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Tsinghua Science and Technology
Tsinghua University, China
ISSN: 1007-0212
Vol. 6, Num. 3, 2001, pp. 231-238

Tsinghua Science and Technology, Vol. 6, No. 3, August 2001 pp. 231-238

Alternative Respiration Induced by Glucose Stimulation and Variation of Adenylate Energy Charge in Glucose-Starved Cells of Green Alga Chlorella Protothecoides*

WU Qingyu , Neil Grant†

Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China;
†Department of Biology, William Paterson University, Wayne, NJ 07470, USA

Supported by the National Natural Science Foundation of China (No. 39870064 and 30070065) and partly by the State Key Basic Research and Development Programs of China (No.G1998010100 and G19990433)

Received: 2000-08-31

Code Number: ts01072

Abstract:   

Effects of inhibitors and glucose on cytochrome and alternative respiration and on adenylate energy charge (AEC) in glucose-starved Chlorella protothecoides were investigated. 1 mmol/L azide (NaN3 ), which immediately caused an increase of O2 uptake by inhibiting the cytochrome pathway and stimulating alternative respiration, resulted in a decrease of AEC value from 0.83 to 0.34 within 3 minutes. When 1 mmol/L salicylhydroxamic acid (SHAM) was added into the cell suspension, there was no apparent variation in AEC. Adding NaN3 and SHAM together into cell suspension to inhibit both cytochrome and alternative pathways showed a same change of AEC as that of adding NaN3 alone.  When 2.0 mmol/L of glucose was added to a suspension of glucose-starved cells, the O2 uptake rate was immediately stimulated from 0.81 up to 1.34 ] mmol/L O2 ·min-1  ·(mL PCV)-1 ] . The respiration stimulated by glucose could be inhibited about 20% by adding 1 mmol/L SHAM. It was found by titration with SHAM in the absence and presence of NaN3 that 53% of O2 uptake went through the cytochrome pathway and 45% of the alternate pathway was operational in enhanced respiration. It implied that induced operation of the alternative respiratory pathway probably resulted from the burst of the electron flux into the electron transport chain by glucose stimulation.  

Key words:  alternative respiration; adenylate energy charge; glucose; Chlorella protothecoides

Introduction   

Cyanide-resistant or alternative respiration has been reported to be widespread in higher plants, phytoplanktons and microorganisms[1-3]. The studies of the  inhibitor-resistant respiration have contributed much to the understanding of the organization and control of complex intracellular respiratory systems and biochemical processes. Grant and Hommersand[4, 5]  examined the inhibitor-resistant respiratory characteristics of the green alga Chlorella Protothecoides grown in high concentrations of glucose to minimize photosynthesis (glucose bleaching process). They found that both the cytochrome pathway and the alternative pathway might exist in C. protochecoides, however, the latter might only be operative when the cytochrome pathway is inhibited by cyanide, NaN3, CO and antimycin. The chemicals and environmental factors including respiratory inhibitors, nutrient deficiency, stress stimuli-cold, excess copper, photofrin, alpha-keto acids, oxidative stress, and  pathogen attack were investigated and suggested to be in relation to the activity of alternative oxidase[6-10].

Respiration and energy metabolism related to the adenine nucleotides (ATP, ADT, and AMP) are closely linked processes, which cytochemistrically couple the metabolic sequences of a living cell. The amount of metabolically available energy that is momentarily stored in the adenylate system is linearly related to the mole fraction of ATP plus half the mole fraction of ADP. This parameter has been termed the energy charge of the adenylate pool[11]. The adenylate energy charge (AEC) has been suggested as a useful index to estimate the physiological state of organisms[12]. Adenylate nucleotides in fungi were also observed on cyanide-resistant and salicyl-hydroxamate-sensitive decylubiquinol oxidase activity and were suggested to control the alternative oxidase[13].

Although the effects of glucose, an exogenous substrate, on respiration as well as the relationship between the respiration rate and adenylate pools have been investigated[14] , little is known about the effects of glucose enrichment on alternative pathway and their relative variation of AEC in glucose-starvation cells of C. protothecoides. The present paper reports the operation of alternative respiratory pathway resulting from glucose stimulation in C. Protothecoides and the variation of AEC in the cells when their one or two respiratory pathway(s) are operative or inhibited.

