
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : January 13, 2015
  • Accepted : May 05, 2015
  • Published : September 28, 2015
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About the Authors
Byung-Hyuk Kim
Sustainable Bioresource Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea
Dong-Ho Kim
Major of Green Chemistry and Environmental Biotechnology, University of Science & Technology (UST), Daejeon 305-806, Republic of Korea
Jung-Woon Choi
Major of Green Chemistry and Environmental Biotechnology, University of Science & Technology (UST), Daejeon 305-806, Republic of Korea
Zion Kang
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea
Dae-Hyun Cho
Sustainable Bioresource Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea
Ji-Young Kim
Bioenergy and Biochemical Research Center, KRIBB, Daejeon 305-806, Republic of Korea
Hee-Mock Oh
Bioenergy and Biochemical Research Center, KRIBB, Daejeon 305-806, Republic of Korea
Hee-Sik Kim
Major of Green Chemistry and Environmental Biotechnology, University of Science & Technology (UST), Daejeon 305-806, Republic of Korea
hkim@kribb.re.kr

Abstract
The potential of microalgae biofuel has not been realized because of the low productivity and high costs associated with the current cultivation systems. In this study, a new low-cost and transparent attachment material was tested for cultivation of a filamentous algal strain, Stigeoclonium sp., isolated from wastewater. Initially, the different materials tested for Stigeoclonium cultivation in untreated wastewater were nylon mesh, polyethylene mesh, polypropylene bundle (PB), polycarbonate plate, and viscose rayon. Among the materials tested, PB led to a firm attachment, high biomass (53.22 g/m 2 , dry cell weight), and total lipid yield (5.8 g/m 2 ) with no perceivable change in FAME profile. The Stigeoclonium -dominated biofilm consisted of bacteria and extracellular polysaccharide, which helped in biofilm formation and for effective wastewater treatment ( viz ., removal efficiency of total nitrogen and total phosphorus corresponded to ~38% and ~90%, respectively). PB also demonstrated high yields under multilayered cultivation in a single reactor treating wastewater. Hence, this system has several advantages over traditional suspended and attached systems, with possibility of increasing areal productivity three times using Stigeoclonium sp. Therefore, multilayered attached growth algal cultivation systems seem to be the future cultivation model for large-scale biodiesel production and wastewater treatment.
Keywords
Introduction
Microalgae are currently produced industrially for a number of niche markets, such as aquaculture and nutraceuticals [33] . In addition to this, there is a great deal of interest in using microalgal biomass as a source of biofuel. Owing to their fast growth rates, it is estimated that microalgae can have areal productivities that are orders of magnitude above those of traditional oil crops, such as canola, soybean, and palm oil [3 , 37 , 39] . Microalgae also have lower nutrient requirements compared with terrestrial crops and can utilize municipal or industrial wastewater as a growth medium, and grow on land not suitable for food production. In spite of these advantages, the production of microalgae for biofuel has not been realized because it is not yet cost competitive in comparison with fossil fuels. The overarching bottlenecks have been the high costs of nutrients, ability of the selected microalgae to withstand environmental conditions, competition, and predation, and low concentration of biomass in the suspended systems as well as the cost and efficiency of existing harvesting techniques [38] .
