The Algal Culture Collection at the University of the Philippines Los ...

Philipp. Scient. 51: 66-103 ©2014, University o f San Carlos Press

The Algal Culture Collection at the University of the Philippines Los Banos-Museum of Natural History (ACC-UPLB-MNH)

Milagrosa R. Martinez-Goss1’2*, Nerissa K. Torreta1, June Owen O. Nacorda1, and Carl Dave S. Mendoza 1 'institute of Biological Sciences (IBS), College of Arts and Sciences (CAS), University of the Philippines Los Banos (UPLB) 2Museum of Natural History, UPLB, College, Laguna 4031, Philippines

ABSTRACT The algal culture collection of the University of the Philippines Los Banos-Museum of Natural History (ACCUPLB-MNH) is one o f the oldest (47 years old) and one of the largest freshwater resources of microalgal cultures in the Philippines. It is supporting researchers, students, and teachers in the academe, as well as those involved in biotechnological applications of microalgae and those engaged in commercial/ economic productions. At present it holds 86 strains distributed in three algal divisions, i.e., the Cyanophyta (15 genera and 32 species and varieties), Chlorophyta (six genera and six species), and Bacillariophyta (two genera and two species). Keywords:

algal culture Chlorophyta, microalgae

collection, Bacillariophyta, Cyanophyta, freshwater

*Corresponding email address: [email protected]. Contact details: Algal Culture Collection, Bioscience Building Room C313, UPLB, College, Laguna 4031, Philippines, telephone nos. +(63 49) 536 3368/2807

Martinez-Goss et al.: Algal culture collection at UPLB

67

INTRODUCTION The history of culturing algae dates as far back as the 1920s when Ernst G. Pringsheim established the first collection of algae in Prague, now known as the Culture Collection of Algae at the Laboratory of Algology (CCALA) in the Czech Republic. Pringsheim later went as a refugee to Cambridge (England, U.K.) where he brought some strains of this collection and put up a similar culture collection in his adopted homeland, now known as the Culture Centre of Algae and Protozoa (CCAP) (George 1976). Algal strains from this collection were later used to establish the culture collection in University of Gottingen when Pringsheim returned to Germany in 1953; this collection became known as the Sammlung von Algenkulturen at Gottingen (SAG) (Day et al. 2004). Pringsheim’s cultures also helped provide the nucleus of cultures that was established in 1953 by Richard C. Starr at Indiana University (IUCC, U.S.A.). The IUCC was later known as the Culture Collection of Algae at the University of Texas (UTEX, U.S.A.) when Starr joined the faculty of the Department of Botany of that university (Starr 1978). Similar collections were later put up in Tokyo (Japan) and in Paris (France); however, the four major service culture collections of algae (CCALA, CCAP, SAG, and UTEX) provide mostly the curatorial and service roles of phycologists globally. At present, the largest holding is found in UTEX which has about 3,000 different strains of living algae (www.utex.org); the rest of the large culture collections hold at least 2,400 strains (www.uni-goettingen.de/en/184982.html, www.ccap.ac. uk/our-cultures.htm, ccala.butbn.cas.cz). Interest in microalgal cultures has tremendously increased over the past decades mainly because of the discovery of some valuable economic products in some of these cultures. These include betacarotene, a pro-vitamin A, from the green alga Dunaliella salina (Borowitzka & Borowitzka 1988), while another green alga, Haematococcus pluvialis, is also a commercial source of astaxanthin, an antioxidant carotenoid that gives a pinkish to red hue to fishes and other food sources (Yang et al. 2011). More common among these microalgae are Spirulina, Chlorella, and Nostoc commune that are used as human food mainly as protein sources (Vonshak 1997, Oh-Hama & Miyachi 1988, Martinez 1988). This paper introduces the algal culture collection at the University of the Philippines-Museum of Natural History (ACC-UPLBMNH) as a comprehensive biological resource of living algae. It is

68

The Philippine Scientist, Volume 51 (2014)

among the largest algal service culture collections in the Philippines although it is younger compared to other algal culture collections. The early beginnings of the ACC-UPLB-MNH can be traced as far back as 1968 when the late Julia B. Pantastico collected and grew microalgae for her research work. This initial collection was housed at the Department of Botany of the College of Agriculture, University of the Philippines (UPCA). Eight years later, the collection became part of the Microbial and Algal Culture Collection of the University of the Philippines Los Banos Museum of Natural History (UPLB-MNH) when it was established in 1976. Pantastico was the first curator, but Milagrosa R. Martinez assumed curatorship in the same year when the former left the university and transferred to the Southeast Asian Fisheries Development Center (SEAFDEC). In its early years, 37 holdings were maintained and its number peaked at 122 in 1992, but over time the number of holdings kept in the collection dwindled. In most cases, the number of cultures depended on the availability of staff members who managed and maintained the cultures and relied on funded research projects. It was not until 2013 when the number of cultures rose from 17 to 86, and this number has been maintained for three consecutive years. The increase of holdings was partly due to a research grant through the Commission on Higher Education (CHED) that allowed Martinez-Goss, Nerissa K. Torreta, and June Owen O. Nacorda to conduct diversity and conservation studies on Philippine microalgae. Capacity building in several State Universities and Colleges (SUCs) for microalgae research also resulted in the establishment of small unit algal culture collections in eight academic and research institutions around the country. The current number of algal cultures (86) is only about 3% of the UTEX collection and yet ACC-UPLB-MNH still strives to support researchers not only in the academe but also in industry who are involved in biotechnological applications of microalgae. The culture collection also serves as an essential infrastructural function for scientific investigation, as well as a repository of knowledge in identifying, isolating, culturing, and ex situ conservation of algae. METHODOLOGY