1 Materials and Methods   

1.1 Organism and its culture conditions

A culture of Chlorella Protothecoides Kruger UTEX strain #25 was obtained from the Collection of Algae at the Department of Botany, University of Texas at Austin. The algal cells were maintained in Petri dishes containing peptone agar and exposed to room light and temperature. They were inoculated into 300-mL Erlenmeyer flasks containing 200 mL of glucose-bleaching medium then shaken in an air bath incubator at  23 °C  for 10 days.

The glucose-bleaching medium contained per liter: KH2PO4, 0.7 g; K2HPO4, 0.3 g; MGSO4 ·7H2O, 0.3 g; FeSO4 ·7H2O, 3 mg; thiamine hydrochloride, 10 mg; glucose, 10 g; glycine, 0.1 g; Arnon's A solution, 1 mL.  Arnon's A solution contained per liter: H3BO3, 2.9 g; MnCl2 ·4H2O, 1.8 g; ZnSO4 ·7H2O, 0.22 g; CuSO4 ·5H2O, 0.08 g; MoO3, 0.018 g. The medium was autoclaved for 20 min at 15 psi.

During the culture, the cell growth was monitored by counting the cell number in a hemacytometer under optical microscope. The mean of twice counts was recorded for plotting the cell growth curve.

1.2 Determination of glucose concentration

The glucose depletion by C. Protothecoides was monitored by measurements of glucose concentration left in the medium. One millilitre of suspension from the culture was filtered through a 0.45 mm millipore membrane to remove algal cells. A single reagent system of enzymatic assay for glucose measurement  was used which contained: ATP, 1 mmol/L; NADP, 0.5 mmol/L; Mg2+ , 2 mmol/L; hexokinase (yeast) 800 U/L, G6PD (glucose-6-phosphate dehyrogenase, yeast) 500 U/L. The measurement was based upon the conversion of glucose to glucose-6-phosphate (G-6-P) by ATP in the presence of hexokinase, which coupled with the subsequent reduction of NADP to NADPH. As NADPH has a high absorption at 340 nm and NADP has no absorption at this wavelength, the reaction can be followed by measuring the increase in A340 . The increase in A 340  due to the formation of NADPH is directly proportional to the amount of glucose present: 

A Gilford Response UV-VIS spectrophotometer was used to measure the absorption of the sample at 340 nm.

1.3 Determination of oxygen uptake rate

Whole cells O2 uptake rate was determined in dark by using a modification of the technique of Estubrook[15]  with a Clark-type O2 electrode and YSI Model 53 Oxygen Monitor. The semi-closed vessel had a capacity of  1.80 mL  and was thermostat at  23 °C . At an early exponential growth phase, the cells of C. protothecoides were harvested by centrifugation at 4000 r/min for 5 min at  4 °C . They were washed once with buffer A and re-centrifuged. The cells were resuspended in a  4-mL  buffer A and packed cell volume (PCV) was measured by hematocrit centrifuge. The buffer A contained KH2PO4,  0.7 g ; 2HPO4,  0.3 g ; MGSO4 ·7H2O, 0.3 g (pH 7.2). Without glucose in the buffer, the algal cells were in a state of glucose starvation. Then 0.2 mL suspension was added into the 1.8 mL of buffer A in the vessel of oxygen monitor. O2 uptake rates were measured from the slope of the recorder trace of O2 tension with respect to time and expressed as mmol/L O2 ] ] ·h-1  ·(mL PCV)-1 . After a control rate (without adding any chemicals) had been established, glucose or inhibitor(s) including azide (NaN3, inhibitor of cytochrome oxidase) or (and) salicylhydroxamic acid (SHAM, inhibitor of alternative oxidase) was added to establish a new linear rate.

1.4 Extraction of adenine nucleotides

A 10-mL cell suspension was spun down and resuspended in 10 mL buffer A in a YSI water bath holder with continual electrode stirring at  23 °C . At this time the algal cells were in the state of glucose starvation. After taking  0.2 mL  of the suspension out to serve as the control group, the inhibitor(s) NaN3, SHAM, NaN3+SHAM and glucose were added into the suspension in the holder. Then starting immediately,  0.2 mL  of the cell suspension was taken out of the holder every minute. These cell suspensions (0.2 mL) were put into a tube with a 5-mL hot tris (10 mmol/L, pH 7.6) on a boiling water bath to extract adenine nucleotides (ATP, ADP, and AMP). After incubation in the bath for 3 min the tubes with the cell extraction were moved out and kept in an ice bath.