Wastewater has been touted as a possible source of nutrients for algal biofuel production. Advantages of using algal-based wastewater treatment as opposed to aerationbased treatment have been documented [21] . However, only a few species of microalgae are capable of effectively treating wastewater, notably Chlorella and Scenedesmus species [14 , 31] . Hence, it is important to unearth new species of microalgae that can treat wastewater as well as produce high-quality biomass while offering other advantages. Currently, the cost of harvesting biomass by various methods such as filtration, flotation, flocculation, sedimentation, and centrifugation has been valued at upto 30% of the total production cost. The high harvesting cost is not only because of the small size of algal cells but also due to lesser dense culture in suspended systems [11 , 23] . To overcome these bottlenecks associated with suspended algal cultivation systems, there is growing interest in using surface-attached algal cultivation systems that are naturally concentrated and more readily harvestable, reducing the costs of harvesting and other downstream processes [7 , 14 , 18] . Attached culture systems have been successfully used for growing microalgae, filamentous cynaobacteria, and macroalgae for removing nutrients from animal wastewater [20 , 29 , 42] . Filamentous algae offer many advantages over smaller, non-filamentous, unicellular algae, including cheaper harvesting options, and high biomass productivity and nutrients uptake [6 , 26 , 29] . However, only a handful of studies have been performed on filamentous green algae with respect to biofuel applications and those studies have found low lipid content in filamentous algae. Considering the diversity of microalgae, more studies on filamentous microalgae for biofuel applications are desirable. In this study, the effectiveness of various attachment materials for growing the filamentous green alga Stigeoclonium sp. HS1 using wastewater was evaluated. Stigeoclonium is a common genus of freshwater algae, with short and laterally branched filaments that end in an acute apex or hair-like extension. Stigeoclonium species are known to tolerate a wide range of water conditions, with ability to grow in waters polluted by heavy-metals and/or organic materials [17 , 27 , 32] . This study also provides insights on the use of a horizontal multilayered open pond algal culture system using attachment materials. Such system not only increased the areal productivity of the open culture three times by efficient use of depth and better light penetration in the attached system but also showed high lipid productivity.
Materials and Methods
- Wastewater Source
The wastewater was obtained at the Daejeon Municipal Wastewater Treatment Facility (latitude: 36 o 22’48.89’’N; longitude: 127 o 24’27.71’’E), which treats the municipal wastewater of Daejeon Metropolitan City. Municipal wastewater was obtained after primary treatment (primary clarifier, before aeration) in the facility. The characteristics of the municipal wastewater were as described before [19] . The municipal wastewater was filtered through a 0.45 μm membrane prior to use. The average total nitrogen (TN) and total phosphorus (TP) levels in the municipal wastewater were 79.41 mg/l and 3.70 mg/l, respectively.
- Strain
The filamentous alga isolated from wastewater was identified as Stigeoclonium sp. HS1 by its green color, cell morphology, and 18S rRNA gene sequence [17 , 27 , 32] . Stigeoclonium sp. HS1 was maintained in 500 ml Erlenmeyer flasks in BG11 medium at 25ºC for 2 weeks with a light intensity of 50 μEm -2 s -1 . Phylogenetic analysis was performed by aligning the 18S rRNA sequence obtained with the reference sequences and constructing the tree using the maximum-likelihood method [16] . The sequences of 18S rRNA gene were aligned by Clustal X [41] and edited by Bioedit [13] with published sequences retrieved from the NCBI database ( http://www.ncbi.nlm.nih.gov ). Phylogenetic trees were reconstructed using the neighbor-joining [36] , maximum-parsimony [9] , and maximum-likelihood [8] algorithms in the MEGA 5 software [40] with bootstrap values based on 1,000 replications [8] .
- Attached Algal Culture
Different materials were tested for attached microalgal cultivation: nylon mesh, polyethylene plate, polypropylene bundle (PB), and viscose rayon (Scotch-Brite, 3M, USA). All these materials were locally procured. The selection of the materials was based on the following criteria: flexible for cultivation and harvest, stable to cultivation conditions, reusable and cheap.
The supporting material was cut into a 10 × 10 cm piece and fixed on the bottom of three identical growth chambers (27 × 11 × 15 cm). The chambers were incubated with 10 ml of algal cell suspension (with 0.5 g dry cell weight L -1 ) and 500 ml of municipal wastewater (after filtering). For multilayered cultivation, the same growth chamber was used with 3 L of municipal wastewater and 60 ml of algal inoculum (0.5 g/l). The growth chambers were placed on a rocking shaker held at 25°C and continuously illuminated with white fluorescent light at 50 μEm -2 s -1 . The rocking shaker provided a rocking motion at 15° from the horizontal plane at approximately 15 tips per minute as mentioned elsewhere [18] . To evaluate if the sedimentation from wastewater contributed to the biomass yield, the same amount of wastewater (without algal inoculums) was placed in the growth chamber and run at the same condition as other systems.
A suspended algal culture was used as a control to benchmark the performance of supporting materials used in this study. The suspended algal culture did not harbor any supporting material but the cells were grown in identical growth chambers as mentioned before. Other experimental conditions such as culture volume, medium, light intensity, and rocking speed were the same as those used in the attached systems.
