Micro Algae Cultivation In A Tubular Bio Reactor And Utilization Of Their Cells

Vol. 16 Suppl.

CHIN. J. OCEANQL. I.IMNOL.

1998

MICROALGAE CULTIVATION IN A TUBULAR BIOREACTOR AND UTILIZATION OF THEIR CEIJIS Koyu Hon-nami, Shunji Kunito ( Energy and Em,tronment R&D Center, Tokyo Electric Power Co. 4 - 1 Egasaki - cho, Tsunuru - ku, Yokohama 230, Japan )

E - ma//: /'0707125 @pma//. tepco, o..,.~

In this study on the possiblities of microalgae technologyas an option for CO2 mitigation, many microalgae were isolated from seawater. Some species of the isolate~, Chlamydomona~ sp. swain YA-SH-1, which accumulates starch in ceils under light and ferment ethanol in dark and anaerobic condition, was grown outdoors by using 50-L tubular bioreactors in batch cultivation and harvested. Using these cells, the performance of ethanol production was examined quantitatively in a 0.5-L scale fermentot. Another species, Tetrase/m/s sp. strain Tt-1, was cultivated in a semi-batch manner by a similar type of tubular bioreactor indoors and examined for its utilization. Tests showed these cells could be used as partial substitute for wood and kenaf pulp for processing into paper. With the idea of making microalgae produce cellulose by genetic engineering in their minds, the. authors studied the structure, of bacterial cellulose synthase genes and the low temperature-,induced, ~versible flocculation in a thermophilic blue green alga (Cyanobacterium), Synechocystis vulcanus in order to examine the feasibility of using these genes as gene source and the cynanobacterium as host.

Key words: microalgae, tubular bioreactor, Tetraselmis, Chlamydomonas INTRODUCtiON Global warming universally recognized to be due to increased atmospheric concentration of greenhouse gases including CO2, is a worldwide problem, essential countermeasures for which should include expansion of CO2 fixation sources and the reduction of fossil fuel combustion. Other countermeasures, such as forest preservation and switching from fossil fuel to renewable energy (including solar energy), are. being tried. It is considered that micnmlgae utilization technology will become one of the options for low-cost CO2 mitigation, if the following problems have been solved : 1 ) the achievement of very high productivities in microalgae cultivation and 2) the development of lowcost cultivation, harvesting and processing techniques (Benemann, 1997). Some micrwalgae, on the other hand, can through photosynthesis convert CO2 into valuable sutrstances, including energycarrying compounds such as ethanol (Gfeller and Gibbs, 1984). In this review we summarize recent studies perfonnext in our group to explore the possibilities of microalgae technology as an option for CO2 mitigation.

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RENEWABLE ENERGY-CARRYING COMPOUNDS AS ALTERNATIVES FOR FOSSIL FUEL FERMENTATIVE MARINE MICROALGAE AND THEIR ETHANOL PRODUCTION It is well known that ethanol is one of the most useful liquid fuels as an alternative for oil (Wyman, 1995). A simple production process and energy-saving system was proposed by our collegues (Hirano et al., 1997) in which a strain of fermentative marine microalgae with superior growth rate and starch accumulation was used to exploit its ability to convert CO-2into ethanol. In the course of screening such superbugs, a green alga, Nanaoch/ortun sp. strain Tit-I (Hirayama et al., 1995) and

Chlamydomonassp.

strain YA-SH-1 (Hirayama et al., 1996) were isolated from

seawater collected at Chichijima Island in the Pacific Ocean and the Red Sea, respectively (Table 1). Table 1 Fermentative mlcroalgae and their some properties Microalga

Productivity [ g/(m~'day) ]

- C h l a m y d o m o n a ~ reinhardtii

N

~

(LTEX2247)

.,~. Tit-I

Ch/am)domonas .~. YA-SH-I

Starch content

Conversion rate~

Reference

(%

(%)

11

45

30

30

25

30

Hirayama et ~. (1995)

30

30

50

Hirayama el al. (1997)

l-limno et al. (1997)

a) Cited values are those, performed m small sealed tubes (20 mL) Ethanol esmversien rate is defined

as

the ratio of mol of pro_

duced ethanol to the tool of those expected from the initial amount of .-,larch by the assumplion that ethanol is produced solely through the Embden-Meyerhof pathway and that ethanol is converted from pyruvate via acetaldehyde. On the basis d this ~

,

each two

moles of ethanol ',rod CO-:.are expected to he t;o~ainod from one tool of #uo,se which composes the starch rese~ed in the cell_s.