1.5 Determination of adenylates

The concentration of each adenylate including ATP, ADP, and AMP was analyzed before and after conversion of ADP and AMP to ATP by enzymatic methods[16]  according to the following reactions:

where MK is   myokinase, PK is pyruvate kinase, PEP is   phosphoenolpyruvate.For ATP determination, 400 mL of the cell extract was added to 100 mL buffer B solution which contained 75 mmol/L potassium phosphate and 15 mmol/L MgCl2 (pH 7.3). For ATP plus ADP determination, 400 mL of the cell extract was added to 100 mL of the buffer B with an extra  3.8 mmol/L  phosphoenolpyruvate and 90 units/mL pyruvate kinase. For total adenylate (ATP + ADP + AMP) determination, 400 mL of the cell extract was added to 100 mL of buffer B with an extra  3.8 mmol/L  phosphoenolpyruvate, 90 units/mL pyruvate kinase and 150 units/mL myokinase. The three mixtures above were kept at  22 °C  for 30 min for enough enzymatic reactions. The concentration of ATP, including original and conversed ATP, was determined by the luciferin-luciferase method[17]  using pure ATP, ADP, and AMP as internal and external standards. The concentration of ATP, ADP, and AMP in original cell suspensions was deduced based on data from the above three mixtures. The values of AEC, as defined by Atkinson (1968) were derived from the equation:

2 Results    

2.1 Cell growth, respiratory level and glucose depletion

The cell growth curve of glucose-bleached C. Protothecoides during the period from 0 to 260 h (Fig.1(a)) shows that after about 70 h a pattern of exponential growth was apparent. This exponential phase lasted till about 120 h, then the cells entered the stationary phase. Correspondingly, the glucose concentration in the medium decreased gradually resulting from the glucose consumption for the metabolism and growth. More than 70% of the glucose was depleted after the cells entered the stationary phase (Fig.1(a)), which reflected a high ability of glucose consumption and mixtrophic growth in C. Protothecoides.

The level of O2 uptake in glucose-bleached C. Protothecoides was very high at its early exponential growth phase (Fig.1(b)). It decreased gradually with the increase of the cell numbers and with the decrease of glucose in the medium. Especially, the decrease rate of the respiratory level was faster in the cell exponential phase than in the stationary phase which corresponded to the increase in cell numbers. According to the results in Fig.1, the cells of C. Protothecoides in the state of early exponential growth were used in the following studies unless others mentioned.

2.2 Effects of inhibitors and glucose on oxygen uptake in glucose-starved cells

Effects of NaN3, SHAM and glucose on O2 uptake rates in glucose-starved  cells are shown in Fig.2. When SHAM (1.0 mmol/L final concentration) was added to a suspension of cells, the O2 uptake rate did not decrease (Fig.2(a)). One millimolar per litre NaN3 immediately stimulated the rate of O2 uptake (Fig.1(b)). The total respiration was inhibited only when SHAM and NaN3 existed together (Fig.2(a), 2(b)). This suggests that there were two respiratory pathways to O2 in C. Protothecoides, one the cytochrome pathway and the other the alternative pathway. However, the latter was only operative when the cytochrome pathway was inhibited. Adding SHAM alone into the suspension of actively-growing cells directly from normal cultures (i. e., the cells were not in the state of glucose starvation) did not decrease their O2 uptake rate either, which also showed that the alternative pathway was not operative under general conditions without adding other chemicals.

It is significant that when glucose (2.0 mmol/L, final concentration) was added to a suspension of glucose-starved cells, the O2 uptake rate was immediately stimulated from 0.81 up to 1.34 [O2 ·mmol/L ·min-1  ·(mL PCV)-1 ] . Afterwards the respiration stimulated by glucose was inhibited by about 20% by adding 1 mmol/L SHAM (Fig.2(c)). The respiration stimulated by glucose was not inhibited by adding NaN3 alone but by adding NaN3 with SHAM together (Fig.2(d)). It can be concluded from Fig.1 that the alternative respiratory pathway in glucose-staved C. protothecoides was apparently induced by glucose stimulation.

