Effects of NaCI and [MgCI.sub.2] on Physiological and Biochemical Changes in Osmoregulation of Chlorococcum hypnosporum L.

Mark G. Hardy [1]

This study investigated the physiological and biochemical changes in cellular osmoregulation when Chlorococcum hypnosporum (L.) was grown in high saline environments. A dramatic decrease in total growth occurred when C. hypnosporum was cultured in increasing concentrations of NaCI. When grown in increasing concentrations of [MgCI.sub.2] a significant decrease in growth of C. hypnosporum was observed only at 10 ppt. Percent sexuality increased at all concentrations of NaCI and [MgSI.sub.2] except at 0.1 ppt. When compared to controls, the amount of dissolved oxygen decreased in all saline concentrations. Growth medium pH of C. hypnosporum was not significantly affected by changes in [MgSI.sub.2] concentrations, with only marginal pH changes in medium containing various concentrations of NaCI. Nuclear magnetic resonance (NMR) analysis indicated that increasing [MgSI.sub.2] concentrations caused increases in cellular osmoregulation by C. hypnosporum resulting in increased production of intracellular [13] C-labe led organic solutes. When C. hypnosporum was grown in increasing NaCI concentrations, similar results were observed only at 0.001 ppt.

Algae have been used as agricultural fertilizers by coastal farmers for a number of years (Booth, 1965). As early as 1980, researchers explored developing fertilizers that do not contaminate ground and surface waters or cause erosion (Waaland, 1981). Conventional fertilizers contain multiple nutrients, nitrogen, phosphate, potash, and secondary micronutrients that can cause degradation in surface water quality due to runoff from upland areas. Fertilizer runoff on the scale of major river drainage basins is thought to be involved in declining surface water quality (Justic et al., 1995).

According to the Association of American Plant Food Control Officials 1996 Annual Report, the United States consumed 50,744,419 tons of commercial fertilizers in 1995. Furthermore, as of June 1996 total usage increased to 53,439,634 tons. The prohibitive costs and environmental consequences of synthetic fertilizer use necessitate a need to investigate microalgae as a "biofertilizer." A potential source of organic nitrogen, microalgae degrade in the soil and drastically reduce the problem of leaching exhibited by other organic and some slow release fertilizers.

Algae are potentially excellent biofertilizers because they have sufficient amounts of nitrogen and potassium but are low in phosphate. If algae are used as alternative fertilizers, they must be supplemented with phosphate for use with most crops. According to Waaland (1981), the advantages of algae as a biofertilizer are that they: (1) are free of weed seeds and fungal spores which can harm terrestrial crops, (2) are good sources of trace elements, and (3) can regulate crop growth and ripening because of the auxin, cytokinin, and gibberellin associated with them. Fertilization with algae may inhibit certain pathogens reducing harm to crops (Waaland, 1981). Deleterious effects of seaweed manures include excess manganese release in low pH soils, water-logging of soils fertilized with Pachymenia, inhibition of plant growth, and low nitrogen availability (Boney, 1965).

The purpose of this research is to investigate the feasibility of improving C. hypnosporum as a biofertilizer by increasing intracellular nitrogenous compounds. In aquatic environments, salinity is considered an important ecological variable, particularly in estuarine regions, salt water ponds, and shore areas where planktonic algae are often subjected to widely fluctuating salt concentrations (Guillard, 1962). Changes in the salinity of seawater usually affect the growth, metabolism, and survival of marine phytoplankton. Guillard (1962) also suggests that the osmoregulatory mechanisms of algae living in marine environments can tolerate a wide range of external saliities. Salinity changes have been explored in only a few microalgal species. Therefore, C. hypnosporum was exposed to five concentrations of two salts, NaCl and Mg[Cl.sub.2] respectively, to assess the physiological and biochemical changes that are involved in increased production of intracellular organic solutes of freshwater microalgae when grow n in high salinities.

