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Influence of different concentrations of sodium bicarbonate on growth rate and chlorophyll content of Chlorella salina

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  74  Journal of the Marine Biological Association of India (2008)  Reeta Jayasankar and K.K. Valsala    J. Mar. Biol. Ass. India, 50  (1) : 74 - 78, January - June 2008 Introduction Inorganic carbon (C i ) dissolved in seawater ismostly composed of high concentration of bicarbonate ion and low concentration of carbondioxide (Israel and Gonzalez, 1996). Themicroalgae utilize bicarbonate as the external sourceof carbon for photosynthesis (Dixon et al. , 1987;Munoz and Morrett, 1989; Beer, 1994). Few algaeare capable of uptake of carbon dioxide directly(Badger, 1985; Raven, 1991) while others convertbicarbonate to carbon dioxide either inside theplasmalemma (Dixon et al. , 1987) or externallyallowing only bicarbonate to diffuse into the cell(Badges et al.,  1980). Photosynthesis in microalgaein a carbon limiting environment displayscharacteristics like C 4  type plants with much higheraffinity to CO 2  but unlike CO 2  enrichment in C 4 plant, the microalgae operate by accumulatinginorganic carbon intracellularly and the uptake isdriven by energy coupled C i  transport system(Yingjun and Martin 2006). Enzyme carbonicanhydrase is associated with the process of reversible hydration of carbon dioxide helping toincrease the efficiency of photosynthesis inmicroalgae (Suzuki et al ., 1994). Chlorella salina  is a single celled green algawith rich source of energy as the photosyntheticefficiency theoretically reaches 8% which iscomparable with the higher crops like sugarcane.It is also an attractive food source of many marinelarvae due to its nutritional package of unsaturatedfatty acids, carbohydrate, minerals and otheressential nutrient content and has great demand inaquaculture industry as live feed. Influence of different concentrations of sodium bicarbonate on growth rateand chlorophyll content of Chlorella salina *Reeta Jayasankar and K. K.Valsala Central Marine Fisheries Research Institute, Cochin-682 018, India.*E-mail: reetajayasankar@yahoo.com Abstract Chlorella salina  was grown in enriched medium supplemented with different concentrations of sodiumbicarbonate to study the influence of inorganic carbon (C i ) on its growth and chlorophyll content. Lineargrowth was observed on the treatment with 0.004 and 0.005 M sodium bicarbonate whereas exponentialgrowth was observed in control and 0.001 to 0.003 M bicarbonate treatment. The growth rate wasmaximum in 0.005 M concentration with a 1/ln value of 0 .270 + 0.01 on 15 days of culture. Increase ingrowth was observed with the increase of concentration of sodium bicarbonate from 0.001 to 0.005 M.Growth rate in 0.005 M concentration was 70% more than that of the control. There was steady decline of photosynthetic pigments (chl a  & chl b ) in both control and treatment in the initial period of growth. Thedecline was up to 4 th  day in the treatments from 0.002 to 0.005 M concentration whereas it was up to 7 th  dayin control and 0.001 M treatment. Chl b  was found to be steady from 7 th  to 15 th  days of treatment.Maximum chl a  was observed in 0.005 M treatment (7.9  µ g/ml) on 15 th  day and minimum during 4 th  dayof culture (0.6  µ g/ml). Chl a  to chl b  ratio showed increase in all the treatments until 13 days of cultureperiod. In control and lower concentration of C i  treatment, the ratio increased to more than 2 after 9 th  day,whereas similar observation was found on 4 th  day of culture in 0.004M and 0.005 M treatments. This resultleads to the conclusion that there is a need to augment inorganic carbon along with the enriched mediumduring the initial period of culture of Chlorella salina  to improve the specific growth rate. Key words: Chlorella salina,  chlorophyll content, sodium bicarbonate, growth rate, inorganic carbon  75  Journal of the Marine Biological Association of India (2008) Sodium