The contribution of the alternative pathway stimulated by glucose to total respiration in glucose-starved cells was determined by titration with SHAM in the absence and presence of NaN3 according to Bahr and Bonner's method[18]  . The effects of SHAM in the presence ] g(i)] and absence (VT) of NaN3 on respiration stimulated by glucose in starved cells are shown in Fig.3(a).  The rates of O2 uptake after the addition of SHAM were calculated and expressed as QO2 /QO2  (control) (%), the percentage of the O2 uptake rate relative to control (the rate before adding SHAM). This may show better a function of concentration of SHAM and rectify the effect of NaN3 in stimulation of O2 uptake. To figure out the contribution of each oxidase to the total O2 uptake, g(i), a function of the concentration of SHAM in the presence of NaN3 was plotted against VT, a function of the concentration of SHAM (without NaN3 ) based on data in Fig.3(a).  A direct linear relationship between the values of g(i) and VT was established (Fig.3(b)). r, the slope of the line, was 0.45 which represented the fraction of the nominal activity of the alternative pathway. And the intercept of the line at the y axis was 53.0, which represents percentage of the activity of the cytochrome pathway in the control. According to Bahr and Bonner[18] , the results of Fig.3 indicate that 53% of O2 uptake went through the cytochrome pathway and 45% of the capacity of the alternate pathway was operational when the respiration was stimulated by glucose in a glucose-starved cell.

2.3 Oxygen uptake and capacity of alternative pathway in different glucose concentrations and different cell growth stages

Since glucose stimulation was the key factor in the operation of the alternative pathway in glucose-starved C. Protothecoides, the response of O2 uptake as a function of glucose concentration in glucose starved cells was investigated. Figure 4 shows that the O2 uptake stimulated by glucose went up within the increase of glucose concentration from 0.001 to 2 mmol/L and went down above 2 mmol/L. Two millimolar per litre glucose, under the conditions of the experiment, was optimal concentration for promoting the operation of alternative respiration in glucose starved cells. Therefore, this concentration was used in all experiments. The increase of O2 uptake stimulated by glucose and changes in capacity of alternative pathways upon different cell growth stages was also investigated and is illustrated in Fig.5. It suggested that the function of glucose stimulating O2  uptake was very high at the early exponential phase of cell growth and it decreased gradually afterward in the cell culture. Glucose was no longer stimulating O2 uptake of starved cells after 260 h in the culture. Correspondingly, the fraction of the nominal activity of the alternate pathway (value of r) was directly proportional to the level of O2 uptake stimulated by glucose. The more the increase of O2 uptake was stimulated by glucose, the larger the capacity of the alternative pathway engaged.

2.4 Effects of inhibitors and glucose on adenylate pool and energy charge

Effects of NaN3 , SHAM, and NaN3 with SHAM together on the amount of adenylates and value of adenylate energy charge are shown in Fig.6. Samples taken at 1 and 3 min as controls before adding any inhibitor(s) showed a high amount of ATP, low amount of ADP and AMP. The AEC value remained at  0.82  to  0.88 , which indicated a normal state of energy metabolism in cells of glucose-starved C. protothecoides. Little variation appeared in the level of the adenylate pool (total amounts of ATP, ADP, and AMP) before and after adding NaN3 , SHAM or NaN3 with SHAM together (Fig.6). It indicated that the inhibition of the cytochrome pathway and alternative pathway had no effect on the level of the adenylate pool. However, after NaN3 was added into the suspension, the value of AEC decreased immediately from  0.83  to  0.34  within 3 minutes resulting from the decrease of ATP and the increase of AMP in cells (Fig.6(a)). There was no apparent variation in AEC value after adding SHAM (Fig.6(b)). After both NaN3 and SHAM was added together into the cell suspension, the value of AEC decreased immediately from  0.84  to  0.42 within 3 minutes resulting from the decrease of ATP and the increase of AMP in cells (Fig.6(c)), which was the same as adding NaN3 alone. The results from Fig.6 indicated that the inhibition of cytochrome pathway did not change the level total adenylate pool but changed the rate of ATP to AMP. The inhibition of alternative pathways changed neither the level of total adenylate pool nor the AEC.The variations in the adenylate pool and the AEC when adding glucose to the suspension of glucose starved cells are shown in Fig.7. It is interesting that the AEC value decreased from  0.85  to  0.68  within 3 min after adding glucose, then it returned to its original level again in another 2 min. There was no obvious effect on the level of the total adenylate pool when the O2 uptake was stimulated by glucose, which resulted in the operation of an alternative pathway.