- Determination of Dry Cell Weight
After the experimental run, the supporting material was removed from the chamber and the used wastewater was drained completely. The biomass was then scraped from the surface of the supporting material using a knife. The biomass was then placed on the pre-weighed filter papers and the dry cell weight (DCW) was measured according to the standard methods [1] .
- Total Lipid and Fatty Acid Methyl Ester (FAME) Analysis
The total lipids were extracted by a chloroform-methanol solvent mixture as described previously [22] . FAME analysis was performed as described previously [35] .
- Microscopy
Optical images of attachment material surfaces were visualized under a light microscope (×100) (Nikon/TMS, Japan). The biofilm in the attached material was examined using confocal laser scanning microscopy (Carl Zeiss GmBH, Germany). The culture was stained with SYBR green for detection of bacteria as mentioned before [5] .
Results and Discussion
Stigeoclonium sp. strain HS1 was isolated from wastewater and its identity was confirmed through genetic and morphologic analyses. Phylogenetic analysis revealed that the strain was member of the class Chlorophyceae. Comparative 18S rRNA gene sequence analysis of Stigeoclonium sp. HS1 with sequences retrieved from the NCBI database (strains CCAC 1901, CCAC 3492, CCALA 500, UTEX 1574, and CCAP 477) showed that HS1 had the highest sequence similarity (99%) with Stigeoclonium sp. CCAC 1901 ( Fig. 1 ). As mentioned elsewhere, Stigeoclonium is a common freshwater filamentous alga but has not been reported so far for biomass production and biofuel application.
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Phylogenetic tree based on the 18S rRNA gene sequence of Stigeoclonium sp. HS1 using the bootstrapped maximumlikelihood method. Bootstrap values based on 1,000 replications are listed as percentages at the branching points.
- Investigations on Different Materials for Attachment
The algal attachment experiment was conducted by culturing Stigeoclonium sp. in real municipal wastewater with five attachment materials: nylon mesh, polycarbonate plate, polyethylene mesh, viscose rayon, and PB. The biomass achieved at the end of the experimental run was 2.08 ± 0.01, 5.80 ± 0.41, 9.20 ± 0.04, 10.66 ± 0.84, and 53.22 ± 0.68 g/m 2 in attached systems with nylon mesh, polycarbonate plate, polyethylene mesh, viscose rayon, and PB, respectively, whereas that of the control (suspended) culture, was 28.44 ± 0.43 g/m 2 . The corresponding biomass productivity was 0.15 ± 0.001, 0.41 ± 0.001, 0.66 ± 0.001, 0.76 ± 0.06, and 3.80 ± 0.05 g/m 2 /day for nylon mesh, polycarbonate plate, polyethylene mesh, viscose rayon, and PB, respectively ( Fig. 2 A). As the results reveal, the attachment material is a very important parameter in biofilm development. It has been demonstrated before that material surface energy influences algal productivity and lipid content [10] . Microalgal attachment in PB was much higher than that of nylon mesh, polycarbonate plate, polyethylene mesh, and viscose rayon, possibly because of PB’s better surface properties. Previous reports using Chlorella and Scenedesmus have indicated polystyrene foam, glass plate, and filter paper as efficient attachment materials [18 , 25] . Mixed microalgal cultivation was reported to have greater growth rates with cotton rope than attached to nylon, polypropylene, cotton, acrylic, and jute [7] . These studies suggest that each microalga has a specific preference for attachment, probably depending upon their cell-wall compatibility, surface tension, and toxicity of the materials [15] . To determine the nature of material and its possible influence on algal attachment, surface properties of the material were examined by microscopy. Both viscose rayon and nylon mesh are composed of thick tube-like fibers, offering probably less surface area for filamentous algae to attach ( Figs. 3 A and 3 B), whereas polyethylene mesh had thinner fibers ( Fig. 3 C). Growth of Stigeoclonium was shunted in these three materials. PB, on the other hand, has extremely thin fibers suitable for attachment as well as provides a higher surface area for filamentous Stigeoclonium , resulting in much higher growth ( Figs. 2 A and 3 D).