bicarbonate on growth rate of Chlorella Material and methods Approximately 100 ml of Chlorella  wasconcentrated by centrifugation and then diluted to4 ml with sterilized seawater. Five hundredmicrolitre of the samples were added to each flask containing 300 ml of seawater enriched withWalne’s medium. Sodium bicarbonate ranging from0.001 to 0.005 M concentration was added todifferent flasks. Duplicate was maintained in eachtreatment. Enriched medium without sodiumbicarbonate was considered as control. Afterinoculation, the flasks were kept under controlledenvironmental conditions having light intensity of 1000 lux, temperature of 24-25 0 C and photoperiodof 16:8 h light and dark cycle. The growth rate wasexpressed in percentage of transmittance bymeasuring the turbidity at three differentwavelength of light in blue, green and red regionat 430, 540 and 678 nm respectively. The growthcurve was drawn by taking the average value of allthe transmittance and converting them to negativelogarithmic value. Photosynthetic pigment wasextracted after centrifuging 5 ml of the Chlorella from the enriched medium and kept overnight in90% acetone. The solution was vortexed and againcentrifuged at 10,000 rpm in Hitachi refrigeratedcentrifuge at 4 0 C. Optical density of the supernatantwas measured at 630, 645 and 663 nm. Estimationsof Chl a  and Chl b  were carried out by followingstandard procedure (Jeffrey and Humphrey, 1975).Two replications were taken for each treatment on2 nd , 4 th , 7 th , 9 th , 13 th  and 15 th  days of culture. Datawere analyzed by Analysis of Variance usingSYSTAT 7.0 and the results were interpretedstatistically. Results There was a steady increase in the growth of  Chlorella  from 2 nd  to 15 th  day of culture in bothcontrol and treatment as observed in the turbiditystudy expressed in the negative logarithmic of therate of transmittance (Fig.1). The growth was foundto be the lowest in the control and was higher withhigh carbon concentration. There was no markeddifference in the growth in all the treatments up to4 days of inoculation. From the 7 th  day onwards,the growth showed linear progress in the treatmentsupplemented with high concentration of C i . At0.005 M enrichment a linear progress of growthwas observed rather than a sigmoid curve. In controland treatments from 0.001 to 0.003 Mconcentration, the steady state growth occurred on9 th  day of culture whereas under the treatment of 0.004 and 0.005 M the growth increased until15 th  day of culture. There was significant variationin growth (p<0.01) between treatment and thecontrol with a maximum negative logarithmicvalue of transmittance of 0.270 at 0.005 M andminimum of 0.218 in controlled condition (Fig. 1).It was observed that both chl a  and chl b declined immediately after the inoculation andpersisted until 7 th  day of culture period in thecontrol and in 0.001 M sodium bicarbonatewhereas in other treatments the decline was onlyuntil 4 th  day of inoculation. Subsequently itincreased. Sharp increase of chl a  was found in thetreatment with 0.005 M followed by 0.004 Msodium bicarbonate till 15 days of culture periodwhereas a steady state was observed in othertreatments and also in the control after 9 th  day of culture. Significant variation was observed amongthe treatments and in control with a maximum chl a  content in treatment with 0.005 M concentrationon 15 th  day of culture (7.9  µ g/ml) and minimum incontrol on 4 th  day of culture (Fig. 2). On 2 nd  dayafter inoculation, the maximum chlorophyll contentwas found in the control followed by treatmentwith lower concentration (0.001 M), which reducedin higher concentration of bicarbonate and was Fig. 1.Growth rate of Chlorella salina  treated withdifferent concentrations of sodium bicarbonate 0.270.260.250.240.230.220.21    1   /  n  a   t  u  r  a   l   l  o  g  o   f   t  r  a  n  s  m   i   t   t  a  n  c  e Control 0.001 0.002 0.003 0.004 0.005   Control 0.001 0.002 0.003 0.004 0.005 247913 15Day of Culture  76  Journal of the Marine Biological