Journal of Environmental Biotechnology
Vol. 14, No. 1, 43–56, 2014

Original paper (regular paper)

Spirulina subsalsa var. salina var. nov.: Thermo-Halotolerant
Cyanobacteria Accumulating Two Kinds of Compatible Solute,
Originated from the Sultanate of Oman
Yoichi Kuroiwa1*, Rashid S. Al-Maamari2, Masaharu Tasaki1, Kazuo Okamura1, Mark
Sueyoshi1, Ayaka Nakashima3, Eriko Yoshida3 and Kengo Suzuki3
1

Shimizu Corporation, Institute of Technology, 3–4–17 Etchu-jima, Koto-ku, Tokyo, 135–8530, Japan
2
Sultan Qaboos University, P. O. Box 33, Al-Khoudh, PC 123, Sultanate of Oman
3
Euglena Co., Ltd., Yokohama Leading Venture Plaza 1,
75-1 Ono-cho, Tsurumi-ku, Yokohama-shi, Kanagawa 230-0046, Japan*TEL: +81–3–3820–5504 FAX: +81–3–3820–5955*E-mail: kuroiwa@shimz.co.jp(Received; 7 January, 2014/Accepted; 22 July, 2014)

Microalgal samples were collected from natural hypersaline sources in Oman. This resulted in the screening of thermohalotolerant cyanobacteria, strain BAH8-10-45, having closed helically coiled trichomes and accumulating glycine betaine
and O-α-D-glucopyranosyl-(1→2)-glycerole as compatible solutes. Morphological observation of BAH8-10-45 suggests that
this strain belongs to the genus Supirulina Turpin ex Gomont and the species of Spirulina. subsalsa Oersted ex Gomont.
Halospirulina has been proposed as a genus for cyanobacteria which has similar morphology to that of S. subsalsa but has
halotolerance in range of 3–13 wt/vol % or higher. For BAH8-10-45, no growth was recorded in the salinity levels of freshwater and seawater, while growth was confirmed in the range of 5–20 wt/vol %, indicating halotolerance different from evencyanobacteria belonging to Halospirulina. Descriptions for Halospirulina as well as the euryhaline character of Spirulina,
were reviewed to examine classification for BAH8-10-45. Based on phycological taxonomy, mainly featuring morphologicaland physiological characteristics, Spirulina subsalsa var. salina var. nov. is proposed.
Key words:	Spirulina subsalsa var. salina var. nov., Halospirulina, halotolerant, glycine betaine, O-α-D-glucopyranosyl(1→2)-glycerole

1. Introduction
The increase in concentration of atmospheric carbon
dioxide (CO2) is considered to be one of the main causesof global warming 63). Microalgae, which are photosynthetic
microorganism with oxygen evolution 11), transform solar
energy to chemical energy through CO2 fixation by photosynthesis and contribute to the reduction of atmosphericCO2 36,60). The transformed energy and carbon are mostly
stored in lipids or carbohydrates in the algae-based biomass
which are utilized as feedstock for bio-fuel 24,26) and finechemical industries 60). Microalgae have the potential not only
to reduce atmospheric CO2 but also to produce biomass on
an industrial scale 24,26).
Much of the Middle East is located in areas geographically suitable for CO2 fixation and algae-based biomass
production, due to high annual solar irradiance 57,59), favorable coastal climate (moderate temperature and low rainfall) 14), and availability of unutilized land (e.g. desert,
sabkha). Additionally CO2 emissions from oil refineries 3)
and produced water from oil wells 12) could be used for suchbiomass production. This study was carried out with a focus
on utilizing the natural resources of the Sultanate of Oman
to reduce atmospheric CO2 and produce algae-based biomassof commercial importance.
Recently, different compatible solutes have been identified in the cells of halotolerant microorganisms 5). Suchcompounds may serve as an energy reservoir, a protectant
from a variety of physical and chemical stresses 30), anda protein stabilizer in their living cells 34,37,46). Compatible
solutes have great potential for further commercial application. For example, their water-holding capacity may allow
them to be used in additives, stabilizers, and sweeteners for
agrichemical 10,48), food 46), cosmetic 34,37), and pharmaceuticalindustries 34,46).
During this study, thermo-halotolerant cyanobacteria(strain BAH8-10-45) accumulating glycine betaine andO-α-D-glucopyranosyl-(1→2)-glycerole (an isomer of glucosylglycerol) were successfully isolated, as the first steps
towards fine-chemicals production from algae-based biomassin the Sultanate of Oman. From the morphological pointof view, the strain BAH8-10-45 is closely related to genus

44

Kuroiwa et al.

Spirulina Turpin ex Gomont characterized by closed helically coiled trichomes, gliding motility, invisible cross-walls,
and no sheaths.
In 1827, genus Spirulina was proposed by Turpin as
Spirulina oscillarioides 58). Gomont then proposed the genus
Spirulina and genus Arthrospila in 1892 21), following theirinitial concideration by Stizenberger in 1852 53), resulting
in the official listing of the genus Spirulina as Spirulina
Turpin ex Gomont 21). Subsequently in 1932, as a resultof a proposal by Geitler, the genus Spirulina and genus
Arthrospila were combined into the single genus of Spirulina(strains of previous genus Spirulina were placed into thesection Euspirulina in the re-established genus Spirulina) 19).
The single genus Spirulina continued for a while based on
this “Geitlerian system” but a new proposal was made to
redivide it into two genera of Spirulina and Arthrospila(Spirulina Turpin ex Gomont and Arthrospila Stizenberger
ex Gomont) as part of the new classification system
proposed by Anagnostidis and Komárek in 1988, taking
into account new morphological knowledge acquired with
an electron microscope 2). In the mid-1990’s, phylogenetic
analysis using the 16S rRNA gene sequence confirmed that
there is a clear distinction between the genus Spirulina andgenus Arthrospila in terms of the phylogenetic lineage 43).
Evaluation results based on the phylogenetic analysis using
the 16S rRNA gene sequence suggested that there would befurther diversity in terms of the phylogenetic lineage within
the genus Spirulina in that Spirulina major and Spirulina
subsalsa would be classified as being from separate phylogenetic lineages. In the latest Bergey’s manual of systematic
bacteriology published in 2001, Spirulina Turpin ex Gomontand Arthrospila Stizenberger were listed separately 8). It wasalso described that some strains in genus Spirulina characterized as halotolerant, formed a tight monophyletic cluster byphylogenetic analysis using the 16S rRNA gene sequence.
Prior to the publication of Bergey’s manual, Nübel et al.
officially proposed in 2000, the new genus of Halospirulina
characterized by halotolerance and forming a tight monophyletic cluster in phylogenetic analysis 45). Halospirulina
is characterized by the use of its physiological specificity
concerning salinity, described as, “Halotolerant, able togrow at salinity between 3–13% or above but not at a
freshwater salinity”. The establishment of the new genus
of Halospirulina subsequently stirred up opposition frommainly the viewpoint of phycological taxonomy on the
grounds that classification of Halospirulina did not follow
the International Code of Botanical Nomenclature becauseof the absence of the diagnosis in Latin and the descriptionof the new genus based on the difference of physiologicalcharacter concerning halotolerance which was extremelyrare for the phycological classification 32). Moreover, in 2003,
Margheri et al. obtained some strains of genus Holospirulina
by 16S rDNA restriction analysis which were able to growat a salinity between 3–13% but also grew in freshwaterof which the salinity is below the lower limit described as
the description of the genus Halospirulina 39). Such findings

