PHYSIOLOGICAL AND BIOCHEMICAL STUDIES ON AN ACID-TOLERANT CHLORELLA VULGARIS UNDER COPPER STRESS

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1 J. Gen. Appl. Microbiol., 39, (1993) PHYSIOLOGICAL AND BIOCHEMICAL STUDIES ON AN ACID-TOLERANT CHLORELLA VULGARIS UNDER COPPER STRESS PRAMODA KUMAR RAT, NIRUPAMA MALLICK, AND LAL CHAND RAI* Laboratory of Algal Biology, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi , India (Received March 15, 1993) This study compared the growth, photosynthesis, electron transport, ATP content, N03 - and NH4+ uptake, Na+ and K+ loss, nitrate reductase and ATPase activities of an acid-tolerant strain and a wild-type strain of Chlorella vulgaris exposed to Cu at varied phs (ph 6.8, 5.0, 4.0 and 3.5). A general reduction in growth, NH4+ uptake, photosynthesis and nitrate reductase activity of both the strains was noticed at decreasing ph. In contrast, the acid-tolerant strain depicted an increased N03 - uptake, insignificant Nat, K+ loss and superactive ATPase at decreasing ph. Both ph and Cu produced a non-competitive inhibition of N03- uptake. An increase in Vmax of ATPase of acid-tolerant Chlorella and inhibition of the Vmax of its wild-type strain at decreasing ph was noticed. Though the magnitude of Cu toxicity was significantly low in tolerant strain, a significant negative correlation (r = -0.99, p <0.01) of Cu uptake with acid ph was noticed. The acid-tolerant Chlorella was less sensitive to Cu and physiologically more efficient than the wild type. Presence of a superactive ATPase and change in membrane permeability are considered responsible for acid tolerance in Chlorella. Acidification and its environmental repurcussions are an area of tremendous biological significance. Though acidity has been found inhibitory for phytoplankton biornass and productivity (6,21), reports on algae surviving in such adverse habitats are numerous. Survival of organisms in such habitats is not only highly complex but fascinating to environmental biologists, especially at a time when very little is known about the physiological and biochemical basis of acid tolerance in algae and other organisms. Gimmler et al. (8,9) observed development of positive membrane and zeta * Address reprint requests to: Dr. La! C. Rai, Laboratory of Algal Advanced Study in Botany, Banaras Hindu University, Varanasi , India. Biology, Centre of 529

2 530 RAI, MALLICK, and RA! VOL. 39 potentials, which were involved in minimizing the influx of protons into the cells in an acid-resistant Dunaliella acidophila. This was suggested to be responsible for maintaining a neutral (ph 7.0) cytoplasrnic ph. In addition to zeta arid membrane potentials, the involvement of H+-ATPase-rediated H+ export was considered responsible for regulating the internal ph of the cells. Enarni et al. (5) further reported ATPase mediated H+ extrusion in the acidophilic alga Cyanidium caldarium. Further, it is also known that ph and Cd individually as well as in combination retard growth and induce ultrastructural changes in cyanobacteria and algae (17, 19). It is worth emphasizing that no comparable data are available on the effects of copper on the physiological and biochemical variables of an acid-tolerant and a wild-type strains of algae in general and Chlorella in particular. Hence, this study was undertaken: (i) to explore the physiological and biochemical variables which enable Chlorella to survive in high acidic environment, and, (ii) to investigate the effects of Cu on such tolerant strain. MATERIALS AND METHODS The unicellular green alga Chlorella vulgaris was grown axenically in modified Chu-10 medium (7) at ph 6.8 under 72,arnol photon m-2 s-1 of light intensity at 14 : 10 LD cycle at 24±2 C. An acid-tolerant strain of this alga was isolated by successive subculturing of the wild type in acidic medium (ph 5.0) buffered with 10 mm MES (morpholinoethane sulfonic acid). Both the strains were subcultured twice in a week into the fresh growth medium and only cultures from logarithmic phase were used for further study. Stock solution of CuCl2.2H2O was prepared in double glass-distilled water and sterilized by passing through millipore membrane filters (0.22,um), before supplementing to the culture medium. Exponentially grown Chlorella (both wild-type and acid-tolerant strain) cells were transferred to the medium having different phs (i.e. 6.8, 5.0, 4.0 and 3.5) buffered with l0 mm MES buffer. To study the effect of Cu, 2 rng l-' of CuCl2 2H2O was spiked into the culture media. Toxicity at different ph was evaluated by taking untreated cultures of respective ph as control and a comparison between both the strains was made. Final yield, in terms of chlorophyll a was measured (2) after 15 days of treatment. The uptake of N03- and NH4+ following 24 h of treatment was estimated colorirnetrically by brucinesulfuric acid (15) and Nessler's reagent (10) methods respectively by measuring their depletion from the growth medium. Nitrate reductase activity was estimated, by measuring the formation of N02- from N03- (3) by using the reagents sulfanilamide and a-(1-1)-naphthylethylene diamine dihydrochloride. Mgt+Adependent ATPase activity was measured as per the method of 0hnisi et al. (16). The ATP extracted in 4% TCA, supplemented with 2 mm EDTA, was measured by luciferin-lucif'erase assay using LKB-1250 luminometer (12). Carbon fixation was measured by recording the uptake of 14C from NaH14C03 (sp. activity 10.5>< 105 Bq) in an LS 7000 liquid scintillation counter as described by Rai amid Raizada (18). Photosynthetic