MATERIALS AND METHODS

Microalgae Culture--A stock culture of C. hypnosporum was maintained in 500-ml Erlenmeyer flasks containing 100 ml of a 1.5% semi-solid agar (FW1) medium (O'Kelley, 1983). Algal cell moculum was prepared from stock cultures by aseptically washing cells from the semisolid agar medium using 5 ml of sterile FW1 medium and shaking flasks forcefully (Hardy and O'Kelley, 1986). The cell suspension was decanted into a 25 ml sterile beaker and diluted by adding 20 ml of fresh FW1. Transmittance readings of FW1 medium and inoculum (9:1, v/v) indicated that a 2 ml volume contained approximately 280,000 algal cells needed for optimal growth initiation.

Thirty-six 250-ml Erlenmeyer flasks containing 50 ml sterile FW1 medium and isotopically labeled [NaH.sup.13][CO.sub.3] (10 [micro]M) were aseptically inoculated with 2 ml of the algal suspension. The initial transmittance of the medium was 100%. The growth medium, initially adjusted to pH 6.8, also contained NaCl or Mg[Cl.sub.2] concentrations of 0.001, 0.01, 0.1, 1, and 10 ppt using 6 replications. Culture flasks were maintained for 21 days at 25 [+ or -] 1 [degrees]C in a Mark III Environmental Growth Chamber. Light intensity was maintained at 13.36 [micro]mol photons [m.sup.-2] [s.sup.-1] using Westinghouse cool-white fluorescent tubes. A day and night regime was set at 12:12 hour light/dark cycle. At the end of each culture period in medium allowing optimal growth, the final packed volume of algal cells was approximately 1.0 ml/L for each liter of culture.

Parameter Analysis and Algal Assessment--After the 21-day growth period, percent sexuality, amount of dissolved oxygen, total growth, pH and cellular osmoregulation by nuclear magnetic resonance (NMR) analysis (General Electric, GN 300 Mhz) was determined. Photosynthetic activity was determined using a YSI model 51B oxygen meter (YSI Inc., Yellow Springs, OH) to measure dissolved oxygen. An oxygen probe was placed in each algal sample and stirred for approximately 2 to 3 minutes. The [O.sub.2] solubility factor was set at 25[degrees]C and dissolved oxygen content measured in mg/L. The probe was rinsed in distilled water and recalibrated after each measurement. Medium pH was determined for each culture using an Orion research digital Jonalyzer/501. Each dissolved oxygen measurement was taken at 9:00 a.m., while each measurement of pH was taken at 10:00 a.m.

Total growth assessments were obtained at 520 nm wavelength using a Baush and Lomb Spectronic 20D spectrophotometer. The spectrophotometer was calibrated to a transmittance reading of 100% using a culture tube containing 10 ml of FW1 medium. Medium (10 ml) containing algae from each experimental flask was placed in a culture tube and spectro-photometrically analyzed.

A Bright-Line hemacytometer and a demoscope, consisting of a light microscope connected to a 19" RCA television was used to determine the percent sexuality of C. hypnosporum. Percent sexuality was calculated by dividing the number of zygotes by the number of zygotes plus vegetative cells in a 1 ml sample, counting at least 150 cells per sample. Zygotes contained "spines" on their cell wall or the appearance of two or more cells in a parent cell wall. After a 21-day growth period, algal cells were collected by centrifugation using a Beckman Centrifuge model J2 1-B. Algal pellets were obtained after centrifugation at 20,000 rpm for 10 to 15 minutes. Pellets were resuspended in 3-5 ml FW1 medium and dispensed into 10 ml test tubes for NMR analysis of 13 C-labeled organic compounds.

Experimental Design--Experimental flasks were arranged in a 1 (inoculation treatment) x 5 (NaCl or Mg[CI.sub.2] concentrations of 0.00 1 to 10 ppt) factorial arrangement in a completely randomized design (CRD) with 6 replications. Each identical 250-ml Erlenmeyer flask containing 2 ml inoculum in 50-ml FW1 medium constituted a treatment replicate. Algal cells were grown in batch culture for 21 days and manually shaken twice a day for adequate dispersal of nutrients.

