Biotechnology Letters, Vol 20, No 8, August 1998, pp.

763–765

Bacteriorhodopsin production by cell

recycle culture of Halobacterium
halobium
Sang Yup Lee*, Ho Nam Chang, Young Soon Um, and Soon Ho Hong
Department of Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of
Science and Technology (KAIST), 373–1 Kusong-dong, Yusong-gu, Taejon 305–701, Korea. Fax: 82–42–869–3910.
E-mail: leesy@sorak.kaist.ac.kr

When Halobacterium halobium R1 was cultured with cell recycle in a bioreactor equipped with an external hollow fiber
membrane unit, the cell and bacteriorhodopsin concentrations reached in 10 days were 30.3 g cell dry weight/l and
282 mg/l, respectively. The productivity of bacteriorhodopsin (1.15 mg/l▪h) was much higher than that (0.16 mg/l▪h)
obtained by typical batch fermentation.

Introduction rhodopsin production. The results obtained from batch and

Halobacterium is a halophilic archaebacterium which
employs its membrane-bound proton pumping protein
bacteriorhodopsin to synthesize ATP under anaerobic con-
dition in the presence of light (Gochnauer and Kushner,
1969; Oesterhelt et al., 1991). Bacteriorhodopsin has
recently been suggested to have several applications in the
design of molecular electron devices and optical computers
since it possesses desirable photophysical/chemical proper-
ties (Birge, 1990). For examples, Birge (1990) developed a
prototype of high speed optical random access memory
using bacteriorhodopsin Langmuir-Blodgett film, while
Miyaska et al. (1992) developed a bacteriorhodopsin based
artifical photorecepter. A large quantity of bacteriorhodopsin
is in demand for research and development, but only a
small amount is available due to the difficulty of culti-
vating halobacterium. Halobacterium halobium has most
frequently been employed for the production of bacterio-
rhodopsin. To date, H. halobium has been cultured only in
batch mode, and cell density typically obtained was optical
density at 660nm (OD660) of 1–2 (corresponding to cell
concentration of 0.9–1.8 g cell dry weight (CDW) /l) in
3–5 days (Kushiner, 1966; Oesterhelt and Stoceckenius,
1974). Little work has been done on the development of
better cultivation system, and bacteriorhodopsin is still
produced mainly, if not in every case, by batch culture. In
order to develop better cultivation condition, we recently
examined the effect of various complex carbon/nitrogen
sources and amino acids for the growth of H. halobium R1
(Um et al., 1997a). Yeast extract as the carbon and nitrogen
source supported best cell growth and bacteriorhodopsin
production among the tested. In this study, we attempted
to grow H. halobium to a high density with good bacterio-
cell recycle culture of H. halobium R1 are reported.
Materials and methods
Bacterial strain and culture condition
Halobacterium halobium R1 (ATCC 29341) was grown at
37 °C in Hb medium (Um et al., 1997a; pH 7.0), which
contains per liter: NaCl, 250 g; MgCl2 6H2O, 20 g; KCl,
2 g; CaCl2 2H2O, 0.2 g; yeast extract (Difco Laboratories,
Detroit, MI). Batch culture was carried out in a 2.5 l jar
fermenter (Korea Fermenter Co., Incheon, Korea) contain-
ing 1.3 l of Hb medium. Seed culture (2%, v/v) was also
prepared in Hb medium. Sterilized air was supplied at
1.3 vvm. A halogen lamp, which does not generate heat,
was used as a light source. Cell recycle culture was carried
out in the same fermenter equipped with an external
membrane filter module. The polysulfone hollow fiber
filter module (MWCO of 500,000, effective surface area
of 650 cm2) was purchased from A/G Technology Co.
(Needham, MA). The operating volume of the system was
1.5 l, consisting of 1.4 l in the fermenter and 0.1 l in the
recycling loop. Cell broth was circulated at 1.2 l/min by
diaphragm pump. The cell-free filtrate stream was drawn
from the filter unit by a peristaltic pump. Fresh medium
was supplied at the same rate as the filtrate flow by a
peristaltic pump. The dilution rate was set at 0.1 h21.
Cells were first grown in a batch mode, and cell re-
cycling was started when cell concentration reached 1.78 g
CDW/l. The dissolved oxygen concentration was main-
tained above 20% of air saturation by increasing the
agitation speed up to 500 rpm and by supplying pure
oxygen when required. Our previous study showed that the
pH increased to 8.0 during cell growth when the initial