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Biomass (A) and lipid productivity (B) of Stigeoclonium sp. HS1 in different attachment materials. SS, suspended system.
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Optical microscope images of the attachment material surface. (A) Viscose rayon, (B) nylon mesh, (C) polyethylene mesh, (D) polypropylene bundle (PB). 100× magnifications. Scale bar represents 50 μm.
PB not only aids the growth of Stigeoclonium , but also heterotrophic bacteria, resulting in the formation of a healthy biofilm. Confocal images of SYBR-stained Stigeoclonium cells scrapped from PB revealed the presence of bacteria and extracellular polysaccharide (EPS), which together form the biofilm ( Fig. 4 ). It has been demonstrated that wastewater aids in the formation of algal biofilm through supply of bacteria and EPS [15] . However, in this study, the bacteria associated with the algae are phycosphere bacteria [34] , perhaps mutualistic, as wastewater was filtered to eliminate microorganisms before use. EPS is shown to serve as the carbon source for algae, and has also been implicated in the formation of algal-bacterial flocs [4 , 5 , 23] .
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Confocal laser scanning image of Stigeoclonium sp., along with SYBR-green stained bacteria (encircled) and EPS (black arrow). The bright green fluorescence is possibly SYBR-green stained nucleus, and red filaments are chlorophyll (Chla) autofluorescence. DIC represents the differential interference contrast image, while the bottom right panel is a merged image of all other panels.
The growth and lipid productivity of Stigeoclonium was much higher in PB and, hence, the performance of attached culture systems from earlier studies was tabulated to compare the performance of the algae and material used in this study ( Table 1 ). Among attached systems studied so far, the biomass productivity achieved in this study was higher than other attached systems, except for two studies, one of that used a rotating bioreactor and the other that used benthic algae for wastewater treatment [7 , 42] . The biomass productivities would differ for several reasons, including the quality of wastewater (treated or untreated), light intensities, and type of algae used. This study used relatively high-strength wastewater and lower light intensities compared with other studies. Moreover, the alga used in this study not only produces higher biomass but also provides higher lipid productivity.
Evaluating the performance of the system under study with that of reported attached culture systems treating wastewater.
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Evaluating the performance of the system under study with that of reported attached culture systems treating wastewater.
- Lipid Productivity, Content, and FAME Profile ofStigeocloniumin Different Attached Systems
The average total lipid content of Stigeoclonium sp. HS1 was 20.39 ± 1.27% of the DCW and did not change significantly between various attachment materials and suspended control. Lipid productivity was 0.13 ± 0.01, 0.08 ± 0.01, 0.08 ± 0.01, 0.14 ± 0.02, and 0.82 ± 0.01 g/m 2 /day for nylon mesh, polycarbonate plate, polyethylene mesh, viscose rayon, and PB, respectively, whereas that of suspended culture was 0.41 ± 0.001 g/m 2 /day ( Fig. 2 B). Early studies have also demonstrated that the lipid content of the attached biomass is similar or lower than the suspended system, possibly because of the presence of bacteria and EPS in the biofilms [10 , 15] . Because the lipid content of the systems was approximately equal, the lipid productivity was highly related to biomass productivity (Fig. S1). The maximum lipid productivity achieved in PB (0.82 g/m 2 /day) was twice higher than that of the suspended system (0.41 g/m 2 /day).
The FAME analysis of Stigeoclonium sp. HS1 showed that the overall fatty acids constituted myristate (C14:0), palmitate (C16:0), oleic acid (C18:1), cis -11-eicosenoic acid (C20:1n9c)2, and lignoceric acid (C24:0)4 ( Fig. 5 ). C16:0, C18:1, and C18:2 comprised 18%, 26.2%, and 5.7% of the total fatty acids, respectively, and the sum of the three fatty acids constituted 49.9% to total fatty acids in the cells. Myristic acid (C14:0), linoleic acid (C18:2), and cis -11-eicosenoic acid (C20:1n9c)2 were the minor fatty acids. It is generally accepted that lipids rich in C16 and C18 fatty acids are suitable for biodiesel. Moreover, biodiesel enriched in oleic acid are desirable, because a high oleic acid content increases the oxidative stability, enabling longer storage [2 , 28] . The FAME profile of Stigeoclonium looks ideal for biodiesel production, as the major fatty acids are C16 and C18 and there is no drastic change in FAME profile in the attached system.
