Acta Biotechnol. 10 (1990) 1, 99-104 Akademie-Verlag Berlin

Heterotrophic Eubscteria Isolated from Cultures of the Cyanobacterium, Spirulina Maxima

Dams, J. E., JONES, L. P., ZAJIC -f, J. E.

Dept. of Biological Sciences The University of Texas at El Paso E l Paso, TX 79968

Summary

Three distinct heterotrophic eubacterial strains were isolated from mixotrophiu cultures of the filamentous cyanobacterium, Spirulina maxiina (Gom) Geitl. Spirulina spp. are considered to be prime candidates for the phototrophic production of biomass protein, particularly in developing countries. These cynnobacteria are extreme alkaliphiles and halophiles, making their production in arid regions promising. Nost previous studies on the eubacteria which live in Spirulina culture systems have focused on determining the possible presence of pathogenic species in biomass protein. Little has been done to understand the symbiotic relationships between the cyanobacterium and its eubacterial cosym- bionts. From the perspective of a lieterotrophic eubacterium, autotrophic cultural systems of Spirulina have limited carbon and energy resources, being limited to cyanobacterial exudates. In this study, three eubacterial strains were isolated and studied. One strain, a Gram-negative, non-sporing, motile rod, grew exceptionally well in a mineral salts medium where only a small amount of a single low molecular weight organic compound (e.g., acetate) was supplied as sole energy source. This strain was also extremely euryresponsive with respect to salinity and alkalinity as well. Two less well-adapted eubacterial strains are also described.

Introduction

Since the 1964 “discovery” of Spirulina [l, 21, several species of the genus Spirulina have been studied worldwide for possible utilization as supplemental sources of biomass protein [3]. Although these photoautotrophic organisms are grown at a pH above 8.5 [4-81, and in media containing little added organic material, significant numbers of heterotrophic bacteria are always found in Spirulina culture systems [3,9-121. These authors have suggested that associated eubacteria use cyanobacterial exudates as their energy sources. Such compounds would likely be low molecular weight substances. OGAWA and TERUI [9] have shown that Spirulina does not require the presence of eubac- terial cosymbionts in order to grow. In autotrophic outdoor cultivation systems, eubac- teria have been reported to occur a t lo5 to 1Og cells per ml [ 131. Spirulina can grow mixo- trophically with the addition of acetate [14, 151. The effect of this on eubacterial popu- lations has not been examined, either in terms of diversity or quantity. Previous studies of eubacteria isolated from Spirulina cultures have focused on determining whether potential human pathogens are present as part of the microbial load of various cyano- bacterial products [9-121.

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100 Acta Biotechnol. 10 (1990) 1

Little is currently known concerning the physiological and ecological nature of the eubacteria that cohabit Spirulina cultures, their relationship to Spirulina, or the effects of different Spirulina cultural systems on eubacterial populations. This paper attempts to answer some of these questions.

Materials and Methods

Source of Spirulina maxima

A culture of S. maxima (Gom) Geitl., UTEX 2342, was obtained from the University of Texas a t Austin Culture Collection of Algae. This strain, a Lake Chad isolate from Africa, arrived suspended in a nonaxenic ,,SpiruEina medium" [16]. Subcultures were made aseptically into sterile media as described by Jeeji Bai and Subramanian [17] supplemented with "A6" solution [18] and were incubated at 32'C in a Conviron Environmental Growth Chamber under a light intensity of 200 W m-2 for 12 hours of each 24-hour period. Subculturing was performed a t weekly intervals to keep actively growing Spirulinu available a t all times.

Eubacterial Strains Three strains of eubacteria, designated D-1, D-2 and D-3 were isolated from actively growing mixotrophic cultures of AS'. maximu. Isolation was made on Difco brand Tryptic Soy Agar (TSA), containing 1% NaCI, and the p H adjusted to 9.2. D-1 and D-2 were isolated from a 1% glucose- enriched Spirulina medium, while D-3 was isolated from a glucose-acetate-enriched mineral salts medium ( 0 . 5 ~ 0 - 0 . 5 ~ 0 ) . All cultures were maintained on TSA a t 32OC, and were subcultured a t appropriate intervals.

Morphology The morphologies the three isolates were described from colonies growing on TSA and Gram- staining.

Oxygen Requirements

Qualitative evaluation of growth under various gas environments was made according to SLACK and GERENSCER [19] using TSA cultures growing at 35OC.

Preparation of Inocula for Substrate Studies

Isolates of D-1 and D-2 were cultured in 125-mI shake flasks containing 50 ml of JEEJI BAI-SUBRA- MANIAN medium, pH 9.2, supplemented with 1% glucose and 0.5% yeast extract (Gibco). D-3 ini- tially did not grow on this medium, so 0.5% sodium acetate was added. After 24 hours of growth at 32"C, cells of each isolate were harvested by centrifugation, washed twice in 10 ml of buffered saline (1% NaCI, 0.25% K,HPO,, pH 9.2), followed by resuspension in saline.

