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Accumulation of poly-?-hydroxybutyrate in : regulation by pH, light?dark cycles, N and P status and carbon sources

Accumulation of poly-?-hydroxybutyrate in : regulation by pH, light?dark cycles, N and P status and carbon sources
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  Accumulation of poly- b -hydroxybutyrate in Nostoc muscorum : regulation by pH, light–dark cycles,N and P status and carbon sources Laxuman Sharma, Nirupama Mallick  * Agricultural and Food Engineering Department, Indian Institute of Technology, Kharagpur 721 302, India Received 25 May 2004; received in revised form 11 October 2004; accepted 16 October 2004Available online 19 December 2004 Abstract Accumulation of poly- b -hydroxybutyrate (PHB) in  Nostoc muscorum  was studied. Cells harvested at stationary phase of growthdepicted maximum accumulation i.e. 8.6% (w/w) of dry cells as compared to lag (4.1%) or logarithmic (6.1%) phases of cultures. Incontrast to alkaline pH, acidic pH, continuous illumination and cells grown in presence of combined nitrogen sources, such asNH 4 Cl and KNO 3 , were found to affect PHB accumulation negatively. However, P-deficiency and addition of exogenous carbonsources (acetate, glucose, maltose, fructose and ethanol) were found stimulatory for PHB accumulation. In this report PHB accu-mulation in  N. muscorum  was boosted up to 35% (w/w) of dry cells when cells supplemented with 0.2% acetate were subjected todark incubation for 7 days. Further studies are needed at metabolic engineering level or to apply genetic engineering techniquesto improve the expression level of PHB photoproduction in cyanobacteria.   2004 Elsevier Ltd. All rights reserved. Keywords:  Acetate; N and P status;  Nostoc muscorum ; pH; Poly- b -hydroxybutyrate 1. Introduction The famous scientist Dr. Alexander Parkes inventedworld  s first synthetic plastic, celluloid, way back in1856. Since then the number as well as the types andqualities have greatly increased, producing superiormaterials such as epoxies, polycarbonates, teflons, sili-cones, polysulfones, etc., and have become one of themost widely used products all over the globe. Durabilityand resistance to degradation are desirable propertieswhen plastics are in use, but they pose problems for dis-posal when out of use. These non-biodegradable plasticsaccumulate in the global environment at a rate of 25  ·  10 6 t year  1 (Dawes, 1990), posing serious threatsto the solid waste management programme. Therefore,today  s demand for biodegradable plastics is one of the most important targets both for basic and appliedresearch.Polyhydroxybutyrate (PHB), the most common rep-resentative of polyhydroxyalkanoates (PHAs), is wide-spread in different taxonomic group of prokaryotes asintracellular storage compounds (Anderson and Dawes,1990; Steinbu¨chel, 1992). The properties of pure PHB,including thermoplastic processability, hydrophobicity,complete biodegradability and biocompatibility withoptical purity suggest that PHB could be an attractivealternative to the common plastics. Till date, PHBaccumulation has been studied in various wild typesand recombinant bacteria in fermentative processes.Nevertheless, the cost of fermentative PHB productionis still a major obstacle for large-scale commercialexploitation. 0960-8524/$ - see front matter    2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2004.10.009 * Corresponding author. Tel.: +91 3222 283166; fax: +91 3222282244. E-mail address: (N. Mallick).Bioresource Technology 96 (2005) 1304–1310  Cyanobacteria, a group of oxygen-evolving photo-synthetic bacteria with a short generation time, needsome simple inorganic nutrients such as phosphate, ni-trate, magnesium and calcium as macro-, and Fe, Mn,Zn, Mo, Co, B and Cu as micronutrients for theirgrowth and multiplication. Further, these organismscan successfully be cultivated in wastewaters due to theirability to use inorganic nitrogen and phosphorus, andwastewaters such as effluents of farm-yards, fish-farms,rubber industries, sewage treatment plants, etc. arerich sources of N and P. Therefore, exploitationof cyanobacteria for PHB production with carbondioxide, wastewaters and sunlight seems