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Fatty acid methyl ester profiles of Stigeoclonium sp. HS1 from different attachment materials.
In addition to high biomass and lipid productivities, Stigeoclonium demonstrated an ability to remove total nitrogen (38% efficiency) and total phosphorus (~90% efficiency) from the untreated wastewater. PB showed relatively higher TN and TP removal efficiencies when compared with the other materials, albeit the suspended culture (control) had a higher TN removal efficiency than all tested materials (Fig. S2). Suspended cultures have better mass transfer and subsequently better nutrient assimilation than attached cultures, hence resulting in better treatment efficiencies [14] .
- Multilayered Cultivation and Harvesting
In addition to providing a higher surface area for attachment, PB was colorless whereas the other materials were opaque and led to inefficient light transfer and reducing the permeability of light, which is important in large-scale experiments. Hence, to exploit the efficient light penetration in the attached system, especially with PB, as well as patchy growth of Stigeoclonium (absence of a visible monolayer of cells, which prevents light penetration), a horizontal three-layered cultivation of Stigeoclonium at different depths was tested (surface, 7.5 cm, and 15 cm). Unsurprisingly, PB showed highest biomass productivity in all three layers. Whereas PB provided higher penetration due to the above-mentioned reasons, resulting in no significant difference between the three layers, the other materials did not allow light penetration from the surface, resulting in negligible biomass production ( Fig. 6 ). This experiment demonstrates that Stigeoclonium could be cultivated along the vertical depth of the reactor with a horizontal design or even a vertical design. Being a filamentous microalga, there is minimum dispersal of cells from the attachment material, leading to a clear penetration of light to the bottom of the chamber.
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Microalgal biomass production in multilayered attached culture system using polypropylene bundle (), polyethylene mesh (■), and viscose rayon ().
One of the major advantages of the attached culture system is the ease of harvesting. In this study, the biomass in attached systems was removed by scraping, whereas in the suspended system, harvesting would only be done efficiently with centrifugation. Although Stigeoclonium is filamentous, the settling process in suspended cultures is determined by various factors like presence of cations, pH, and bacteria [23] . Since the Stigeoclonium biofilm possesses bacteria and is filamentous in nature with extensive branching, the attachment or growth of the algae on the surface of PB was facilitated. Moreover, it is known that both bacteria and EPS help in settling of algae by the formation of settleable flocs [23] , which would have enhanced attachment, ultimately resulting in the formation of the biofilm. The water content of the algae harvested from the attached culture was 1.5% higher than that harvested from a suspended culture. The water from the attached system in the experiment was removed by dripping, whereas in suspended culture, the biomass is separated by centrifugation, which is highly energy intensive [18] . Because of the higher water content in attached systems, sun-drying has been proposed, combining the two cost-effective systems of scraping and sun-drying that would reduce about 20-25% of the total production cost, as harvesting consumes 30% of the total algal production costs. Besides this, such approaches would help in reducing the energy inputs for dewatering [24] .
In summary, the microalgal attached system offers several advantages over conventional culture systems. Here, we propose a multilayered attached system for cultivating a filamentous microalga, Stigeoclonium , using wastewater. The maximum biomass productivity and lipid productivity of Stigeoclonium sp. using PB were 3.8 g/m 2 /day and 0.82 g/m 2 /day, respectively, which was two times higher than the suspended culture system. Furthermore, the algal biomass harvesting and dewatering process can be made more streamlined and cheaper in the attached system. In such multilayered systems, there is chance to increase the areal productivity of the culture system using the same resources, by efficient use of depth, as the light penetration is not a hindrance.
Acknowledgements
This work was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Korea Government Ministry of Science, ICT and Future Planning (ABC-2011-0031351), 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 & Energy (No. 2012T100201665), and a grant from the KRIBB (Korea Research Institute of Bioscience and Biotechnology) Research Initiative Program (http://www.kribb.re.kr).
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