In order to obtain more precise data on ethanol productivity with Ch/amydomonas sp. strain YA-SH- 1, 50L scale batch-cultivation outdoors and a following fermentation test of 0.5 L scale in darkness were done in the Hiroshima R&D Center, Mitsubishi Heav Industries, Ltd., where our collaborating group worked. Algal cells were. cultured in F/2 medium (Ong et al., 1984; Castenholz, 1988) with an artificial seawater agent according to an ordinary technique. Precuhivation in a 10 L carboy with working volume of 8 L was done with illumination by surrounding fluorescence lamps and light intensity of about 7 klx at the outer surface of the vessel in a room controlled at 25 o(.. Almost 7 days culture yielded algal concentration of 0.5 g/L for innoculation. In the outdoor cultivation, concentrations of nutrients including nitrate, phosphate, metals, and vitamins were enriched several times. The working volume was 50 L in the 53 L tubular bioreactor used. Sterilization of culture medium and reactor inside were done as follows: tap water was used as medium after filtration with a kind of filter (TWC-IN-PPS, Advantec Toyo Ltd. ), and the reactor was filled with sodium hypochloride solution (10 rag/L), circulated overnight before use. Cultivation started in general by innoculation of 16 L of the preculture (ca. 30% volume of the working volume). Air containing 1.8% CO2 was supplied at 1.7 L/min to drive the media circulation of 0.3 m/s. Keeping culture media under 35 ~ was essential for good growth, and water was sprayed on tubes if necessary. Illumination of sunlight measured with a digital illuminometer ( M-3, Topkon Corporation) was recorded. Outdoor cultivation was carried out from July 19 to October 30, 1996. Winter seemed

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BIOREACqY)R CULTURE OF MICROALGAE

77

to be Ioo cold for the cells to grow. A reactor system consisted of: polyacryl tubes, air compresser, flow meters for CO2 and air, temperature ~ n s o r s for c u h u ~ medium and the atmosphere..

Fig. 1

Fig. 3

Microscopic photograph of a fermentative mafine mieroalga, Ch/amydomonas sp. strain YA-SH-I, which stores starch in cells and ferments alcohols including ethanol

Fig. 2

Microscopic photograph of a marine microalga, Tetraselmis sp. strain Tt - 1

A 50 L sc',de tubular bioreactor set in a room having a temperature controlling system with metal haloid lamps

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The algae growth was excellent. Well dispersed cells, with little tlocculation and adhesion to inner walls of the tube, and little contamination by other microorganisms, were obse.rved. The reactor system operated well, except that the flow-out of the medium was almost filled with foams from the upper part of the reactor. A series of batch cultivation for 10 to 12 days produced about 80 g dry mass of cells when good growth conditions were kept. Starch content reached maximtm of 39% at 10 days cultivation, which corresponded to the fourth or fifth day after nutrients starvation, followed by gradual decrease to 35% (Table 2). The favorable results of the cultivation showed that this tubular bioreactor system and strain of cells are suitable for microalgal cultivation outdoors. Table 2

A typical growth of a fermentative mkrealga, Odamydomonas sp. YA-SH-I, in a 50 L tubular bioreactor outdoors

Time (day) Cell c~acentration (g/L) Starch omtent (%) Nitrate eoncentratiota(rag/L)

0

5

7

10

I1

12

0.16

0.55

0.78

1.5

1.6

1.6

nd

15

10

39

35

35

340

140

ND

ND

ND

ND

nd: not determined. ND: not de~ected (less than l rag/L)