Establishment of Microalgal Cultures Sample collection. Algal samples were obtained from various aquatic and soil samples. Soil samples were collected following the

Martinez-Goss et al.: Algal culture collection at UPLB

69

method of Roger & Reynaud (1976). Soil samples from wetland rice fields were sampled using a modification of the method of Roger & Reynaud (1976), wherein composite samples consisting of 10 subsamples each of rice field soil and flood water were obtained using a core sampler (corer), which is a cylindrical tubing, made of either glass or plastic, that is about 10 cm long and 3 cm in diameter. A transect line of 10 m was drawn diagonally across a rice plot. Soil (5 g) and floodwater (5 mL) samples were obtained at every 1-m interval of the transect line. The subsamples were obtained only from the upper layer of the soil and water. The same protocol was used in coring for dry soil except in this case the upper 5 mm of the soil surface was scraped with a knife blade or metal spoon. Water samples were scooped with a 600-mL plastic dipper tied to a bamboo pole and transferred to a wide-mouth plastic bottle. All samples were processed within 4 h after collection for isolation and cultivation, and/or preserved for museum storage. Samples that were transported from long distances were kept inside a cooler prior to processing. Isolation o f microalgae. Isolation of microalgae was carried out by spread plating on solidified media with 1% agar. The medium that was frequently used was BG11 (Stanier et al. 1971), while BG11 minus N (BG11-N) was obtained by removing NaN03 from the medium. The medium used for cultivating diatoms was based on Tanada’s medium (Tanada 1951) or BG11 plus sodium metasilicate to give a final concentration of 100 mg/L. The present culture of Arthrospira (Spirulina) platensis was obtained from the raceway pond in Geofarm in Bayambang, Pangasinan in 2005; however, in earlier culture studies A. platensis was successfully isolated from rice paddies in Pampanga using BG11 medium. Soil samples were diluted up to 10~6 using distilled water. Aliquots of 0.1 mL from 10 2to 10 6 dilution of each sample was spread plated onto the appropriate solid medium for the target organism. For isolation of microalgae from water samples, the same method was performed except that water samples were only diluted up to 10 5 using sterile distilled water, and 10 1to 10 5 dilutions were plated. The plates were then incubated at ambient room temperature (20-25°C) and under illumination using three 40-W fluorescent lights providing an irradiance of 120-150 pmol photons m 2s ’ for at least three weeks. The Nostoc commune samples were obtained from the field, where they occurred in bloom. These were cleaned by washing with

70


sterile distilled water to remove other algal contaminants, and then inoculated into liquid BG11-N and illuminated for about three weeks. Colonies from plates were picked and streaked onto solid BG11 and BG11-N to remove contaminants. The plates were then incubated again under the same conditions above. Re-streaking was repeated until the algae were free from contaminants. Purification o f microalgae. Purification of green algae and diatoms was carried out using the antibiotic streptomycin following a modification of the method of Baldia et al. (2007). Nystatin (mycostatin) was used as an antifungal agent, as needed, at a dosage of 3-10 pg/mL of the medium (Ferris & Flirsch 1991). The alga was inoculated into 10 mL of liquid BG11 medium and was incubated for at least a week to allow sufficient growth of the alga. Filter-sterilized streptomycin and/or nystatin were/was added into the medium that resulted in concentrations of 15 mg/L and 10 mg/L, respectively, after which the culture was incubated again. After 24 h, a loop of sample from the culture was transferred into an antibiotic-free liquid BG11, BG11-N or BG11 plus sodium metasilicate and was incubated again with illumination until visible growth of the alga was observed. Confirmation of the purity of the alga was done microscopically under bright-field and phase-contrast microscopy using Carl Zeiss Axio Scope Al (Carl Zeiss, Gottingen, Germany). Characterization and identification o f microalgae. The isolates were characterized based on cultural characteristics and morphocytological features. Cultural morphology of the organisms was based only on their growth on solid BG11 and BG11-N. Their ability to grow in the two media differentiated a nitrogen-fixing alga from the non nitrogen fixers. Cyto-morphological characteristics of the isolates were observed microscopically using a bright-field and phase-contrast microscope (Carl Zeiss AxioScope A l, Carl Zeiss, Gottingen, Germany) and all photomicrographs of the specimens were taken under immersion oil objective (100x) with an attached camera (AxioCam ERc5S, Carl Zeiss, Gottingen, Germany). Cell dimensions were taken with a calibrated micrometer eyepiece. Identification of the algae was carried out using the combination of cultural and cyto-morphological characteristics with the aid of available references (Smith 1950, Desikachary 1959, Prescott 1962, Rippka et al. 1979, John et al. 2002, Lee 2008, Wehr & Sheath 2003).