Nitrogen Requirements

The medium used for preparation of inocula was modified to contain a single source of nitrogen (Tab. 1). The amounts of nitrogen salts used contained equivalent amounts of nitrogen. Growth was monitored using optical density (560 nm) measurements on a BAUSCE and LOMB Spectronic 20 for cultures at 32°C. The initial p H w w 9.2-9.4. Cultures were shaken by hand four times a day.

DAVIS, J. E., JONES, L. P. et al., Heterotrophic Eubacteria 101

Tab. 1. Substrates for nitrogen utilization by eubacterial isolates

Nitrogen source w-7

Ammonium chloride 1.6 Ammonium nitrate 1.2 Vitamin-free casamino acids (Difco) 1.0 Sodium nitrate 2.5 Urea 0.9 Yeast extract, Gibco 1 .o

pH Tolerance Growth of each isolate was monitored in a series of tubes containing Difco brand Tryptic Soy Broth (TSB), the pH’s of which were adjusted to 3.8, 4.7, 5.4, 6.4, 7.2, 8.1, 8.6, 9.6 and 10.6, res- pectively, using sterile 1N HCl or 2N NaOH. Incubation was a t 32°C with agitation provided by handshaking four times a day.

Sodium Chloride Tolerance Sodium chloride tolerance was evaluated in shake flask cultures of the basal medium (See pre- paration of inocula), pH 9.2. Levels of NaCl used were 1, 5, 9, 13, and 17%, respectively. Incuba- tion was a t 32°C.

Temperature Tolerance Growth of each isolate was evaluated by cultivation in TSB, pH 9.2, a t 5, 15, 25, 35, 45, 55 and 65°C. Cultures were agitated four times daily.

Fermentation of Carbohydrates Difco brand Phenol Red Carbohydrate Fermentation Media was used to test each isolate’s ability to produce acid and/or gas from the following substrates: sodium acetate, arabinose, fructose, galactase, glucose, lactose, maltose, mannitol, rhamnose, sucrose and xylose. Incubation tem- perature was 35OC, and pH after sterilization was adjusted to 9.2.

Miscellaneous Tests Presence of catalase was determined by dropping 3% H,O onto colonies growing on TSA. Urease production was determined on Urea Medium (Difco) for cultures growing a t 35°C. Oxidase tests also performed.

Use of Acetate, Butyrate and Palmitate Each isolate’s ability to utilize fatty acids as sole energy sources was tested in the basal medium (pH/9.2) a t 35°C in shake cultures.

Sensitivity of D-1 to Different Amounts of Acetate

The effects of varying acetate concentration on the D-1 strain was studied because it was the dominant strain occurring in acetate-enriched cultures of S. mazimu. Shake flask cultures of basal medium contained 0.01, 0.02, 0.1, 0.2, 1.0 and 2.0% acetate as the only energy source were grown a t 35°C. I n addition to the original amount of acetate present, an equal a m p n t of acetate was added after 24 hours of growth. Dilution plating was used to estimate eubacterial numbers in relation to acetate feeding.

102 Acta Biotechnol. 10 (1990) 1

Growth on Other Organic Molecules by D-1 Shake flask cultures containing basal medium, and 0.1% of the following compounds as the only energy source inoculated with D-1, and incubated a t 35OC: methanol, ethanol, n-propanol, isopropanol, formaldehyde, acetaldehyde, propanaldehyde, formic acid, sodium propionate, vitamin-free casamino acids (Difco), 8-aminobutyrate, 3-hydroxybutyrate, 4-hydroxybutyrate, isobutyrate, isovaleraldehyde, B-ketobutyrate, sodium lactate, oxalacetate, tryptophan, alanine, aspartate, cystine, glutamic acid, glycine, homoserine, isoleucine, leucine threonine, and valine.

Results

The general characteristics of three eubacterial isolates are given in Tab. 2. None of the three isolates was a sporeformer. Isolate D-1 grew well on JEEJI BAI-SUBRAMANL~X Spirulina medium supplied with glucose and inorganic nitrogen, but this isolate also grew well on basal medium containing each of the following compounds added singly as the sole energy source : ethanol, n-propanol, propionate, vitamine-free casamino acids, 3-hydroxybutyrate (the substrate for polyhydroxybutyrate, Spirulina's chief storage product), isobutyrate, isovaleraldehyde, lactate, oxalacetate, aspartate, alanine, glutamate, butyrate, palmitate and acetate. Non-utilized substrates included methanol, isopropanol, acetaldehyde, formaldehyde, formate, 8-aminobutyrate, 4-hydroxybuty- rate, B-ketobutyrate, tryptophan, cysteine, glycine, homoserine, isoleucine, leucine, threonine, and valine. Any nitrogen sources could be utilized.