After the harvest of the culture (ca.2 g of cells/L) by a centrifugation system with capacity of more than 50 L culture medium containing ca. 100 g of cells. The packed cells were transferred for a following fermentation in a system consisting of a fermentor, a cooler for volatile substances, and a gas collector (TedlarR Bag for 1 L). The fermentation vessel used was a flat bottom, rotating, cylindrical glass flask with separable cover and automatic pH controlling system. After suspending the ceils with an appropriate solution to a suitable concentration ( 150 to 200 g of cells/L) and air removal by flushing with N2, the microalgal slurry was maintained automatically at a desired temperature ( 15 - 40~ ) in a water bath and to the neutral pH region by intermittent addition of 2 mol/L NaOH solution coupled with a pH sensor and a controller under constant rotation (60 r/min) by a moving U-shape vane with a shaft. Since micrtmlgal ethanol fermentation depends in general on pH and is especially sensitive to the acidic region below pH 7 (Hirano et al., 1997c), pH control is crucial for development of effective fermentation.

Chlamydomonassp.

strain YA-SH-A, however,

did not seem to respond well this treatment, probably due to less production of organic acids (Hirayama et al., 1996). By keeping the microalgal suspension of less 500 mL in the dark and under anaerobic condition for 48 h, most of the starch, about 80%, was reserved in the cells and converted into fermentative products. In addition to ethanol, CO2 and HCOs ion, 2,3-butanediol was produced (unpublished results). Organic acids including acetate we~ just minor products. Ethanol conversion rate was 61%, which means that 41% of C in the starch contained in cells was fixed to ethanol although one fifth of C still remained in the starch. In summary, fermentative microalgae with higher growth, higher starch content and higher ethanol conversion rate may be potential biotransformers used in microalgae technology for CO2 miti-

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79

gation. CONTRIBUTION FOR THE PRESERVAION OF FORESTS AS A CARBON SINK 1. A marine microalga and its ufili7ntion as paper material

One of the most cost-effective mitigation, and environmentally beneficial measures for coping with the increasing CO 2 issue is recognized to be reducing global C02 emissions from forest destruction and unsustainable agricultural and land use practices (Hughes and Benemann, 1997). Forest protection and afforestration will be of significance as options for CO2 mitigation. From this point of view, we have tried to elucidate the possibility of using microalgae as wood pulp substitute. For this p~,

test micro,algae must have the capacity for strong CO2 fixation to its cell body. In the

course of screening such algae resulting from natural selection, eight types of microalgae were obtained from seawater collected along the coasts of Sagami Bay and Seto Inland Sea in Japan. One of them was Tetraselmis sp. strain Tt-1, which was unicellular, had size of 8 - 10/an, productivity of 10 - 15 g/(m2"day), and non-adhesive tendency. This strain was used to study the performance of

a tubular bioreactor (Hon-Nami et al., 1997; Samejima et al., 1997). A tubular bioreactor set in a temperature-controlled room was used for stable, semi-batch cultivation for more than 60 days un-

der 30 klx irradiation for 12 hours a day. Analyses of obtained data showed that optimal initial cell concentration was around 0.5 g/L and that its productivity was more than 20 g/(m2"day) at the flow rate of the culture medium (Hon-Nami et a l . , 1997; Samejima et a l . , 1997). Some algae have been used as paper feedstocks. In addition, microfibers in wood pulp can greatly improve surface features related to suitability for printing. We studied the effectiveness of adding microalgae to wood pulp. Cells obtained by cultivation mentioned above were used since this strain is relatively large among unicellular microalgae stocked in our group. The quality of papers made with a hand-made paper making machine was evaluated for density, gas p e ~ i l i t y ,

smoothness, ink absorptivity and tensile index. In addition, the paper deteriora-

tion characteristics over time were also examined. The resulting increased paper density with increased algae content showed that the algae ceils filled up the voids of the paper. The effectiveness of the algae as additive was confirmed by the observation that paper qualities including gas permeability, smoothness, and ink absorptivity were improved. As shown in Fig. 4, on the other hand, with increase of algae content, the tensile index decreased slightly, but in a range satisfying standards. This is also the case with whole kenaf pulp paper (Samejima et al., 1997). This strain, Tetraselmis sp. Tt-1, therefore, can be used as a partial substitute for pulp including wood and kenaf pulps. 2.