© 1998 Chapman & Hall Biotechnology Letters ⋅ Vol 20 ⋅ No 8 ⋅ 1998 763

S.Y. Lee et al.
pH was between 5 and 8 (Um et al., 1997b). Attempt to
control the pH at the desired value of 7.0 required
addition of much acid, resulting in cell lysis (Um et al.,
1997b). Therefore, pH was not controlled in this study,
and was left to increase from the initial pH of 7.0 to
ca. 8.0.
Analytical methods
Cell growth was monitored by measuring the OD660. The
OD660 of 1.0 corresponds to cell concentration of 0.89 g
CDW/l. The concentration of bacteriorhodopsin was meas-
ured as described by Shand and Betlach (1991). Cells from
2 ml culture broth were harvested by centrifugation, and
were lysed by resuspending in 1 ml of diH2O containing
DNase (0.01 mg). The lysate was well mixed until it
became homogeneous and no longer viscous. The lysate
was mixed with 4 M NaOH and 4 M NH4OH in the ratio
of 9:0.5:0.5 (lysate:NaOH:NH4OH) in the dark. The
absorbance at 568nm (A568) was first measured in the dark
(A5680). The mixture was exposed to light for 24 h to
remove retinal from purple membrane, and again the A568
was measured (A56824). As the molecular weight of
bacteriorhodopsin is 26 kDa and the molar extinction
coefficient is 63,000 M21cm21, the concentration of bac-
teriorhodopsin was determined by the following equation:
bacteriorhodopsin (g/l) 5 26,000 3 (A5680 2 A56824) /
63,000. Acetic acid concentration was measured by
enzyme assay kit purchased from Bohringer (Mannheim,
Germany).
Results and discussion
Batch culture
Cells were first cultured in a batch mode to examine
the growth characteristics (Fig. 1). Cell growth reached
stationary phase in 60 h. Bacteriorhodopsin production
Figure 1 Time profiles of cell and bacteriorhodopsin
concentrations during the batch culture of H. halobium
R1.
764 Biotechnology Letters ⋅ Vol 20 ⋅ No 8 ⋅ 1998
continued even after cells entered the stationary phase. The
cell and bacteriorhodopsin concentrations obtained in 85 h
were 1.87 g CDW/l and 12 mg/l, respectively. The
maximum bacteriorhodopsin productivity was 0.16 mg/l▪h
at 69 h. To see the possibility of growing cells to a higher
density by fed-batch culture, varying amounts of fresh
medium were added at the end of exponential growth.
However, cells did not grow further (results not shown). To
examine if there is growth-inhibitory product produced
during cell growth, newly prepared actively growing cells
were transferred into the supernatant of culture broth
obtained at the end of batch culture supplemented with
varying amounts of salts and yeast extract. Again, cells did
not grow. These results suggested that cells produced
growth-inhibitory product(s) during growth, and there-
fore, fed-batch culture seems to be difficult to carry out
until this compound is identified and eliminated. One
possible inhibitory product was thought to be acetic acid
since its concentration at the end of batch culture was
0.6 g/l. However, the addition of acetic acid at the
concentration of 0.5–2.0 g/l in the initial medium did not
inhibit cell growth, which suggested that acetic acid is not
the one inhibiting cell growth.
Cell recycle culture
Since cells could not be grown to a higher density by
feeding more nutrient due to the presence of unknown
growth-inhibitory product, cell recycle culture with con-
tinuous supply of fresh medium (and continuous removal
of filtered broth) was considered as an alternative (Chang
and Furusaki, 1991). The time profiles of cell growth and
bacteriorhodopsin production during the cell recycle culti-
vation of H. halobium R1 are shown in Fig. 2. Cell growth
was much improved compared with batch culture, and the
Figure 2 Time profiles of cell and bacteriorhodopsin
concentrations during the cell recycle culture of H.
halobium R1.
 Bacteriorhodopsin production

cell concentration reached in 224 h was as high as 30.3 g
CDW/l. By simply removing the unidentified growth
inhibitor(s) from the culture broth with continuous supply
of fresh medium, H. halobium could be grown to a high
density. The final bacteriorhodopsin concentration obtained
in 245 h was 282 mg/l, which was 23.5 times higher than
that obtained in batch culture. The bacteriorhodopsin
productivity was 1.15 mg/l▪h.
In this paper, we showed for the first time that H. halobium
could be grown to a relatively high density by cell recycle
culture, resulting in the production of bacteriorhodopsin
with high productivity. To date, it has been difficult to
grow H. halobium to a density greater than 1.5 g CDW/l
even though there has been much interest on this bac-
terium as a source of bacteriorhodopsin. H. halobium does
not utilize typical carbon sources such as glucose and
sucrose, and its growth relies on the complex carbon/
nitrogen source such as peptone and yeast extract. The
strategy described in this paper is just the beginning of
the effort towards the efficient production of bacterio-
rhodopsin. Optimization of the medium composition and
operation condition will allow us to achieve higher cell
density as well as higher bacteriorhodopsin concentration
and productivity.
Acknowledgement
This work was carried out as a part of Samsung Multi-
media Frontier Project.
References
Birge, RR (1990). Annu Rev Phys Chem 41: 683–733
Chang, HN and Furusaki, S (1991). Adv Biochem Eng Bio-
technol 44: 27–64
Gochnauer, MB and Kushner, D J (1969). Can J Microbiol 15:
1157–1165
Kushner, DJ (1966). Biotechnol Bioeng 8: 237–245
Miyasaka, T, Koyama, K and Itoh, I (1992). Science 255:
342–344
Oesterhelt, D and Stoceckenius, W (1974). Methods Enzymol 31:
667–678
Oesterhelt, D, Brachle, C and Hampp, N (1991). Quaterly Rev
Biophys 24: 425–478
Shand, RS and Betlach, MC (1991). J Bacteriol 173:
4692–4699
Um, YS, Park, JT, Lee, SY and Chang, HN (1997a). Kor J
Biotechnol Bioeng 12: 304–308
Um, YS, Park, JT, Lee, SY and Chang, HN (1997b). Kor J
Biotechnol Bioeng 12: 309–313

Received: 24 April 1998

Revisions requested: 27 May 1998
Revisions received: 22 June 1998
Accepted: 23 June 1998

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