3 1993 Acid-Tolerant Chlorella vulgaris 531 e of 02-evolution of the test alga exposed to Cu was measured with a polarographic oxygen electrode enclosed in a 10 ml air-tight reaction vessel coin.nected to an oxygen analyzer (digital oxygen system, Model-10, Rank Brothers, U.K.). The photosynthetic electron transport activity was measured as per the method of Lien (13) as detailed in Rai et al. (20). Metal uptake by Chlorella cells over a 2 h incubation period was measured by the method of Martin (14) with the help of a Perkin-Elmer-2380 atomic absorption spectrophotometer. The loss of Na+ and K+ from cells exposed to Cu at different ph for 2 h was measured following the method of Rai and Raizada (18). The efflux was calculated by subtracting the Na+ and K+ content of the treated cells from untreated control. To study the kinetics of N03- and NHS+ uptake, dig ml -1 of N03- and NH4+ were added to the culture media. After 30 min of incubation the samples were withdrawn, centrifuged and the depletion of nitrate and ammonium from the medium was measured as per the methods described above. For the study of nitrate reductase kinetics, 2-12 mmol of N03 was added to both the cultures and the NR activity was measured as per the method of Cam.m and Stein (3) after 4 h. Algal cells (both acid tolerant and the wild type) pre-grown for 24 h at different ph were used for studying the kinetics of ATPase. A 2-12 mmol ATP was added to the extracted ATPase and the P043- liberated after 1 h was measured (1). The values Km and Vmax were obtained by plotting the double reciprocal plots. For measuring the lipid, approximately 100 ml cultures of both the strains were harvested by centrifugation (7,000 X g for 10 min). Lipids were extracted from samples using chloroform : methanol (2: 1, v/v) and dried under a flash of nitrogen at 40 C. The total lipid obtained was estimated gravimetrically. The results were verified by Student t-test and correlation coefficient (r). RESULTS Table 1 compares the ph-induced toxicity of Cu on chlorophyll a content of acid-tolerant and wild-type strains of C. vulgaris after 15 days of treatment. As compared to the wild-type Chlorella, where a complete inhibition of growth was noticed at ph 4.0 and 3.5, the acid-tolerant strain showed only 39 and 62% reduction in growth as compared to ph 5.0 respectively at the above ph values. Compared to ph 6.8 the growth reduction was, however, 72 and 11%, respectively for wild-type and tolerant strain at ph 5.0. Likewise, 62% inhibition of growth was observed at LCSO concentration of Cu in the wild-type strain at ph 6.8. But in acid-tolerant strain this reduction was only 47%. At ph 5.0, 4.0 and 3.5 these reduction percentages were respectively 71, 75 and 100 as compared to complete death of wild type at above phs. Compared to ph 5.0, 11 and 28% decreases in NH4+ uptake were noticed respectively at ph 4.0 and 3.5 in acid-tolerant strain. In wild type, however, this reduction was about 32 and 39% in the above order compared to ph 6.8 (Table 1). Both the strains depicted higher NH4+ uptake at ph 6.8 in comparison to ph 5.0.