3 Discussion   

It was reported that inorganic phosphate (Pi) enrichment of the Pi-limited green alga Selenastrum minutum in the dark caused a 2.5-fold increase in the rate of O2 consumption[19]. However, if it involved an alternative respiration is still unknown. Figure 2 indicated that glucose bleached C. Protothecoides possesses both azide-sensitive and SHAM-sensitive respiratory pathways, but the SHAM-sensitive (alternative) pathway does not operate under general conditions. The alternative respiratory pathway is apparently operational during an O2 uptake burst by adding glucose to glucose-starved cells. Therefore, both glucose starvation and stimulation are necessary for the operation of the alternative pathway. Our other experiments showed that when the cells were not in a state of glucose starvation, O2 uptake couldn't be stimulated by exogenous glucose and accordingly was not SHAM sensitive (data not shown). The questions addressing the mechanism of alternative respiration induced by glucose stimulation are still open.

Theolois and Laties[20]  have found that in the respiration of aged potato slices there was no contribution by the alternative pathway (r=0). Whereas r became 1 when an uncoupler of oxidative phosphorylation was added and later almost doubled the rate of O2 uptake and thus saturated the capacity of electron flux through the cytochrome pathway. It therefore appears that in vivo the intensity of electron flux through the cytochrome pathway is probably the factor that determines the apportioning of electrons between the two pathways. Grant and Hommersand[4, 5]  suggested that stimulation of the O2 uptake by NaN3 in C. protothecoides cells was not due to a stimulation of the alternative oxidase directly. The stimulation was the result of an increase of the electron flux into the electron transport chain, perhaps by releasing constraints on the glycolysis by lowering the level of ATP in the cell. In the present experiments, it was found that the enrichment of glucose almost doubled the rate of O2 uptake in glucose-starved C. protothecoides (Fig.2). According to the former work by Theolois and Laties (1978) and Grant and Hommersand (1974), we suggest that the stimulation of O2 uptake by glucose in C. Protothecoides is probably not due to a stimulation of the alternative oxidase directly. And increase of the electron flux suddenly into the electron transport chain by glucose stimulation is one of key factors for operation of alternative respiration.

The energy charge in living cells tends to stabilize in the range between 0.75 to 0.95 and remains quite constant[21]. Several reports were focused on variation of adenylate energy charge corresponding to cellular nutrition. Riemann and Wium-Anderse[22]  reported that pure cultures of lake algae showed decreased AEC during P limitation but not during N limitation. The exhaustion and starvation of the carbon source leads to a decrease in energy charge in some organisms[21]. In present experiments there was no obvious decline in the value of AEC in C. protothecoides when all glucose was removed from the culture medium. The AEC values remained stabilized in the range of 0.8 to 0.9 in all control samples  (Figs. 6, 7), this perhaps resulted from utilization of endogenous carbohydrate or endogenous respiration, which seems to be one of the characteristics in glucose-bleached C. Protothecoides.

Fader and Koller[12]  investigated the relationship between respiration rates and adenylate and carbohydrate pools of soybean fruit and demonstrated that AEC remained relatively constant in the pod wall, seed coat and cotyledons during changes in respiration rate. And the total adenylate pool in seed coat and cotyledons increased with increasing respiration rates of the fruit. However, the studies of the relationship between respiration rates and adenylate energy, particularly those concerning inhibitors resistant respiration are still limited. In the present study, it has been shown that when NaN3 was used to inhibit the cytochrome pathway, while the respiratory rate increased with the flux of electrons through the alternative pathway (Fig.2), the AEC value dropped immediately (Fig.6). This implied that the AEC value could be used to judge whether the cytochrome pathway is operative in the presence of inhibitors when cellular respiration is still at a high rate. In other words, if AEC is low while the O2 uptake is high, the alternative respiration might be engaged.

It was interesting that AEC values went down immediately after adding glucose in glucose-starved cells and then returned to its original level (Fig.7). A possible explanation is that glucose-induced operation of alternative pathways probably might deplete certain adenylate energy to overcome some obstacle and results in a decrease of AEC at first. However, more studies are needed to confirm or revise these explanations.