indicated that there might be a problem with the descriptionof the genus Halospirulina according to concerning halotolerance. At the XVIIIth International Botanical Congress(IBC) held in Melbourne in 2011, International Code of
Nomenclature (ICN) eliminated the requirement of Latinnames for algae, fungi and plants 31). Currently, some organizations related to phycological taxonomy have adopted thegenus Halospirulina 22). However, the problem concerning
halotolerance, for diagnosis of the genus Halospirulina, hasremained.
The strain BAH8-10-45, isolated in this study, was closely
related to the genus Halospirulina because of its morphological characteristics and its halotolerance in a high salinity
conditions. Detailed analysis has, however, indicated thatstrain BAH8-10-45 has a specific halotolerance which differsfrom the “ability to grow at a salinity between 3–13% orabove” as description of the genus Halospirulina or “growth
range including fresh water of which the salinity is 3 wt/vol% or below” described by Margheri et al. in 2003 39).
The present study aimed to screen microalgae whichwere producing compatible solutes, and to also identify theclassification of the glycine betaine and O-α-D-glucopyranosyl(1→2)-glycerole producing strain BAH8-10-45 which was
discovered during this search. During the work, the existing
diagnosis of the genus Halospirulina 45) were re-examined
and the euryhaline described in the diagnosis on Spirulina
Turpin ex Gomont was reviewed 21). The classification of
BAH8-10-45 has been clarified based on the phycologicaltaxonomy mainly featuring the morphological and physiological characteristics of the strain.

2. Materials and Methods
2.1. Sampling and algal screening
Environmental samples (soil, water, microbial mat andsalt crystals) were collected from the shoreline at Shannah(20°44’N/58°41’E) and the surface of salt pan in Barr
Al-Hikman (20°34’N/58°20’E).
These samples were collected in sterile plastic bottles
and stored in a dark environment at ambient temperature.
For screening of thermo-halotolerant microalgae, aliquotsof environmental samples were suspended in 20 ml sterile
IMK medium (Wako Pure Chemical Industries, Ltd., Japan)
containing NaCl at 10–30 wt/vol %, and incubated undercool white light at 45°C. After the growth of microalgae
was confirmed visibly (within 1–5 days), liquid culture wasrepeated for faster growth and screening of microalgae. Pure
cultures were obtained after several rounds of serial dilution
in liquid media to eliminate contamination, and inoculated
onto solid media. Solid media were prepared with IMK
medium containing NaCl at 10–20 wt/vol % with 1 wt/vol% Difco agar (Becton, Dickinson and Company) on sterilized petri dishes (Becton, Dickinson and Company). After
1–2 weeks, the isolates formed colonies on solid media.

Spirulina subsalsa var. salina var. nov.

2.2. Halotolerance and thermotolerance assays
For halotolerance assays, the isolated strain was inoculated
into 100 ml flasks containing 20 ml of IMK medium at ten
different salinity concentrations (0, 0.5, 1, 3, 5, 10, 15, 20, 25
and 30 wt/vol %). For thermotolerance assays, the isolated
strain was inoculated into 100 ml flasks containing 20 ml of
IMK medium at nine different temperatures (20, 25, 30, 35,
40, 45, 50, 55, and 60°C) under the optimum growth salinityof each of the isolates. Triplicate cultures were grown undercool white light. Biomass was measured every two days using
the dry weight method to determine the maximum growthdensity.
Evaluation of halotolerance of Halospirulina tapeticola CCC Baja-95. Cl. 2T which is type strain of the genus
Halospirulina followed the method used by Nübel et al. 45).
The modified 1/2 Provasoli’s enriched seawater culture
medium 51) was adjusted by NaCl to different total salinities,
was used for halotolerance assays. Algal cultures took placeat 40°C and the dry weight was measured every other day.
2.3. Light microscopic observation
Photomicrographs of Fig. 1 were taken using a digital
camera (Nikon DS-L2) with a microscope (Nikon BX-51)
equipped with Nomarski interference contrast (DIC) illumination.
For comparison of the cell size among strains of S.
subsalsa and relates under the microscopic observation.
Spirulina sabsalsa NIES-27 was cultured in MA medium 27)
at 25°C, and H. tapeticola CCC Baja-95. Cl. 2T was cultured
in modified 1/2 Provasoli’s enriched seawater medium 51) was
adjusted to 7 wt/vol % in total salinities with NaCl 45).

45

2.4. Extraction of compatible solutes and measurement of
13
C-NMR
Compatible solute was extracted from BAH8-10-45 cells,grown in IMK medium at 15 wt/vol % NaCl, essentially as
described by Borowitzka et al. 5). The solute was freeze-dried
and dissolved in D2O for further NMR analyses. Naturalabundance 13C NMR spectra of the solute were recorded
at 100 MHz on a JEOL JNM-400A spectrometer. The
chemical shifts were recorded in ppm using 3-(trimethylsilyl)
propionate-2,2,3,3-d4 (TSP-d4) as an internal standard. The
recorded chemical shifts were then compared with those ofliterature values for O-a-D-glucopyranosyl-(1→2)-glyceroland glycine betaine to determine the components 44).
2.5. PCR amplification
To determine a partial 16S rRNA gene sequence ofisolates, a small number of cells were collected from a microbial colony on solid media using a needle and suspended ina PCR solution. This PCR solution (50 ml) contained 1.25 U
AmpliTaq Gold DNA polymerase (Applied Biosystems),
0.5 ml 10×PCR buffer, 0.2 mM of each dNTP, and 50 pmolof each primer (primers 27F and 1492R 61)). Amplification
conditions were as follows: 2 min activation of the polymerase at 95°C, followed by 35 cycles consisting of 30 s at
94°C, 30 s at 50°C, and 2 min at 72°C, and finally 7 min of
extension at 72°C. The PCR product was electrophoresed
through a 0.8 wt/vol % agarose gel with TBE buffer, andthen purified with a QIAquick gel extraction kit (QIAGEN).
A nucleotide sequence of a PCR product was determinedusing a Bigdye terminator v3.1 cycle sequencing kit (Applied
Biosystems) and a model 3730 DNA sequencer (Applied
Biosystems).
2.6. Phylogenetic analysis
The 16s rRNA nucleotide sequences determined werealigned with reference sequences obtained from the
GenBank ®4) database using Clustal W version 1.83 55) andedited manually using BioEdit Sequence Alignment Editorver. 7.0.9. 23) A phylogenetic tree was constructed using theneighbor-joining method with K2 (Kimura-2-parameter 29))
+I+G model in MEGA ver. 5.0 54). Bootstrap values were
calculated based on 1000 replicates.