RESULTS

Cell growth of C. hypnosporum, measured as transmittance, was 2 times greater in Mg[Cl.sub.2] compared to NaCl at all concentrations except 10 ppt (Table 1). Growth inhibition was highest in cultures grown at 10 ppt Mg[Cl.sub.2]. Zygospore production in algal cells grown in NaCl increased as NaCl increased, with the exception of cells grown in 0.1 ppt NaCl. All NaCl concentrations demonstrated significantly higher zygospore production than algal cells in controls. There was a significant difference in zygospore production of controls versus experimental cultures of C. hypnosporum at all [MgCl.sub.2] concentrations, with the exception of 0.1 ppt (Table 2). There was no significant difference in oxygen evolution of C. hypnosporum cultures grown in [MgCl.sub.2] when compared to NaCl. Oxygen evolution decreased in cells grown in [MgCl.sub.2] and NaCl at all concentrations tested, with the exception of 0.001 ppt [MgCl.sub.2] (Table 3). Medium pH in experimental cultures was significantly different at 0.001, 0.01, and 0.1 ppt NaCl. However, no differences in medium pH were observed in algal cells grown in all concentrations of [MgCl.sub.2] (Table 4).

NMR spectra of [[blank].sup.13]C-labeled C. hypnosporum indicated that the accumulation of organic solutes, although significantly higher than controls, was fairly constant at 0.001 to 1 ppt [MgCl.sub.2]. However, there was a dramatic increase in organic solutes at 10 ppt [MgCl.sub.2] (Table 5). Organic solute accumulation fluctuated in algal cells grown in increasing NaCl concentrations. The highest accumulation of organic solutes was observed at 0.001 ppt NaCl.

DISCUSSION

After 21 days of growth, C. hypnosporum grew significantly better in all [MgCl.sub.2] concentrations except at 10 ppt when compared to NaCl. Although algal cells grown in 10 ppt NaCl grew better than cells in 10 ppt [MgCl.sub.2] all other NaCl concentrations had a deleterious effect on algal growth with at least a two-fold decrease compared to cultures grown in [MgCl.sub.2]. While NaCl exhibited the greatest inhibitory effect on growth, there was no significant difference in algal growth with increasing [MgCl.sub.2] concentrations compared to controls except at 10 ppt. In Chlorella autotrophica, the inhibition of growth at high salinities was associated with decline in turgor pressure (Ahmad and Hellebut, 1984). The decline in total growth was due mainly to a decrease in vegetative cells with a corresponding increase in zygospores as salinity increased. These findings agree with the observations of Sze (1993) where sexual reproduction was often induced by stressful conditions.

Herbst and Bradley (1989) observed that growth and persistence of algae in a saline environment required physiological tolerance andlor resistance stages in the life cycle. Chlorococcum hypnosporum demonstrated a greater physiological tolerance to Mg[Cl.sub.2] than NaCl as indicated by higher growth measurements after 21 days. Increases in the number of zygospores with corresponding reductions of vegetative cells indicated that both salts promoted sexuality and persistence of C. hypnosporum. Thirteen percent zygospore production in controls was increased to over 50% in cultures grown in NaCl and Mg[Cl.sub.2] This demonstrated a dramatic shift in the life cycle of C. hypnosporum. Previous studies by Setter and Greenway (1983 ) showed evidence of osmotic regulation control in Chiorella emersonii using two endogenous osmotic solute concentrations of proline and sucrose which resulted in large changes in cell life cycle.