Structure analyses of cellulose synthase gene from bacteria: a gene source for production

of cellulose in blue green algae (cyanobacteria)

We hope to produce cellulose by microalgae even in waste lands where plants cannot be grown. Microalgae producing cellulose at high efficiency have not yet been identified or isolated. Some bacteria, Acetobaterium species, are well known to produce high quality cellulose fiber. Structural

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CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY

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analyses of the cellulose synthase gene was started by using Acetobacter xylinum strain JCM7664

40

35

25 0

Fig. 4

!

I

I

5

|0 Algac content ( % )

15

2u

Dependencyof the. tensile index in microalgae-added paper on the algae content, rq : Taraselmissp. "It-1 ;C~: a marine green alga, strain T c - l ; v : a fresh green alga, strain M-3. ,MI these algae were mentioned in (Hon-Nami, et al., 1997).

since it is one of the best in bacterial cellulose production in vivo among JCM collections (Obata et a l . , 1993). With PCR and Southern hybridization techniques three positive DNA fragments were cloned from this strain (Umeda et a l . ,

1997 ). One of them was highly homologous to the type I

genes of bacterial cellulose synthase,

Ms/i,

bcsB, bcsC and bcsD from another strain 1306-3

(Wong et a l . , 1990). Unlike the fusion gene acsAB in A. xyliman strain ATCC53582 (Saxena et a l . , 1991) and strain AY201 (Saxena et a l . ,

1994),

bcsA and bc.sB genes are separated in the

JCM7664 strain and in the strain 1306-3. We named these, genes bcsA/, bcsBl, bcsCl and bcsDI. In addition, this strain also carried genes for another type. of cellulose synthase, type I] enzyme, found in strain AY201 (Saxena and Brown, 1995). These genes named as bcsABll- A and bcsAB 1] -B were extensively homologous to each other (more than 99% equal at nucleotide level), although upstream regions were. distinctly different. In l-x)th bcsABll - A and bc~dB II - B , domains of bcsA homolog and bcs B homolog were fused to comprise a single gene. Further searching bcsDl

Type I

bcsAl

bcsBI

>{>

bcsCl

I

Type 11

t

1 t

t

l

I ]

1

\,\\\\\

'

bcsABII-A

bcsCI1

bcsX tx'sY l"i~,. 5 The hactereial cellulose. ~wnthase t,ene ooeron of ,4. xvlimun JMC 7644 (Umeda et at., 1997)

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81

of the flanking regions revealed that an ORF homologous to bcsC! is located in the downstreeun of

bcs ABII-A. This novel ORF was named as bcsCll.

Between bc-

sABII-A and &'sCll,

two more

OFRs, bcsX and bcsY, were detected with eodon typically used for A.

xylinum; the latter was

significantly

homologous

to

tran~cylase gene, while the other had no clear homology in the database.

No biochemical evi-

dence for these gene products have t ~ n obtained at present. In summary, genes related to cellulose. synthase seem to be distributed in at least three locations on the chromosome of JCM strain. The main two of them are shown in Fig. 5. 3. Lower-temperature induced flocculation

in

thermophilic

blue green algae (cyanobateria): a candidate to be trans-

Fig. 6 ~anning electron mierographof flocculated, thermophiliefresh blue green alga (Cyanobaeterium), Syr~chocysti.~ vulx.an~s (Hirano et al., 1997)

formed by recombination In microalgae technology, cell harvest is suppo:~ed to be one of the most energy - requiring proeesses because unicellular mienvalgal cells are well - dispersed in general, especially under good conditions for growth. If algal cells aggregate into flocs, they can be conveniently harvested. In .some thermophilie blue green .alga (cyanobacterium), reversible floceulation induced by lower temperature under light have been observed, as shown in Fig. 6 (Flirano et al., 1996; Hirano et al., 1997c). The flocculation occurs below 30 ~ in S)nechocystis rub'anus, which temperature is 20 ~ lower than its optimum temperature for growth. These 'algae may be one of the candidates in which value - added substances are produced through gene engin~ring technology. CONCLUSION ,Some mieroalgae can with high efficiency convert CO, into valuable substances. Basic research on development of a low-cost bioreactor with high performance in microalgae euhivation as well as

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systematic utilization of cell bodies and their products will become more important. More studies including processing techniques are required to elucidate possibilities of employing mieroalgae technology as an option for C02 mitigation. ACKNOWLEDGEMENTS We thank members of our laboratory for helpful discussions. We are also grateful to Shin Himyama, Ryohei Ueda, Yasuyuki Ogushi, Kazuhiko Masuda, Makio Hasuike, Yuichi Tsuyuki, Hideo Akiyama, Takuo Onizuka, Masahiko Ikeuchi, and Yorinao Inoue.