Association of India (2008)  Reeta Jayasankar and K.K. Valsala lowest in 0.005 M concentration. Thus the lowestchl a  content was observed in the 0.005 Mtreatment on 4 th  day after inoculation. But in thecontrol the decline persisted for 7 days. There wasnot much significant variation in chl a  contentamong the treatments but the variation wassignificant in different days of culture period(p<0.01).Chl b  also showed a similar trend with aninitial decline until 4 th  and 7 th  day but there wasnot much difference in the subsequent days. Amongthe treatments the chl b  was higher in higherconcentration (0.005 M). Significant variation wasobserved in chl b  among the treatments and control(Fig. 3) and between different days of culture period(p<0.01).a decline on 7 th  day. From 9 th to 13 th  day it increasedmarginally and then declined on 15 th  day. It wasobserved that the ratio of chl a  /chl b  showed adeclining trend in all the treatments on 15 th  day(Fig. 4). In all the other treatments excluding thecontrol, the ratio increased until 15 th  day of culturewith a maximum value on 13 th  day. Significantdifference was observed between the treatmentsand between the days of culture (p<0.01). Fig. 2.Chlorophyll a  content of Chlorella   salina  treatedwith different concentrations of sodium bicarbonate The Chl a/b ratio sharply increased in thecontrol and low concentration of sodiumbicarbonate (0.001 M) from 7 th  to 13 th  day of inoculation, but declined subsequently. In thetreatment with 0.004 and 0.005 M concentration,the ratio sharply increased on 4 th  day followed by Fig. 4.Chl a  /chl b  ratio of Chlorella salina  subjected todifferent treatment of sodium bicarbonate While comparing the growth with control, itwas observed that there was 5 to 70% increase ingrowth in all the treatments with the highest valuein 0.005 M concentration of sodium bicarbonatein 15 days of culture period. Discussion Growth rate of microalgal population is ameasure of increase in biomass over time and isdetermined from the exponential phase. Theduration of the exponential phase depends uponthe size of the inoculums, their growth rate, culturecondition and the enriched medium where it grows.The growth of microalgae not only depends on thetemperature, light and nutrient availability, but alsohas a direct impact on the available carbon in theculture medium. The carbon to chlorophyll ratio isa sensitive indicator of the physiological state of microalgae (Geider, 1987). In the presentexperiment on growth, the pigment constituentswere taken as the indicator of the physiology of microalgae influenced by inorganic carbon. It isreported that at low cell density, the addition of inorganic carbon will reduce the pH therebyreducing the growth in the initial phase of inoculation (Geider, 1987). Similar observation wasmade in the present experiment as the cell density Fig. 3.Chlorophyll b  content of Chlorella salina  treatedwith different concentrations of sodium bicarbonate 2 4 7 9 13 15Day of culture Cont 0.001 0.002 0.003 0.004 0.005 9876543210    C   h   l  o  r  o  p   h  y   l   l  a  c  o  n   t  e  n   t   (     µ   g   /  m   l   )   Control 0.001 0.002 0.003 0.004 0.005 543210    C   h   l  a   /  c   h   l   b  r  a   t   i  o 247913 15   Cont 0.001 0.002 0.003 0.004 0.005 Day of culture   Control 0.001 0.002 0.003 0.004 0.005 247913 15Day of culture Cont 0.001 0.002 0.0030.004 0.005   Control 0.001 0.002 0.0030.004 0.005 14121086420    C   h   l  o  r  o  p   h  y   l   l   b  c  o  n   t  e  n   t   (      µ   g   /  m   l   )  77  Journal of the Marine Biological Association of India (2008) Sodium bicarbonate on growth rate of Chlorella was very low (88 x 10 4  cells) when diluted to 300ml of culture medium with a concentration of lessthan 1500 cells/ml. This could have influenced achange in the pH and lowered the pigmentconcentration in higher C i  enrichment in the initialperiod.Pesheva et al. (1994) observed that the growthof the marine microalga, Chlorococcum littorale suppressed for