3. Results and Discussion
3.1. Selection of thermo-halotolerant microalgae

Fig. 1. Photomicrographs of isolated thermo-halotorerant algal
strains originated from the Sultanate of Oman.
a: strain BAH12-20-45 (at 20 wt/vol % total salinities); b:
strain BAH13-20-45 (at 20 wt/vol %); c: strain SHN3-30-45(at 20 wt/vol %); d–f: strain BAH8-10-45 (at 10 wt/vol %).
Bar=10 mm

From the results of thermo-halotolerant screening, four
algal mixed cultures (SHN3, BAH12, BAH13 and BAH8)
were screened, and algal strains (SHN3-30-45, BAH12-2045, BAH13-20-45 and BAH8-10-45) were isolated fromeach mixed culture (Table 1). Photomicrographs of the four
isolates are shown in Fig. 1a-d. All isolates are simply identified using the “Key to the form-genera of Subsection I or III”
in Bergey’s Manual of Systematic Bacteriology 8).
BAH12-20-45 and BAH13-20-45 are unicellular, ovoid,Kuroiwa et al.

46

Table 1. Characteristics of isolated thermo-halotolerant cyanobacteria originated from the Sultanate of Oman.
Characteristic
Growth temp. range(optimum temp.); °C
Growth salinity range(optimum salinity); %Width(Mean<±SE>); mm
Length(Mean<±SE>); mm
Shape
Morphological
identification
Isolation source

SHN 3-30-45
30–50 (35)

BAH 8-10-45
25–45 (30)

BAH12-20-45
30–45 (40–45)

BAH13-20-45
25–45 (35)

3–30 (10)

5–20 (10)10–30 (15)

10–30 (20)

3.6 <±0.5>

4.1 <±0.2>

3.3 <±0.4>

3.9 <±0.8>970 <±420>

530 <±210>

5.4 <±0.9>

7.6<±1.7>

Filamentous, straightcylindrical
Geitlerinema sp.

Filamentous, closed helically Unicellular, ovoid without
mucilarinous envelope
Spirulina sp.
Halothece sp.

Unicellular, ovoid without
mucilarinous envelope
Halothece sp.

Stagnant hyper saline pond
Microbial mat fromMicrobial mat from surface Microbial mat from surface(salinity 19%)
heliothermal shallow saline
soil in salt pansoil in salt pan
shore (salinity 6%)

without mucilaginous envelope, and with binary fission in
one plane in subsequent generations. BAH12-20-45 has thecell width of 2.5–4.2 mm and the cell length of 4.1–7.1 mm.
BAH13-20-45 has the cell width of 2.9–5.5 mm and thecell length of 5.5–10.6 mm. These morphological featuresindicated that BAH12-20-45 and BAH13-20-45 are closely
related to genus Cyanothece. According to the descriptionof the Bergey’s manual, genus Cyanothece are divided into
three clusters on the phylogenetic tree of cyanobacteria basedon 16s rRNA gene sequences. Cluster 3 contains strains PCC
7418, PCC 9718, MPI95AH11, MPI95AH13, MPI96AL06,
MPI96P408, MPI96AL03, and Syn C1 P22, differing fromcluster 1 and cluster 2 in growing well at salinity greater thanthat of seawater (3 wt/vol % NaCl) and tolerating salinities
as high as 20 wt/vol % NaCl, reflecting their original habitat,
in a solar evaporation pond 8). According to the description by Garcia Pichel et al., the strain MP195AH13 hasdiverse at the cell shape, the fusiform (Dactylococcopsis-likeshape) is sometimes obserbed as one of its cell shape 18). The
fusiform also obserbed at the strain BAH12-20-45 showing
by the arrow in Fig. 1a. After the publication of latest
Bergey’s manual (2001), Margheri et al. proposed the new
genus Halothece in 2008, as the new classification for thestrain MPI96P605 in the genus Cyanothece 40). The genus
Halothece is considered to consist of a group of strains,
ranging from those similar to the genus Cyanothece to thoseof which the similarity of the 16S rRNA gene sequence isabove 91% with Halothece californica MPI96P605 as thetype strain 40).
Morphological characteristic of BAH12-20-45 andBAH13-20-45 has similar to those of the Cyanothecementioned above, and similarity with H. californica MPI
96P605 was higher than 91% (data not shown). According
to some morphological features, BAH12-20-45 and BAH1320-45 were identified Halothece sp..
BAH8-10-45 is characterized by filamentous, closed helically coiled trichomes, with invisible cross-walls, no sheaths,
gliding motility, helix widths between 3.7 and 4.5 mm, andthrichome widths between 1.2–1.6 mm. These morphologicalfeatures indicated that BAH8-10-45 is closely related togenus Spirulina Turpin ex Gomont 8). According to above
mentions, BAH8-10-45 was identified as Spirulina sp..
SHN3-30-45 is characterized by filamentous, straightcylindrical trichomes, with gliding motility, and absence ofconstrictions between adjacent cells, and trichome widths
between 2.9–4.0 mm. These morphological features indicatedthat SHN3-30-45 is closely related to genus Geitlerinema(Anagnostidis & Komárek) Anagnostidis 8). SHN3-30-45 was
identified as Geitlerinema sp. from the mentions above.
Growth characteristics of the four isolates are shown in
Table 1. Temperature tolerance for BAH12-20-45 ranged
from 30–45°C, with an optimum at 40–45°C. Salinity
tolerance ranged from 10–30 wt/vol % total salinity, with
an optimum of 15 wt/vol %. Temperature tolerance for
BAH13-20-45 ranged from 25–45°C, with an optimum at
35°C. Salinity tolerance ranged from 10–30 wt/vol % totalsalinity, with an optimum of 20 wt/vol %. Temperature
tolerance for SHN3-30-45 ranged from 30–50°C, with an
optimum at 35°C; no growth was observed at 55°C. Salinity
tolerance ranged from 3–30 wt/vol % total salinity, with an
optimum of 10 wt/vol %. Temperature tolerance for BAH810-45 ranged from 25–45°C, with an optimum at 30°C; nogrowth was observed at 50°C. Salinity tolerance ranged from
5–20 wt/vol % total salinity, with an optimum of 10 wt/vol%; no growth was observed at <5 wt/vol %.
According to the description of Borowitzka & Borowitzka 6),
Dunaliella species are known as halophilic chlorophytes,
and their commercial production was started in Australia,
Israel and USA during the 1970s and 1980s. The optimum
temperature for Dunaliel salina has been reported to be in
the range 20–40°C, and growth salinity at <approx. 32 wt/
vol % saturation point. D. salina is the first micro alga to
be used commercially to produce fine chemicals, because
its extreme salinity tolerance simplifies maintenance of aunialgal culture, relatively free of competitors, pathogens
and predators 6). All our isolates are also characterized asthermo-halotolerant similar to Dunaliella species. This indicates that these isolates are also suitable for simplifying
maintenance of a unialgal culture for the commercial mass
production. In addition, commercial production from D.