Referetm~

Benemann, J. R., 1997. CO2 mitigation with microalgae systems. Energy Convers. Mgrm 38(suppl. ) : 475 - 479. C~astenholz, R. W., 1988. Culturing methods for cyanobacteria. Methm~ in Enzymol. 167: 6 8 - 92. Gfeller, R. P., Gibbs, M., 1984. Fermentative metabolism of Chlamydomonas reinhardt//. 1. Analysis of fermentative products from starch in dark and light. Plant Physiol. 75:212 - 218. Hirano, A. 0 lnoue Y., Ikeuchi, M., 1996. Characterization of cytoplasmic membrane proteins frona a thermophilic cyanobacterium, Syn~hococcus t,udcanu.~. Plant Cell Physiol. 37(Suppl): 50. ttirano, A. , Kunito, S., Inoue, Y. et al., 1997a. Light and low temperature induced cell flocculation of a thermophilic cyanobaterium, Synechococcustndcaaus. Plata Cell Physiol. 38:(Suppl): 37. Hirano, A., Samejima, Y., Hon-Nami, K. et al., 1997b. Carbon Dioxide Fixaafion and Ethanol Production by Femaentative Microalgae: Outdoor cultivation in a 50-L Tubular Bioreactor and Co-fermentation with Propionate in Dark. In: Prt~eedings of the 3rd Biomass Conference of the Americas, Montreal, Canada, August. Hirano, A., Ueda, R., Himyama, S. et al., 1997c. CO2 fLxationand ethanol production with mieroalgal photosynthesis and intracellular anaerobic fermentation. Energy 22:137 - 142. Hirayama, S., Ueda, R., Nakayama, Y. H. et al., 1995. Ethanol production by a micrtudga isolated form seawater. in : Abstracts of the Annual Meeting of the Japanese Society for Marine Biotechnology, Tokyo, Japan, p. 60. (in Japanese) Hirayama, S., Ueda, R., Ogushi, Y. et al., 1996. Ethanol production by a microalga newly isolated form seawater.

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the 4th International Conference on Carbon Dioxide Utilization, Kyoto, Japan. ~"Saxena, I. M., Lin, F. C., Brown, R. M., 1991. ldentificalion of a new gene in an ooperon for cellulose biosynthesis in Acetobaterxylitmm. Plant Mol. Biol. 16:9.47 - 954. Saxena, I. M., Kudlicka, K., Okuda, K. et al., 1994. Characterization of genes in the eellulose-synthesizzing operon ( acs operon) of Acetobater xyliruan : Implications for cellulose crystallization. J. Bacter/ol. 176:5735 -

5752.

Saxena, I. M., Brown, R. M., 1995. Identification of a second cellulose synthase gene (ac~d/I) in Acetobater

xylinum. J. Bacteriol. 177: 5276- 5283. Umeda, Y. , Hirano, A., Hon-Nami, K. et al. , 1997. Conversion of CO2 into ellulose by gene manipulation of microalgae: cloning of cellulose synthase gene form A c ~

xylinum. In : Alrstraets of the 4th International Con-

ference on Ca.dmn Dioxide Utilization, Kyoto, Japan. Wong, H. C., Fear, A. L., Calhoohn, R. D. et al., 1990. Genetic organization of the cellulose synthase operon in Acetobaterxyliman. Proc. Natl. Acad. Sci. USA. 87: 8130-8134. Wyman, C. E., 1995. Biomass-derived oxygenates for transportation fuels. In: Proceechnss of the 2nd Biomass Conference of the Americas: Energy, Environment, Agriculture, and Industry. Portland, U . S . A . , August. p. 966 - 975.