the first 3-4 days after inoculationwith medium supplemented with 40% carbondioxide. However logarithmic growth was observedafter that period. According to them, the growthrate was influenced by the suppression of PSIIactivity whereas the PSI activity increased (Iwasakiand Miyachi, 1996). Increase of PSI/PSII ratio willsupport the energy supply for ATP synthesisthrough cyclic photophosphorylation. Pronina et al . (1993) also observed photosynthesis byincreasing the concentration of carbon dioxide withintracellular acidification. Similar observation wasmade in the present experiment. Increase in biomasswas due to linear growth than exponential growthwith respect to time and is directly proportional tothe input of carbon dioxide as observed in thetreatment with 0.004 and 0.005 M carbon dioxide.Carbon to chlorophyll ratio is also a sensitiveindicator of the physiological state of microalgae(Geider, 1987). With constant supply of light andtemperature, there is a linear relationship betweencarbon input and chlorophyll content. Similar resultwas obtained in the present experiment where thechl a content showed a linear increase at 0.005 and0.004 M concentration of sodium bicarbonate.Photosynthetic pigments in green algaeconstitute mostly chl a and chl b . The physiologicalcondition of microalgae can immediately influencethe pigment constituents. The ratio betweenchlorophyll a  and chlorophyll b  indicate thephysiological status of the microalgae. In thepresent experiment, an immediate decline of chl a and chl b  in all the treatments including controlwas found, which may explain the change in pHwhile acclimatizing the plant to new enrichedmedium .The decline was more in control and0.001M treatment of sodium bicarbonate andpersisted until 7 th  day of culture. The decline wasless as the concentration of sodium bicarbonateincreased. According to Shiraiwa et al.  (1993), thereare several microalgae, which have CO 2 concentration systems that maintain pH balanceintracellularly and generally increase pHextracellularly. Chlorella salina  showed directinteraction with inorganic carbon by increasingthe growth rate and the reduction in chlorophyllpigments from 0.002 M to 0.005 M sodiumbicarbonate treatment. This might be due to theneutralization of the plant to overcome the changein pH extracellularly for at least 4 days to overcomethe intracellular changes of pH.Israel and Gonzalez (1996) reported thatenriched medium led to decline in pH with theformation of carbonic acid due to extracellularcarbonic anhydrase. Ion of sodium bicarbonate maynot be able to neutralize it. Similar observationswere made in the present experiment. The ratio of chl a  /chl b  showed a very low value for thecontrol and the treatment with 0.001 M sodiumbicarbonate corresponding to high concentrationof chl a  and chl b  . This increase of chlorophylldid not influence the growth considerably. Furtherthere was significant difference in growth indifferent treatments with control. Perhaps there wascarbon limitation while culturing Chlorella salina in control and with low carbon input. Perceptiblechanges in growth were observed in all thetreatments after 4 days of inoculation. Thus inputof carbon intermittently along with enrichment of seawater will enhance the growth to a greater extentin situations of carbon limitation. References Badger, M. R. 1985. The fluxes of Inorganic carbonspecies during photosynthesis in cyanobacteria withparticular reference to Synechococcus  sp.  In : W. J.Lucas and J. Berry. (Eds.) Inorganic carbon uptake byaquatic photosynthetic organisms.  Am. Soc. Plant Physiol. , Rockville, MD, p. 39-52.Badger, M. R., A. Kaplan and J. A. Berry. 1980. Internalinorganic carbon pool of Chlamydomonas reinhardtii: evidence for a carbon dioxide concentratingmechanism. Plant Physiol. , 66: 407-413Beer, S. 1994. Mechanisms of inorganic carbon acquisitionin marine macroalgae (with special reference to thechlorophyta).  In : D. J. Chapman and F. Round (Eds.)
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