Spirulina subsalsa var. salina var. nov.

47

Table 2. Diagnostic and descriptive features or observed features of cyanobacteria having with tightly coiled trichomes.
Organisms
Spirulina subsalsa NIES-27
Halospirulina tapaticola CCC Baja-95 Cl. 2T
Spirulina subsalsa BAH8-10-45
Spirulina subsalsa Oersted ex Gomont 21,33)
Spirulina labyrinthiformis Kützing ex Gomont 21,33)
Halospirulina Nübel, Garcia-Pichel et Muyzer 45,33)
Halospirulina tapaticola Nübel et al. 45,33)

Trichome width (mm)1.5 <±0.1>
1.5 <±0.1>
1.4 <±0.1>(0.8) 1–2 (2.2)
0.8–1.4
1.5
1.5–3

Helix width (mm)4.2 <±0.3>
4.2 <±0.2>
4.1 <±0.2>(2) 3–5 (5.6)(1.5) 2–2.7 (3)4
4–6

Trichome length (mm)390 <±160>
530 <±250>
540<±210>
150–500 (–700)
75–120 (–160)
NA
NA

Mean <±SE> mean values of at least 50 samples. NA: information not available.

Fig. 2. Natural abundance 13C NMR spectrum of an aqueous extract from cells of BAH8-10-45.
Each resonance is identified as to the particular solute molecule, B: glycine betaine; G: O-α-D-glucopyranosyl-(1→2)-glycerole (details of
the identification, see reference 5,44)).

salina also requires a suitable, low-cost harvesting method 6).
Shelef et al. 49) also pointed out that cell size is an important factor since low-cost filtration procedures are presently
applicable only for harvesting fairly large cell size (e.g.
Coelastrum, Spirulina).
Among our four thermo-halotolerant isolates (SHN330-45, BAH12-20-45, BAH13-20-45, and BAH8-10-45),
BAH8-10-45 was found to have fairly large cell size (Fig.
1d and Table 2) suitable for low-cost filtration procedures
for mass production. In addition, cells of BAH8-10-45
accumulated glycine betaine and O-α-D-glucopyranosyl(1→2)-glycerole (Fig. 2) as compatible solutes, indicating
potential for production of fine chemicals of commercial
importance.
Gabby-Azaria et al. pointed out glycine betaine accumulating under hypersaline growth conditions in S.
subsalsa 16) and Mackay et al. also described glycine betaine

in halotolerant species of Spirulina 38). There had beenreported glycine betaine accumulation in other species of
Spirulina, but BAH8-10-45 accumulated glycine betaine
and O-α-D-glucopyranosyl-(1→2)-glycerole as compatible
solutes. It was suggested that BAH8-10-45 have characteristic compatible solute accumulation different from other
halotolerant species of Spirulina.
Glycine betaine and O-α-D-glucopyranosyl-(1→2)-glycerolehave great potential for further commercial application.
Glycine betaine have been known to function as osmoprotectant, for reduction of plant stress from salt 30) anddrought 10,48), as skin protection from ultraviolet induced
cell damage 34), and as inhibitor of b-amyloid formation in
Alzheimer’s disease 34), used in agricultural, cosmetic, andpharmaceutical industries. O-α-D-glucopyranosyl-(1→2)glycerole is also expected to have potential in cosmetic, food,and pharmaceutical industries 37,46).

48

Kuroiwa et al.