References

  1. Eriksen N T, Lewitus A J. Cyanide-resistant respiration in diverse marine-phytoplankton - evidence for the widespread occurrence of the alternative oxidase. Aquatic Microbial Ecology, 1999, 17: 145-152.
  2. Shugaev A G. Alternative cyanide-resistant oxidase in plant-mitochondria - structure, regulation of activity, and presumable physiological-role. Russian Journal of Plant Physiology, 1999, 46: 262-273.
  3. Lodolo E J, Oconnorcox E S C, Axcell B C. Evidence of antimycin-insensitive respiration in a commercial brewing yeast. Journal of the Institute of Brewing, 1999, 105: 35-43.
  4. Grant N G, Hommersand M H. The respiratory chain of Chlorella protothecoides, I. Inhibitor responses and cytochrome components of whole cells. Plant Physiology, 1974, 54: 50-56.
  5. Grant N G, Hommersand M H. The respiratory chain of Chlorella protothecoides,  II. Isolation and characterization of mitochondria. Plant Physiology, 1974, 54: 57-59.
  6. Devars S, Hernandez R, Covian R, et al. The content of alternative oxidase of Euglena mitochondria is increased by growth in the presence of cyanide and is not cytochrome O. Journal of Eukaryotic Microbiology, 1998, 45: 122-130.
  7. Vianello A, Braidot E, Petrussa E, et al. ATP synthesis driven by alpha-keto acid-stimulated alternative oxidase in pea leaf mitochondria. Plant and Cell Physiology, 1997, 38: 1368-1374.
  8. Salet C, Moreno G, Ricchelli F. Effects of photofrin(R) photodynamic-action on mitochondrial respiration and superoxide radical generation. Free Radical Research, 1997, 26: 201-208.
  9. Vanlerberghe G C, Mcintosh L. Alternative oxidase - from gene to function. Annual Review of Plant Physiology and Plant Molecular Biology, 1997, 48: 703-734.
  10. Reyes E, Jennings P H. Effects of chilling on respiration and induction of cyanide-resistant respiration in seedling roots of cucumber. Journal of the American Society for Horticultural Science, 1997, 122: 190-194.
  11. Atkinson D E. The energy charge of adenylate pool as a regulatory parameter; interaction with feedback modifier. Biochemistry, 1968, 7: 4030-4035.
  12. Fader G M, Koller H R. Relationships between respiration rate and adenylate and carbohydrate pools of soybean fruit. Plant Physiology, 1984, 75:  694-699.
  13. Sakajo S, Minagawa N, Yoshimoto A. Effects of nucleotides on cyanide-resistant respiratory activity in mitochondria isolated from antimycin A-treated yeast Hansenula-anomala. Bioscience Biotechnology and Biochemistry, 1997, 61: 396-399.
  14. Kamiya A. Effects of light on growth and glucose consumption in colorless Chlorella-valgaris mutant cells. Plant Cell Physiology, 1985, 26: 151-160.
  15. Estabrook R W. Mitochondria respiratory control and the polar-graphic measurement of ADP: O ratios. Methods in Enzymology, 1967, 10: 41-47.
  16. Jaworek D, Gruber W, Hans U R. Adenosine-5'-diphosphate and adenosine-5'-monophosphate. In: Hans U B (Ed). Methods of Enzymatic Analysis. Verlag Chemie Weinhei Academic Press, Inc., New York, 1974. 2127-2129.
  17. Tillberg J E, Rowley J R. Physiological and structural effects of phosphores starvation on the unicellular green alga Scendesmus. Physiologia Plantarum, 1989, 75: 315-324.
  18. Bahr J T, Bonner W D. Cyanide-insensitive respiration II, control of the alternate pathway. Journal of Biological Chemistry, 1973, 248:  3446-3450.
  19. Gauthier D A, Turpin D H. Inorganic-phosphate (Pi) enhancement of dark respiration in the Pi-limited green-alga Selenastrum minutum- interactions between H+/Pi cotransport, the plasmalemma H+-ATPase, and dark respiratory carbon flow. Plant Physiology, 1994, 104:  629-637.
  20. Theolois A, Laties G G. Relative contribution of cytochrome mediate and cyanide-resistant electron transport in fresh and aged potato slices. Plant Physiology, 1978, 62: 232-237.
  21. Otto R. Energy metabolism in streptococcus-cremoris during lactose starvation. Arch Microbiology, 1985, 141: 348-352.
  22. Riemann B, Wium-Andersen S. Predictive value of adenylate energy charge for metabolic and growth states of plankton communities in lakes. Oikos, 1982, 39: 256-260.

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