4 532 RAI, MALLICK, and RA! VOL. 39 Table 1. ph-altered growth and nutrient uptake of a wild-type and an acid-tolerant strain of C. vulgaris : Interaction with Cu. Supplementation of 2mgl-' Cu caused about 29, 61, 81 and 85% inhibition, respectively at ph 6.8, 5.0, 4.0 and 3.5 in wild-type strain. But for tolerant strain the inhibition was 26, 56, 61 and 63% in the above order. In contrast to NH4+ uptake, the N03- uptake of tolerant strain registered a 2-fold increase at ph 3.5 as compared to ph 5.0. However, the wild type showed a declining trend in N03 - uptake with decreasing ph (Table 1). Cu supplementation inhibited N03- uptake by 35, 69, 75 and 86% respectively at ph 6.8, 5.0, 4.0 and 3.5 in wild-type Chlorella; in tolerant strain, the reductions were about 32, 42, 46 and 50% at these phs. Kinetics of N03- uptake as affected both by ph and Cu are shown in Fig. 1. A constant Km and change in Vmax of N03 - with decreasing ph clearly demonstrated that ph effects were of non-competitive type. Likewise the inhibition produced by Cu was of non-competitive type. In contrast to this an unchanged Vmax and a variable Km of NH4+ uptake of both strains in presence of Cu demonstrated a competitive type of inhibition (Fig. 2). In spite of these general trends, the tolerant strain showed an increased Vmax (26%) and a low (37%) Km for N03- uptake at ph 6.8 compared with the wild-type strain. The Vmax of tolerant strain was found to increase with decreasing ph. A reverse situation was, however, observed in case of wild-type Chlorella. Table 2 summarizes the interactive effect of Cu with ph on photosynthetic carbon fixation, 02 evolution and ATP content of wild-type and tolerant strains of C. vulgaris. A general decrease in the rate of carbon fixation and 02 evolution was noticed at acid ph. But the percent reduction in tolerant strain was significantly lower than the wild type. The electron transport system was also inhibited in a

5 1993 Acid-Tolerant Chlorella vulgaris 533 Fig. 1. Interactive effect of acid ph and Cu on the kinetics of N03- wild-type (A) and tolerant strain (B) of C. vulgaris. Control, ; Cu, 0. uptake of similar way (data not shown). The pattern of inhibition ATP pool was similar to other parameters. In the case of wild type the reduction was 53, 60 and 63% respectively at ph 5.0, 4.0 and 3.5. These percentages were, however, 35, 41 and 43 for tolerant strain in the above order. Cu inhibited the ATP content by 32, 75, 82 and 94% at ph 6.8, 5.0, 4.0 and 3.5, respectively in wild strain and 28, 40, 44 and 47% in tolerant strain in the above order. Effects of acid ph and Cu on activity of ATPase are given in Fig. 3. The ATPase of wild-type Chlorella showed a gradual decrease with decreasing ph, though the magnitude of inhibition was quite low (about 12, 12 and 20% decrease respectively at ph 5.0, 4.0 and 3.5). Interestingly, compared to ph 5.0, a 3-fold higher ATPase activity of tolerant strain was noticed at ph 3.5. Cu inhibited the ATPase activity by 35, 40, 45 and 63% respectively at ph 6.8, 5.0, 4.0 and 3.5 in wild type. But in tolerant strain the inhibition percentage was 26, 32, 35 and 40 in the above order. It is also interesting that nitrate reductase was found highly sensitive to acid ph. A complete inhibition of its activity was noticed at ph 4.0 for wild type and at ph 3.5 for the tolerant strain (Table 4). The kinetics of nitrate reductase (Fig. 4) and ATPase (Fig. 5) of both the strains showed a variation in Vmax and constant Km at varying ph and Cu

6 534 RAI, MALLICK, and RAI Vo~. 39 Fig. Z. Kinetics of NH4+ uptake as influenced by wild-type (A) and arid-tolerant strain (B) of C, vulgaris. Control, 0; Cu, 0. copper and acid ph in Fig. 3. ph-induced strain of C. vulgaris. effects of Cu on ATPase activity of wild-type and tolerant

7 1993 Acid-Tolerant ChIorella vulgaris 535 Table 2. Effect of Cu on carbon fixation, 02 evolution and ATP content of wild-type and acid-tolerant strains of C. vulgaris at different ph values. Table 3. ph-induced effect of Cu on Na+ and K+ efflux and Cu uptake by wild-type and tolerant strains of C. vulgaris. concentration. As compared to wild type an increase in Km and Vmax values of NR of tolerant strain was, however, noticed. Though Km and Vmax of ATPase were found to be the same in both the strains at ph 6.8, the tolerant strain depicted an increase in Vmax at acidic ph. A significant difference in Na+ and K+ loss was noticed in wild-type and

8 536 RA!, MALLICK, and RA! VOL. 39 Table 4. Copper-induced inhibition of nitrate reductase activity of C. vulgaris at different ph values after 24 h. Fig. 4. ph-altered effect of copper on the kinetics of nitrate wild-type (A) and acid-tolerant strain (B) of C. vulgarisa Control, ; Cu, 0. reductase of