Tab. 2. Characteristics of three eubacterial isolates from Spirulina maxima cultures

D- 1 D-2 D-3

Cell morphology

Colonies on TSA

Gram reaction Motility Catalase Oxidase Urease Gas requirement,

pH range NaCl tolerance Temperature Acetate Butyrate Palmitate Acid from:

bacillus 2.0 x 0.6 pm singly & clumps

creamy white smooth, entire negative Yes positive positive positive aerobic anaerobic 7.0- 10.6 0-13% 15-45 "C Yes Yes Yes arabinose fructose galactose glucose maltose mannose sucrose

bacillus 1.0 x 0.7 pm filaments, pairs, & v-shapes red smooth, flat positive no positive positive negative aerobic anaerobic 7.0-9.5 0-5% 15-35 "C no no no arabinose fructose galactose glucose lactose maltose mannitol sucrose

coccus 1.0 x 1.0 pm pairs, tetrads, & clumps yellow to orange smooth, flat positive no positive positive positive aerobic

5.4 -9.5 0-5% 15-45 "C Yes Yes no arabinose fructose galactose glucose maltose mannose sucrose

DAVIS, J. E., JONES, L. P. et al., Heterotrophic Eubacteria 103

Strains D-2 and D-3 utilized all nitrogen sources (except urea for D-Z), however, best growth occurred in the presence of organic nitrogen. In a separate unpublished study comparing the effects of acetate supplementation on Spirulina maxima growth, total eubacterial numbers was consistently 107 ml-1 in the absence of of acetate with the D-1 strain predominant. When small amounts of ace- tate were added, eubacterial number increased to 101O ml-l, and only the D-1 isolate was recoverable.

Discossion

Until the present study, the biological and ecology properties of the eubacteria living as cosymbionts in cultures of the alkaliphilic cyanobacteria Spirulina have not been closely examined. Most frequently isolated species from cultural systems have included Bacillus, Micrococcus, enterococci, as well as Gram-negative rods [9- 121. Strain D-1 did share some physiological characteristics with certain strains of WATA XABE et al. [20], and HORTXOSHI and AKIBA [2l], but could not be considered a pseudo- monad, as D-1 is a facultative anaerobe. Nor does it closely resemble any genus already described within the Enterobacteriaceae [ 221. This organism was extremely euryrespon- sive [21] in terms of many environmental parameters, and utilization of small mole- cular substrates for energy. Strain D-2 differed from the coryneform alkaliphilic isolates of HORIKOSHI and AKIBA [21], and KOBAYASHI and HORIKOSHI [23] in that this strain possessed red colonies, could not grow above a NaCl concentration of lo%, had a narrow range of pH tolerance, could use ammonium salts as sole nitrogen source, and grew anaerobically. Carbo- hydrate fermentation patterns also differed considerably. GEE et al. [24] reported on five strains of alkaliphilic corynebacteria. However, their description made no mention of v-shaped morphologies. In addition, their isolated could not ferment arabinose or lactose, nor could they use ammonium or nitrate. SOUZA and DEAL [25] have described a new alkaliphilic coryneform strain, but their isolate is a strict aerobe, without v-shaped morphology. Strain D-3 closely resembled the Micrococcus strain 31-2 of HORIKOSHI and AKIBA [21], as well as the description given by BAIRD-PARKER [26]. In unpublished experiments comparing autotrophic and mixotrophic (acetate) growth of Spirulina maxima, the total number of eubacteria in autotrophic cultures was very similar to previously published reports ( lo7 ml-l), however, strain D-1 was much more numerous than either D-2 or D-3. When acetate was added, eubacterial numbers in- creased three-fold (lolo ml-l), and only the D-1 isolate was recovered. I n conclusion, D-1 and D-2 may represent new taxa. The taxonomic position of many alkaline isolates is difficult to ascertain because their physiology is probably quite diffe- rent from their relatives in more moderate environments. As alkaline environments are more extensively studies, it can be expected that many new taxa will be discovered.

Received June 19, 1989

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104 Acta Biotechnol. 10 (1990) 1

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Book Review

M. S. S. PAIS, F. MAVJTUNA, J. M. NOVAIS

Plant Cell Biotechnology (NATO AS1 Series H: Cell Biology, VOL 18)

Berlin, Heidelberg, New York, London, Paris, Tokyo: Springer-Verlag. 1988 500 pp., 92 figs., DM 298.00, ISBN 3-540-18556-9

Plant Cell Biotechnology offers significant potential benefits in the areas of plant improvement and production of fine chemicals. The book is the Proceedings of a NATO Advanced Study In- stitute held in Portugal 1987 and includes the review and research lectures given, papers baaed on poster presentation of current research and notes on some of the discussion sessions. It covers many of the diverse topics in this field: applications in agriculture and plant breeding, plant gene- tic engineering, biochemical and engineering aspects of biotransformation and production of the fine chemical by plant cell and tissue cultures. A big part of the book contains the large-scale culti- vation of plant cells, bioreactors of plant cell culture and engineering aspects. That’s why the book should appeal to the researchers, both experts and novice in the field, and professionals from agri- cultural, pharmaceutical and chemical sectors with a current or potential interest in what plant cell biotechnology can offer.

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