Both NaCl and Mg[Cl.sub.2] caused a decrease in oxygen evolution in C. hypnosporum. A significant decrease in oxygen was observed in all experimental flasks except 1 ppt Mg[Cl.sub.2] when compared to controls. The greatest reduction in oxygen production was in medium containing 10 ppt NaCl. In studies with Chiamydomonas eugametos and Chiamydomonas moewusii, Lewin (1956) found that production and maintenance of gametes in the light required the presence of oxygen, and that gametic activity is lost more rapidly in the dark under anaerobic conditions than in the presence of oxygen. Their findings could explain why we observed that as oxygen evolution decreased, zygo spore production increased in NaCl. This inverse relationship between oxygen evolution and zygospore production was not distinctly demonstrated in cultures grown at various levels of Mg[Cl.sub.2]. Stifer (1959) also suggested that the presence of oxygen affected mating in algal cultures.

After 21 days of growth, growth medium of C. hypnosporum increased from an initial pH of 6.80 to as high as 8.52 at 0.1 ppt NaCl compared to 8.11 in controls. There was no significant difference in medium p11 when algal cells were grown in various concentrations of Mg[Cl.sub.2]. Since FW1 medium contains NaH[CO.sub.3] this carbonate salt and the production of [CO.sup.2] by C. hypnosporum and the resulting [CO.sub.2] equilibrium probably caused the pH changes. Coleman (1962) suggested that within the bounds of physiological neutrality there were no striking effects of pH in the mating reactions of algal gametes. Mating has been reported in cultures ranging in pH from 4 to 8.5. In this experiment, medium pH as high as 8.52 had no appreciable effect on zygospore production in cultures containing NaCl or [MgCl.sup.2]. There was no correlation between medium pH and zygospore production in cells grown in NaCl or [MgCl.sub.2]. Biebi (1962) suggested that an increased zygote yield correlated with a slight rise in pH value of the medium, but the effect was rarely as much as twofold. According to Gerloff et al. (1952) growth and survival of many algae seemed to be endangered only at pH values exceeding 10.

Microalgae naturally accumulate a variety of intracellular organic compounds. The halotolerant alga Dunaliella baradawii grown in NaCl medium contained glycerol, proteins, lipids, carbohydrates, carotene, and chlorophyll (Ben-Amotz and Avron, 1982). Avron (1986) found that Dunaliella osmo-regulates by producing glycerol. NMR spectra peak measurements of C. hypnosporum indicated that it increased levels of [[blank].sup.13]C-labeled organic solutes when grown in medium containing increasing concentrations of NaCl or [MgCl.sub.2]. Ben-Amotz and Avron (1982) suggested that an alga producing massive amounts of useful organic compounds by osmoregulation make it a natural organism for cultivation. These enhanced algal cells thus provided valuable products and a high protein-containing feed material (Ben-Amotz and Avron, 1983).

NMR spectra peak measurements increased from 32 mm in controls to 108 mm in cells grown in [MgCl.sub.2]. Highest NMR spectra peak measurements was observed in 0.001 ppt NaCl. Based on these increases, C. hypnosporum is potentially a useful alga when employed as a biofertilizer. The presence of introduced [[blank].sup.13]C in all but one of our experimental flasks at levels much higher than the controls suggests that C. hypnosporum may be osmoregulating using carbon-containing compounds such as glycerol. However, further analyses must be done to determine if glycerol or other organic compounds were produced by C. hypnosporum in high salinities. Adverse changes in other parameters investigated also indicate that high concentrations of NaCl and [MgCl.sub.2] induced stress in C. hypnosporum resulting in osmoregulation. If C. hypnosporum is found to contain high quantities of nitrogenous compounds, it will be an excellent alternative to conventional fertilizers. If this alga could be grown economically and suppli ed to crops, eutrophication of lakes and streams could be drastically reduced and potential leachates from synthetic fertilizers would be virtually eliminated.

ACKNOWLEDGMENTS

We gratefully acknowledge the financial support of the Environmental Science Ph.D. Program in the School of Science and Technology at Jackson State University and the Department of Biology.

(1.)Author for correspondence

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