9 1993 Acid-Tolerant Chlorella vulgaris 537 Fig. 5. Inhibition of ATPase activity by Cu in wild-type (A) and tolerant strain (B) of C. vulgaris at different ph. Control, ; Cu, 0. tolerant strain with decreasing ph (Table 3). In wild type the loss of Na+ was 21, 24 and 31% respectively at ph 5.0, 4.0 and 3.5. These percentages were 4, 6 and 7 in tolerant strain. After Cu supplementation the Na+ loss increased to 42, 68, 72 and 89% respectively at ph 6.8, 5.0, 4.0 and 3.5 in wild-type strain, while in tolerant strain the loss of Na+ was 37, 46, 51 and 57%. K' loss was, however, 20, 25 and 29% for wild type at ph 5.0, 4.0 and 3.0. In tolerant strain, however, the ph-induced K _t loss was not so significant. Cu supplementation, however, increased the loss up to 28, 52, 59 and 60% and 18, 35, 40 and 43%, respectively in wild-type and tolerant strain at ph 6.8, 5.0, 4.0 and 3.5. Table 3 contains data on Cu uptake by both the strains. Compared to ph 6.8, the wild type registered a 2, 4.8 and 5-fold increase in Cu uptake respectively at ph 5.0, 4.0 and 3.5. Though a similar trend prevailed for tolerant strain, the magnitude of Cu uptake was significantly low. Approximately 67, 25, 48 and 40% lower Cu uptake was noticed respectively at ph 6.8, 5.0, 4.0 and 3.5 in tolerant strain. Likewise the lipid content (on % dry weight basis) of the acid-tolerant Chlorella (15.2 ±0.05) was approximately 60% higher than the wild type (9.5 ±0.03).

10 538 RAI, MALLICK, and RAI VOL. 39 DISCUSSION A significant reduction in growth, NH4+ uptake, photosynthesis, Nat, K+ efflux and ATP content at decreasing ph (see Tables 1-3) offered testimony to our contention that ph causes disastrous impact on the physiological and biochemical behavior of living organisms including algae. The failure of wild-type Chlorella to survive at ph 4.0 and 3.5 might be due to its inability to regulate internal ph (5), when exposed to acid stress. In contrast to wild type, the acid-tolerant Chlorella was found to be physiologically more efficient to regulate the internal ph. Enami et al. (5) suggested that regulation of intracellular ph in an acid-tolerant C. caldarium was brought about by H+-ATPase-mediated extrusion of H + ions. Gimmler et al. (9) also reported the presence of an extremely efficient ATPase system in acid-tolerant D. acidophila, which exports H+ from the cells and regulates the cytoplasmic ph. Thus the presence of a superactive ATPase as found in our study (Fig. 2) not only finds support from these studies but seems to be responsible for acid tolerance and survival of Chlorella in acidic environment. The development and functioning of this superactive ATPase was further confirmed from our kinetic data; when we did not find any change in Vmax and Km values of wild-type and tolerant strain at ph 6.8 (Fig. 5). However, when ph was changed to 5 and 3.5, an increase in Vmax and an unaltered Km of acid-tolerant strain was observed. This increased reaction velocity demonstrated the presence of a superactive ATPase. In the case of wild-type Chlorella however, a decrease in Vmax with increasing acidity was noticed. It is therefore confirmed that the ATPase of acid-tolerant Chlorella was stimulated by acid phs. Besides, another physiologically interesting event was the observation of a significantly higher N03- uptake in tolerant strain. The enzyme nitrate reductase of this strain was almost inhibited at low ph (only 9% activity at ph 4.0 and complete inhibition at ph 3.5). This observation is supported by earlier studies (11) where acid-tolerant higher plants were found to contain a very low NR activity. Gimmler et al. (8) demonstrated the development of positive membrane and zeta potentials in the acidophilic D, acidophila. These were implicated in regulating the internal ph either through repelling of cations or enhanced uptake of anions. Hence, an accelerated uptake of N03- in absence of its assimilatory enzyme NR points towards the accumulation of anions inside the cell, which in turn may help in regulating the cytoplasmic ph of the cell. A higher uptake of anions (C032-, N03-) by the acid-tolerant Chlorella, could also be due to a positive membrane potential as found in case of D. acidophila (9), which facilitates increased uptake of anions for bringing the internal ph to neutrality (4). If this is true, it will be necessary for the cells to minimize the uptake of cations. This point has been clearly demonstrated by reduced uptake of NH4t and Cu2+ at decreasing ph. This reduced uptake could be due to repelling of cations by positive zeta and membrane potentials of the acid-tolerant strains (9). Hence a low Cu toxicity in

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