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 mitiga-
tion, 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 fermen-
tot. 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 cel-
lulose 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 (in-

cluding solar energy), are. being tried. It is considered that micnmlgae utilization technology will be-

come 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 low-

cost cultivation, harvesting and processing techniques (Benemann, 1997). Some micrwalgae, on

the other hand, can through photosynthesis convert CO2 into valuable sutrstances, including energy-

carrying 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.
 76 CHINESE JOURNAL OF OCEANOLOGY AND LIMNOI/._)GY Vol. 16

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 col-
legues (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 Starch content Conversion rate~ Reference
[ g/(m~'day) ] (% (%)
- C h l a m y d o m o n a ~ reinhardtii (LTEX2247) 11 45 30 l-limno et al. (1997)
N ~ .,~. Tit-I 30 25 30 Hirayama et ~. (1995)
Ch/am)domonas .~. YA-SH-I 30 30 50 Hirayama el 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; Casten-
holz, 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 en-
riched 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 fil-
tration 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. Keep-
ing culture media under 35 ~ was essential for good growth, and water was sprayed on tubes if nec-
essary. 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
 Suppl. 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 Microscopic photograph of a fermentative ma- Fig. 2 Microscopic photograph of a marine microalga,
fine mieroalga, Ch/amydomonas sp. strain Tetraselmis sp. strain Tt - 1
YA-SH-I, which stores starch in cells and
ferments alcohols including ethanol

Fig. 3 A 50 L sc',de tubular bioreactor set in a room having a temperature controlling system with metal haloid lamps
 78 CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY Vol. 16

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 reac-
tor 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) 0 5 7 10 I1 12

Cell c~acentration (g/L) 0.16 0.55 0.78 1.5 1.6 1.6

Starch omtent (%) nd 15 10 39 35 35
Nitrate eoncentratiota(rag/L) 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, cylin-
drical 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 re-
moval by flushing with N2, the microalgal slurry was maintained automatically at a desired tempera-
ture ( 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 (Hi-
rayama 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-
 Suppl. BIOREAC3'OR CULTURE OF MICROAI.GAE 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 destruc-
tion 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 ob-
tained 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 in-
creased 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 perme-
ability, 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 stan-
dards. 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 ke-
naf 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 bac-
teria, Acetobaterium species, are well known to produce high quality cellulose fiber. Structural
 80 CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY Vol. 16

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

40

35

25 ! I I
0 5 |0 15 2u
Algac content ( % )

Fig. 4 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), al-
though 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
t
1
t
\,\\\\\
t l
I
] 1 '
Type 11 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)
 Suppl. BIOREACTOR (35 [,TURE OF MICROA I ,(;AE 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 de-
tected 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 cellu-
lose. synthase seem to be distribut-
ed 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 Fig. 6 ~anning electron mierographof flocculated, thermophiliefresh
blue green algae (cyanobateri- blue green alga (Cyanobaeterium), Syr~chocysti.~ vulx.an~s
a): a candidate to be trans- (Hirano et al., 1997)
formed by recombination
In microalgae technology, cell harvest is suppo:~ed to be one of the most energy - requiring pro-
eesses 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 tem-
perature 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
 82 CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY Vol. 16

systematic utilization of cell bodies and their products will become more important. More studies in-
cluding processing techniques are required to elucidate possibilities of employing mieroalgae technol-
ogy as an option for C02 mitigation.

ACKNOWLEDGEMENTS

We thank members of our laboratory for helpful discussions. We are also grateful to Shin Hi-
myama, Ryohei Ueda, Yasuyuki Ogushi, Kazuhiko Masuda, Makio Hasuike, Yuichi Tsuyuki,
Hideo Akiyama, Takuo Onizuka, Masahiko Ikeuchi, and Yorinao Inoue.

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