BAH8-10-45 was selected from isolates, to accumulate compatible solutes and to have fairly large cell size
suitable for low-cost filtration procedures for mass production 49). Subsequently, the identification of BAH8-10-45 was
performed.
3.2. Identification of BAH8-10-45
3.2.1. Morphology of BAH8-10-45
Morphology of BAH8-10-45 (Fig. 1d–f) is characterized
as follows: filamentous closed helically coiled trichomes,
with invisible cross-walls and no sheaths. Trichome width is
between 1.2–1.6 mm, and helix width is between 3.7–4.5 mm(Table 2). While the helix width is almost constant up to thehead, the body is straight or slightly bent (as indicated by
the arrow in Fig. 1e). Trichomes coiled counter-clockwise
attach to one another at certain intervals, forming a helix(Fig. 1d). These trichomes are viscous and the cells are either
solitary or aggregated (Fig. 1d). Trichomes regularly turnaround spirally with little forward motion. Morphologicalcharacteristics are similar to Spirulina Turpin ex Gomont 21).
According to above mentions, BAH8-10-45 was defined
genus Spirulina.
Further morphological comparison was conducted with thediagnosis of S. subsalsa in the original article by Gomont 21).
Gomont described the morphological characteristics of S.
subsalsa as “Trichomala pallide aeruginosa, in stratum
saturate aerugineum aut aerugineo-lutescens agglomerate,
vel inter varias Oscilliarieas sparsa, ambitu irregulariter
tortuosa, rarissime recta, in spiram densam subirregularem,
passim laxiusculam, aut rarius regularem, diametro 3 m
and 5 m aequantem contorta, 1 m and 2 m crassa; anfractus
contigui vel subcontigui (v.s.)”. The morphological characteristics of BAH8-10-45 agreed with those of S. subsalsa
described above. The trichome of BAH8-10-45 shown in Fig.
1 is either blue-green or greyish white (showing by the arrow
in Fig. 1d) and inside of the trichome is homogenous (Fig.
1f). The helix is almost straight or slightly bent (showing by
the arrow in Fig. 1e). Trichomes coiled regularly closed (Fig.
1d–f) and moves in a specific direction. The helix width is
3.7–4.5 mm compared to the standard width of 3–5 mm while
the trichome width is 1.2–1.6 mm compared to the standard width of approximately 1–2 mm. Further morphologicalcomparison was made with the description of S. subsalsa by
Komárek and Anagnostidis (2005) 33). Their article described
the morphological diversity of S. subsalsa and correctly
determined the trichome width and length to be (0.8) 1–2(2.2) mm and 150–500 (700) mm respectively. The helix widthwas (2) 3–5 (5.6) mm. The morphological characteristics of
BAH8-10-45 are similar to those of S. subsalsa.
Based on the above results, BAH8-10-45 was identified as
Spirulina subsalsa Oersted ex Gomont.
Table 2 shows the helix width and trichome width of
Halospirulina tapeticola CCC Baja-95 Cl. 2T and relates.
The results were showing that helix and trichome widths ofgenus Halospirulina or H. tapeticola CCC Baja-95 Cl. 2T
closed to those of S. subsalsa. It is indicated that all of strainof H. tapeticola CCC Baja-95 Cl. 2T. are identified as S.
subsalsa, same as strain BAH8-10-45 and S. subsalsa NIES27, based on the morphological identification described by
Komárek and Anagnostidis (2005) 33).
3.2.2. Salinity response of BAH8-10-45
There are two groupings of Spirulina according to halotolerancy (Fig. 3); a higher tolerance group (growth at >15 wt/
vol %) and a lower tolerance group (no growth at >15 wt/
vol %). The lower tolerance group consists of S. subsalsa,
Spirulina labyrinthiformis and S. major. The higher tolerance group composed of Halospirulina strains and S.
subsalsa including BAH8-10-45. Among the higher tolerance group, all strains were able to grow at around seawatersalinity (approximately 3–4 wt/vol %) except BAH8-10-45(Fig. 3.). Around freshwater salinity (approximately less than
0.5 wt/vol %), Halospirulina sp. IR21 and PE1 strains wereable to grow as described by Margheri et al. 39). The strainof Spirulina subsalsa BAH8-10-45 was unable to growthin freshwater, brackish-water and seawater salinities, and its
halotolerance ranged from 5–20 wt/vol %.
By Gomont’s re-establishment of the genus Spirulina in
1892, S. major, S. labyrinthtiformis and S. subsalsa wereincorporated into the genus Spirulina 21). According to thediagnosis of Gomont in 1892, the genus Spirulina, especially S. subsalsa, is characterized by its habitat in diverse
environmental conditions around the world as suggested
by its expression of “cosmopolitan” by many researchers 21).
Gomont’s original diagnosis of the distribution and habitat of
Spirulina Turpin ex Gomont also mentioned its euryhaline
using the expression of “hydrophilae vel halophilae” after
the description of the diagnosis composed of its morphological characteristics 21). Fig. 3 sammarized halotoleranceof these three Spirulina species, i.e. S. major, S. labyrinthiformis and S. subsalsa including BAH8-10-45. The
halotolerance range of S. major and S. labyrinthiformis was
generally between freshwater and salinity of 13 wt/vol %. In
contrast, S. subsalsa was found to generally grow between
freshwater and salinity of 20 wt/vol %. These results indicatethat the euryhaline of the genus Spirulina, its distribution
and habitat, ranging from freshwater, brackish-water, andseawater includes a much higher salinity of at least around20 wt/vol %, at least. In the diagnosis of Gomont 21), it wassuggested that S. subsalsa has a typical euryhaline, meaning
higher halotolerance than S. major or S. labyrinthiformis,
the former described as “aquas salsas aut subsalsas” while
the latter two were described simple as “aquas subsalsas”.
Halotolerance of the S. subsalsa distinguished from otherswith an expression of “salsas”, is inferred to be between atleast 13–15 wt/vol % and approximately 20 wt/vol % (Fig.
3).
3.2.3. Taxonomic consequences
Taxonomic account
Spirulina subsalsa var. salina Kuroiwa et Tasaki var. nov.
Spirulina subsalsa var. salina (sa.li’na. L. adj. salina,
salted, saline). Fig. 1d.
Description: halophilic, no growth below 5 wt/vol % andSpirulina subsalsa var. salina var. nov.

49

Fig. 3. Growth yields of the Spirulina/Halospirulina strains in response to salinity.
The growth was defined as optimal; 75–100%, sub-optimal; 50–74%, weak; up to 49%. Originated from a: Nübel et al. (Salinity tolerance
tested at total salinity 0–25 wt/vol %) 45), b: Margheri et al. (Salinity tolerance tested at total salinity 0–18 wt/vol %) 39) and c: This study(Salinity tolerance tested at total salinity 0–30 wt/vol %). Classification based on originated articles or trichome morphology, generic andspecies names given are sensu Castenholz 8), Komárek & Anagnostidis 33) and Gomont 21).

above 20 wt/vol % able to grow at least 13–15 wt/vol %as total salinity. No growth at freshwater, brackish-water,and seawater. Morphological characteristics are the sameas Spirulina subsalsa Oersted ex Gomont. The optimum
growth temperature is observed at 30°C while no growth isobserved at 20°C or 50°C. It accumulates glycine betaine
and O-α-D-glucopyranosyl-(1→2)-glycerole as compatible
solutes at 15 wt/vol % in total salinity.
Habitat: heliothermal hypersaline environment.
Type Locality: sere algal mat on the surface of marine
sabkha (salt pan) in Barr Al-Hikman, the Sultanate of Oman
(20°35′24.72′′N/58°16′8.69′′E).
Type: preserved specimen number NIES-3373, depositedin the Microbial Culture Collection at the National Institutefor Environmental Studies (NIES), Ibaraki, Japan.
Type Strain: BAH8-10-45, isolated by Yoichi Kuroiwa.
Castenholz et al. described some species belonging togenus Spirulina having worldwide distribution in freshwater,brackish-water, and seawater 8). Spirulina species are also
common in inland saline lakes and in some hot springs at
temperatures as high as 50°C 7). The halotolerant morphospecies of Spirulina have been observed in North America 8),
South America 41), Europe 17), Australia 13), Africa 35), andAsia 1,9,42). Many ecological studies clearly indicate that the
distribution of morphospecies of Spirulina is widespread in
hypersaline environments throughout the world.