Martinez-Goss et a l Algal culture collection at UPLB

71

The isolates were identified up to the lowest taxon possible. Current names were based on algae base (Guiry & Guiry, 2015). Classification at the division level was based mainly on the combined schemes of Lee (2008), Round et al. (1990), and Van den Hoek et al. (1995). Maintenance of Culture Collection Clean isolates were picked up (single colony) from streaked agar plates (Fig. 1A) and maintained by serial transferring on agar slants, preferably using screw-capped test tubes, with BG11 and BG11-N for nitrogen-fixers (Fig. IB). Diatoms were maintained in BG11 plus sodium metasilicate or in Tanada’s medium (Tanada, 1951) to give a final concentration of 100 mg/L. Some vitamins, such as Vitamin B12, Vitamin B1 and biotin, were added to the modified diatom medium to induce sexual means of reproduction, following the formulation of Guillard (1975). Arthrospira (Spirulina) platensis was maintained in Zarrouk’s medium (Zarrouk 1966, Amara & Steinbiichel 2013). After six weeks of incubation under illumination of three 40-W lamps (at about 120-150 pmol photons m 2s '), the number of lamps was reduced to one 40-W fluorescent lamp giving an estimated irradiance of 40-50 pmol photons m 2s ' at an ambient temperature of 20-25°C and under a light: dark regime of 8:16 h. At least 1 mL of the liquid medium was also added to each of the cultures to prevent rapid desiccation. After 10 weeks of incubation, the cultures were re-streaked on the appropriate agar slants. The cycle was repeated to maintain the cultures. Microscopy and regular streaking on plates were also conducted to check the purity of the cultures. Cultures that were frequently requested were maintained in 100 mL liquid medium in 300 mL bottles on illuminated culture benches (Fig. 1C). RESULTS The algal culture collections at UPLB-MNH started as a product of research activities of the Phycology Laboratory I, hence most of the activities for culturing and maintenance were carried out by staff members of various projects. Project leaders in that laboratory had to pursue grants for research projects to sustain the algal cultures. The number of cultures started with 37 isolates in 1968 as part of the project of Pantastico on diversity and conservation studies of algae in Laguna de Bay, however, the number of isolates was not sustained over time.

72


Figure 1. The Algal Culture Collection at the University of the Philippines Los Banos Museum of Natural History (ACC-UPLB-MNH). (A) Culture plate with microalga; (B) Isolates in screw-capped test tubes; and (C) Isolates in 100-mL liquid media stored in 300-mL cotton-plugged ketchup bottles.

At present the ACC-UPLB-MNH maintains a total of 86 unialgal cultures representing 56 strains in the division Cyanophyta (blue-green algae, BGA), two in the division Bacillariophyta (diatoms), and 28 in division Chlorophyta (green algae) (Table 1-3; Plates I—VII). These 86 strains belong to 40 species, of which 32 species and varieties are in the Cyanophyta, two species in Bacillariophyta, and six species in the Chlorophyta. Among the cyanophytes about 59% of the species and varieties are heterocystous forms that are presumed to be nitrogen fixers. All the strains are isolates of the Phycology Laboratory I, Institute of Biological Sciences and the ACC-UPLB-MNH except for Arthrospira (Spirulina) platensis (Cy 7) and Chlorella vulgaris (Cl 15), which were obtained from Geofarm in Bayambang, Pangasinan and the UTEX algal culture collection (# 2714), respectively. These isolates were mostly obtained from freshwater habitats in the country, mainly during the conduct of the research project funded by Commission on Higher

M artinez-G oss et al.\ A lgal culture collection at U PLB

o

W D 03 IS Oh C

>

'9 i a o o U

l ^7 o m' 8 ^ >

MO Q > ^ T3 > rP U l-S " Vt/ o 2 U m O 1 60 U S_

« a J2 o PL, *-C

-9 -g

samoensis Wille

O X5 c. *C

cd .a, g !S T3 -S C3 ■- < & a °° 8 > | o -g

•'d>> U

O

&

o & 8 t &

vo

o

field soil from hemispherical colonies; enclosed by a clear, Mangatarem, thick envelope; cells yellowish to bluish-green, ____________________ Pangasinan___________ ellipsoidal, 7.3 pm long, 4-5.3 pm wide.

o PQ

O

73


OA « -Z — O ^©

43 U) cd 43 _ co 23 ~ > O ' ' t r p

S g

kb

cd

o -a O 43 so ao T3 (lT

I §

CO

43 w>3

g>

• § 1 6) 0) S 2^ *s > 42

■g,l cd

bO

9

u a u

& o P « 0i-U 2> u

-O

cd

o £ q 52 'g PQ

11 o m

u

M artinez-G oss et a l.: A lgal culture collection at U PLB

75

a CQ B P. •O

43

00 £ h

00

po

O C t3

S

arjj tS «

o 1 1 O O 6 - a S .g O c3 rn W W) »Cfl W^ p »| O « 2 pc

X? u S 3 aJ > 2 P « •§ ctja 'o*