In a hypersaline endoevaporitic microbial community in
Eilat (Israel), morphospecies of Spirulina, both Halothecelike and Phormidium-like cyanobacteria, were identified by
microscopic observation 49). Nübel et al. also described that
Halospirulina (characterized morphospecies of Spirulina),
Oscillatoria limnetica-like, and Euhalothece group species
were detected by DGGE analysis, in the microbial mat inevaporation ponds of a saltern in Guerrero Negro, Baja
California, Mexico (No. 6 pond: ca. 14% wt/vol totalsalinity) 45). Ecological studies report the distribution of
Spirulina morphospecies in hypersaline environments at
Solar Lake, Eilat in Israel 49), Yallahs Salt Ponds in south
Jamaica 20), salterns of Alicante in Spain 47), coastal salt lakes
in Western Australia 28), salt pans of southeastern coast in
India 42), hypersaline microbial mats in Sultanate of Oman 1),
and solar salterns of Petchaburi in Thailand 9). These
ecological studies clearly show that phycological identification based on ‘genus or species’ phenotypes has not changed
since establishment of genus Spirulina Turpin ex Gomont
and related species 21).
The proposal for the new genus of Halospirulina by Nübel
et al. 45) meant that at least two genera and two species (H.
tapeticola and S. subsalsa) exist in the grouping describedas “Spirulina morphospecies in hypersaline environments”
as pointed out by Komárek and Anagnostidis 33). It is difficult
to separately identify them based only on morphological

50

Kuroiwa et al.

observation, possibly creating a problem that multi-step identification process, such as evaluation using the 16s rRNA genesequence (phylogenetic analysis etc.) or evaluation of thehalotolerance after the isolation of a strain, may be required.
In this study, the results of the morphological observation of
H. tapeticola CCC Baja-95 Cl. 2T have re-confirmed that
this strain is morphologically identified as S. subsalsa 32), andthe same as S. subsalsa NIES-27 or S. subsalsa var. salina
BAH8-10-45.
Nübel et al. commented on the conventional classification of cyanobacteria that, “morphological classification
may provide insufficient taxonomic resolution and cyanobacteria with similar or identical morphology may haveslightly different physiology”. They proposed a new genus
of Halospirulina based on the extreme halotolerance as aphysiological characteristic can be used to define a phylogenetically coherent group 45).
Many phycological researchers have attempted over a
period of some 100 years to clarify the diversity of thehalotolerance of S. subsalsa Oersted ex Gomont, startingin 1892 by Gomont who used the words “aquas salsas aut
subsalsas” to describe its distribution and habitat 21). In 1932,
Geitler wrote, “In stehenden salzhaltigen Gewässern, in
Meer, in Brackwasser, in Thermen, auch in Hochmooren;
Kosmopolitisch” 19). In 2005, Komárek and Anagnostidis
wrote, “In marine biotopes and in inland salty and brackish
stagnant waters, frequently also in mineral and thermal
springs, sometimes (?) in freshwaters, bogs …; possibly
distributed worldwide, cosmopolitan” 33). As a result of theirwork, it is now clearly established that diversity in termsof halotolerance as a morphological characteristic of S.
subsalsa. If it is necessary to present such character as aclassification, the diversity of halotolerance of the genus
Spirulina must be taxonomically evaluated and classified
with variety (or subspecies) or even lower taxonomic groups
than the genus level where the morphological classification has been established. The findings of the present study
suggest that as far as the halotolerance of those classified in
the genus Spirulina is concerned, any effort to improve theclassification accuracy should be based on a more detailed
classification of the variety or subspecies, etc. mainly below
S. subsalsa.
After the description of a new genus by Nübel et al. 45),
Margheri et al. in 2003 verified the growth of Halospirulina
strains (Strains IR21 and PE1) in freshwater and a salinityof 3 wt/vol % or lower (Fig. 3) 39). It is indicated that there
are some strains, having the defining halotolerance of thegenus Halospirulina, in this Halospirulina cluster. In thisstudy, it was confirmed that BAH8-10-45 was also includedin the Halospirulina cluster as described later. In addition,
it is worth noting that our isolate BAH8-10-45 showed no
growth below 5 wt/vol % total salinity (Fig. 3), although
growth salinity response of genus Halospirulina, describedas one of the character of the genus, ranged from 3–13 wt/
vol % total salinity or above 45). This strongly suggests that
there are at least three genotypes, based on growth salinity

response, in the Halospirulina cluster/genus.
Based on the above, it is evident that taxonomical statusof genus Halospirulina and the taxonomical positions ofstrains in the Halospirulina cluster/genus are unclear. In
addition, the taxonomical status of genus Halospirulina
should be re-examined by further studies without making
exceptions on the phycological taxonomy. From the diagnosisof the euryhaline characteristics of S. subsalsa in terms of its
distribution and habitat by Gomont in 1892 21), to the more
recent description by Komárek and Anagnostidis in 2005 33);
it was steadily established within the framework of conventional physiological taxonomy that there was a diversity ofS. subsalsa in terms of a physiological characteristic, namely
its halotolerance. Based on the results this study’s originaldescriptions, we propose that the halotolerant BAH8-10-45
having phenotype of S. subsalsa, should be placed under the
species S. subsalsa, and we also propose Spirulina subsalsa
var. salina for this strain. The description of this new taxonomical status is given above.
3.2.4. Molecular phylogenetic relationship of BAH8-10-45
among traditional morphospecies of Spirulina
The following evaluation using the 16S rRNA gene sequence
has two purposes. One is to clarify the phylogenetic position of BAH8-10-45 which has been identified as Spirulina
subsalsa var. salina. The other is to clarify the phylogenetic
status of the “Highly halotolerant/Halospirulina cluster”(sensu Nübel et al. 45)) as a new cluster that should be
included in the genus Spirulina.
Phylogenetic analysis of the 16S rRNA gene sequencealso included comparable long sequences (from 101 to 1450
corresponding to Escherichia coli str. K-12 substr. MG1655[U00096] numbering) of particularly well defined strains
belonging to cyanobacteria available in GenBank ®4) (Fig.
4a). The strains identified as Spirulina and available in
GenBank were divided into two clusters (tentatively named
Spirulina cluster A and Spirulina cluster B) in the phylogenetic tree of cyanobacteria (Fig. 4a). The branching of theseclusters was supported by high bootstrap values (96 or 100%)in Neighbor-Joining analysis (Fig. 4a).
Fig. 3b shows the phylogenetic analysis of the 16S rRNA
gene sequences having a sequencial similarity to Spirulina(from 78 to 1469 corresponding to E. coli numbering). Five
Spirulina clusters (Spirulina cluster 1.1, 1.2, 1.3, 2 and 3)
were formed in the phylogenetic tree among Spirulina andrelated cyanobacteria. Cluster A was subdivided into fourclusters (Spirulina cluster 1.1, 1.2, 1.3 and cluster 3). Cluster
1.1 contained traditional morphospecies of S. subsalsa. S.
subsalsa CCAP 1475/1 [HF678502], Spirulina sp. MPI S4[Y18792], Spirulina sp. P7 [AF091109], uncultured bacterium clone GBI-65 [GQ441246], GBI-66 [GQ441247], andGBII-78 [GQ441342] were isolated from marine and brackish
water 4,45). Cluster 1.2 contained traditional morphospecies ofS. subsalsa. S. subsalsa NIES-27 [AB003166], S. subsalsa[AF329394], S. subsalsa FACHB351 [FJ826621], anduncultured bacterium clone SA 82 [JQ738968] were isolated
from coastal and sediment on surface rocks 4). S. subsalsa

Spirulina subsalsa var. salina var. nov.

strains in Cluster 1.2 exhibit drought resistance 4). Cluster
1.3 was “Highly halotolerant/Halospirulina cluster” (sensu
Nübel et al. 45)) containing traditional morphospecies of S.
subsalsa. Spirulina sp. EEW1 [HQ008224], Halospirulina
sp. CCC Baja-95 Cl. 3 [Y18790], Halospirulina sp. MPIS3 [Y18789], H. tapeticola CCC Baja-95 Cl. 2T [Y18791],
and strain S. subsalsa var. salina BAH8-10-45 [AB873003]
were isolated from hypersaline environments 4,45) and exhibit

51

a hyper salt tolerance 4,45). Cluster 2 mainly contained traditional morphospecies of S. major strains. Spirulina sp.
PCC 6313 [AM709631], S. major OBB36S18 [AJ639890],
S. major 1LT27S0 [FM177505], S. major OBB22S09[AJ635436], Spirulina sp. GLS010 [FJ546714], Spirulina sp.
[X75045], and S. subsalsa CCAP 1475/2 [HF678507] wereisolated from a wide salinity range of habitats (freshwater,
blackish and marine) 21,45). Cluster 3 contained traditional

Fig. 4a. Neighbor-Joining phylogenic tree of cyanobacteria based on 16S rRNA gene sequences.
Numbers at nodes indicate bootstrap percentages from 1000 replicas obtained with distance and parsimony analyses, respectively; values
were reported only at nodes where both methods gave bootstrap percentages as 100%. Accession codes of sequences retrieved from Gen
Bank 4). The position of the 16S rRNA gene sequence of cyanobacteria with helical, tightly coiled trichomes were marked in bold. The
scale bar indicates 1% estimated sequence divergence. The analysis involved 46 nucleotide sequences of 16S rRNA gene. There were a totalof 1284 positions in the final dataset.

52

Kuroiwa et al.

Fig. 4b. Neighbor-Joining phylogenic tree of Spirulina and related cyanobacteria based on 16S rRNA gene sequences.
Numbers at nodes indicate bootstrap percentages from 1000 replicas obtained with distance and parsimony analyses, respectively; values
were reported only at nodes where both methods gave bootstrap percentages as 100%. Accession codes of sequences retrieved from Gen
Bank 4). The position of the 16S rRNA gene sequence of BAH8: 10–45 was marked in bold. The scale bar indicates 2% estimated sequence
divergence. The analysis involved 32 nucleotide sequences of 16S rRNA gene. There were a total of 1325 positions in the final dataset.

morphospecies of S. labyrinthiformis strain exhibiting athermotolerance. Spirulina sp. CCC Snake P. Y85 [Y18793],
uncultured cyanobacterium clone SM2A06 [AF445707], andSM1D12 [AF445678] were isolated from a hot spring 4,45).
BAH8-10-45 formed a distinct branching at the peripheryof the Spirulina cluster 1.3 (Fig. 4b). The branching of
BAH8-10-45 at the base of this group was supported bybootstrap values of 100% in the neighbor-joining analysis.
The percentage of 16S rRNA gene sequence similarity among
BAH8-10-45 and traditional morphospecies of Spirulina

were 90.8–97.8% (H. tapeticola CCC Baja-95 Cl. 2T:
97.8%, S. subsalsa NES-27: 92.1%, Spirulina sp. PCC
6313 as S. major 45): 90.9% and Spirulina sp. CCC Snake
P. Y85 as S. labyrinthiformis 45): 90.8% showing in Table3). The percentage of similarity among BAH8-10-45 andrelated strains clearly justifies the definition of a new species
according to common bacteriological practice 50).
However, when the similarity within each cluster is evaluated regarding the current species belonging to the genus
Spirulina, the percentage of similarity within each species is

15
92.4
92.7
92.1
92.5
91.8
91.8
91.7
91.2
91.3
90.9
99.4
99.2
99.4
98.8
100.0
14
92.5
92.9
92.3
92.5
91.9
91.8
91.9
91.4
91.5
91.1
99.5
99.4
99.5
100.0
12
13
92.3 92.7
92.6 92.9
92.5 92.6
92.7 92.7
91.8 92.0
91.6 91.9
91.8 92.0
91.3 91.5
91.8 91.7
91.2 91.2
99.9 100.0
100.0 99.9
100.0
Spirulina subsalsa CCAP 1475/1
Spirulina sp. strain MPI S4
Spirulina sp. P7
Spirulina subsalsa IAM M-223
Spirulina subsalsa
Spirulina subsalsa FACHB351
Halospirulina sp.CCC Baja-95 Cl. 3
Halospirulina sp.MPI S3
Halospirulina tapeticola CCC Baja-95 Cl. 2T
Spirulina subsalsa var. salina BAH8-10-45
Spirulina major 0BB36S18
Spirulina major 1LT27S06
Spirulina major 0BB22S09
Spirulina subsalsa CCAP 1475/2
Spirulina sp. PCC 6313
Spirulina sp. CCC Snake P. Y-85

HF678502
Y18792
AF091109
AB003166
AF329394
FJ826621
Y18790
Y18789
Y18791
AB873003
AJ639890
FM177505
AJ635436
HF678507
AM709631
Y1879312
3
1 100.0 97.8 97.5
2
100.0 99.4
3
100.0
4
5
6
7
8
9
10
11
12
13
14
15
16

4
92.7
93.0
92.9
100.0

5
92.8
92.8
92.6
94.9
100.0

6
92.8
92.8
92.5
94.7
99.2
100.0

7
91.7
91.8
91.6
92.6
92.2
92.1
100.0

8
91.2
91.3
91.1
91.9
91.6
91.5
98.3
100.0

9
91.6
91.6
91.6
92.1
92.1
91.8
98.7
97.8
100.0

10
91.0
91.0
91.2
92.1
91.7
91.9
98.0
97.3
97.8
100.0

11
92.7
92.9
92.6
92.7
92.0
91.9
92.0
91.5
91.7
91.2
100.0

Spirulina cluster 2
Spirulina cluster 1.3
Spirulina cluster 1.2
Spirulina cluster 1.1
GenBank Accession
NumberStrainTable 3. 16S rRNA gene sequence similarities among the morphospecies of Spirulina.

91.0–99.4% for S. subsalsa (Cluster 1.1: 97.5–99.4% (n=3),
Cluster 1.2: 94.7–99.2% (n=2) and Cluster 1.3: 97.3–98.7%(n=4)), 98.8–100% for S. major (Cluster 2, n=5), and 100%for S. labyrinthiformis (Cluster 3, n=1). It is clear that thesimilarity threshold of the species belonging to the genus
Spirulina is equal to or slightly lower than threshold rangeof 98.7–99% for new species of common bacteriologicalpractice 50). In other words, there appears to be a situation where clusters including species of the genus Spirulina
in the phylogenetic analysis based on the 16S rRNA genesequence correspond to the genus (or higher) which is abroad concept bundling species of most common bacteria.
The similarity within S. subsalsa having three sub-clusters
is low (91.0–99.4%). The range of similarity within eachof the sub-clusters forming S. subsalsa is generally similar
to that of S. major (Cluster 2), suggesting a possibility thatthe future progress of research may divide S. subsalsa into
several species. Of the three sub-clusters forming S. subsalsa,
Cluster 1.3 appears to show a different character in terms of
halotolerance of its strains from strains forming other clusters of S. subsalsa. It must be noted that this difference wasused by Nübel et al. to propose the genus Halospirulina 45). If
halotolerance described above are re-arranged (for example,
such condition pointed out by Margheri et al. in 2003 that astrain must be resistant to a salt concentration of 15 wt/vol% or higher 39)) with related strains for the further advancement of research, there is a possibility of new developmentsin the near future. Such developments may include a changeof the diagnosis of S. subsalsa in the genus Spirulina Turpin
ex Gomont based on plant taxonomical evaluation and theestablishment of a new species as a morpho/ecospecies.
Another possibility is the introduction of a new genus withthe condition that species grow with a salinity of approximately 13–15 wt/vol % or higher or re-definition of thegenus Halospirulina based on classification and analysis
with emphasis on phylogenetic analysis using the 16S rRNA
gene sequence.
Accordingly, until a new definition of this new taxonomic
group is given to Cluster 1.3, this cluster is considered to bea S. subsalsa cluster based on the classification of the genus
Spirulina which is firmly established at present. If the strainsin this cluster are to be further classified using significant
characteristics, a taxonomic group below S. subsalsa shouldbe established in respect of the classification conforming tophycological taxonomy as in the case of BAH8-10-45 so thatits status is clear. As far as evaluation of the phylogenetic
analysis of the genus Spirulina is concerned, there appears to
be a gap in the sense that the species and variety (or subspecies) in the classification based on phycological taxonomy
generally correspond to such higher taxonomic groups as
genus and species in the phylogenetic analysis and evaluationof the similarity of most common bacteria using the 16S
rRNA gene sequence.
This gap between phylogenetic analysis using the 16S
rRNA gene sequence and the conventional classification also
exists within the taxonomy of most common bacteria. While53

Spirulina
cluster 3
16
91.5
93.0
92.3
93.5
91.8
92.2
91.0
90.8
91.1
90.8
92.2
91.9
92.2
92.0
92.0
100.0

Spirulina subsalsa var. salina var. nov.

54

Kuroiwa et al.

the 16S rRNA gene is used as the most effective molecularmarker for the taxonomical study of the bacteria, it has been
pointed out that resolution regarding the taxonomy of species
is limited for some existing taxonomic groups of bacteriaof which the taxonomy has been finely established. In suchcases, this limitation is sometimes compensated for by phylogenetic analysis using the sequence of a specific housekeeping
gene, such as the gyrB gene, of which the evolutionary speed
is faster than that of the 16S rRNA gene 25,52,56,62). Contrary to
the situation described above, phylogenetic analysis using the
16S rRNA gene sequence with the genus Spirulina suggests
a possibility of analysis to the variety (or subspecies) level
beyond the species level dealt with the existing classification
system because of the slow evolutionary speed of the 16S
rRNA gene. As such, this evaluation approach is inferred tooffer an effective resolution when the evolutionary relationship of species/strains belonging to the genus Spirulina is tobe evaluated.
The taxonomic work this time was conducted primarily withcyanobacteria obtained in the screening process of halophilic
microalgae which autotrophically produce highly value-added
compatible solutes from the viewpoint of their commercial production for industrial use. When this screening wasplanned, it was difficult to find effective screening conditions
based on the existing taxonomical knowledge of microalgae
which was largely confirmed to morphological information.
For the identification of BAH8-10-45 this time, as
the historical background and definition of the genus
Halospirulina were somewhat problematic, the taxonomyof this strain was clarified.
From the viewpoint of industrialists, we are hoping to see
the establishment of at least the species and preferably thevariety (or subspecies) as well, within the current phycologicalclassification system so that diverse physiological data canbe accumulated and systematized through the classification
work, as in the case of most common bacteria to facilitate
the utilization of cyanobacteria in a number of different
industrial fields. In the case of most common bacteria, industrialists themselves can identify and classify them without
the involvement of highly specialized experts. It is hopedthat a similar environment allowing the easy classification of
cyanobacteria can be established in the near future. In otherwords, this environment will allow the establishment of a new
phycological classification system in which new genus, species
and variety (or subspecies) can be backed by simple identification results through readily available and convenientphylogenetic analysis using the 16S rRNA gene sequence.
Although phylogenetic analysis using the 16S rRNA genesequence as the starting point, has become a common practice today in the case of cyanobacteria; the present situation
appears to be that the phycological classification system forhigher classes than genus to form the background has not yet
been fully established.

Acknowledgments
This study has been generously supported by the Japan
Cooperation Center, Petroleum (JCCP) under the auspicesof the Ministry of Economy, Trade and Industry, Japan.
We are grateful to Dr. Y. Kasai, MBI Chair of Marine
Biosciences, Kamaishi Research Laboratory, Kitasato
University for his valuable suggestions and discussions on algal
isolation and phylogenetic analysis. We would like to thankDr. R. W. Castenholz, Culture Collection of Microorganisms
from Extreme Environments, for his generous gift of thestrain H. tapeticola CCC Baja-95 Cl. 2T. Our thanks also
go to Dr. M. Kawachi (National Institute for Environmental
Studies) and President T. Hasegawa (Ecorenaissance, Co.,
Ltd.) for their many helpful suggestions regarding taxonomic
research.
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