highlypromising as it has the combined advantage of produ-cing polymer and treating the wastewaters.Prof. N.G. Carr, University of Warwick, UK, wasthe first to report the presence of PHB in the cyanobac-terium,  Chloroglea fritschii  , where the method of ana-lysis was based on the conversion of PHB to crotonicacid, and subsequent detection of crotonic acid byUV spectroscopy (Carr, 1966). Since then few research-ers have studied the intracellular accumulation of PHBin some selected cyanobacterial species. Detection of PHB was done electron microscopically by Rippkaet al. (1971) and Jensen (1980), respectively in  Gleo-capsa  PCC 6501 and  Nostoc  sp. under photoautotro-phic conditions. Oval structures resembling PHBgranules were also detected by means of ultrastructuralanalysis in  Microcystis aeruginosa  (Jensen and Baxter,1981) and  Trichodesmium thiebautti  , collected from adepth of 15 m in the northwestern Sargasso Sea (Sid-diqui et al., 1992). However, these publications, report-ing the presence of PHB in cyanobacterial strains onthe basis of microscopic observations, not supportedby chemical analyses, are difficult to interpret in orderto establish the actual presence of the polyester, owingto the possibility of mistaking other cell inclusions asPHB granules. On the other hand, Stal (1992) detectedthe presence of PHB in  Oscillatoria limosa  and  Gloeo-thece  sp. PCC 6909 by Gas–liquid chromatography.Recent reports of  Wu et al. (2001) and Nishioka et al. (2001) also demonstrated the presence of poly- b -hydroxybutyrate in the cyanobacteria,  Synechocystis sp. PCC 6803 and  Synechococcus  MA19. However, itis noteworthy here that in spite of the increasing num-ber of cyanobacterial strains that are reported to con-tain PHB, most of the papers deal with other aspectsof cyanobacterial physiology and biochemistry; thepresence of polyester being reported without anyfurther investigation. In this report, PHB biosynthesisin the culture of   Nostoc muscorum  has been studiedwith an aim to establish the actual potential of theorganism, and also how its biosynthesis is regulatedby various factors such as pH, light–dark cycles, Nand P status, and also in presence of different carbonsources. 2. Methods  2.1. Organism and growth conditions Axenic cultures of   Nostoc muscorum  were grownin 150 ml Erlenmeyer flasks containing 50 ml BG-11medium (Rippka et al., 1979). The medium constituentswere citric acid: 0.006 g, ferric citrate: 0.006 g, EDTA(disodium magnesium salt): 0.001 g, Na 2 CO 3 : 0.02 g,MgSO 4 Æ 7H 2 O: 0.075 g, CaCl 2 Æ 2H 2 O: 0.036 g,K 2 HPO 4 : 0.04 g, MnCl 2 Æ 4H 2 O: 1.81 mg, Na 2 MoO 4 :0.039 mg, H 3 BO 3 : 2.86 mg, CuSO 4 Æ 5H 2 O: 0.079 mg,Co(SO 4 ) Æ 7H 2 O: 0.04 mg and ZnSO 4 Æ 7H 2 O: 0.222mg/l. The pH of the culture medium was maintainedat 8.0 with Tris–HCl (4.0 mM) buffer. The cultures wereincubated in a temperature-controlled incubator at25 ±1   C under a photoperiod of 14:10 h at light inten-sity of 75  l  mol photon m  2 s  1 PAR.  2.2. Growth measurement Growth was measured in terms of chlorophyll  a . Aknown volume of algal suspension was centrifuged andthe pellet so obtained was suspended in 80% acetone.After incubation at 4   C overnight, it was again centri-fuged and the resultant supernatant was used formeasurement of chlorophyll  a  at 663 nm using a spectro-photometer (Specord S 100, Analytic Jena, Germany),and quantified following Mackinney (1941).  2.3. Estimation of dry weight Cell dry weight was determined gravimetrically. Aknown volume of algal culture was filtered through 0.4nm polycarbonate filters and dried under vacuum at45   C till a constant weight was obtained (Rai et al.,1991).  2.4. Extraction of poly-hydroxyalkanoates (PHA) A known amount of algae was suspended in metha-nol at 4   C (overnight) for removal of pigments. The pel-let obtained after centrifugation was dried at 60   C. Thepolymer was extracted in hot chloroform followed byprecipitation with cold diethyl ether. The precipitatewas centrifuged at 10000 rpm for 20 min to get the pel-let. The pellet was washed with acetone and the samewas dissolve in hot chloroform again following Yelloreand Desia (1998).  2.5. Assay of poly- b -hydroxybutyrate (PHB) The spectrophotometric assay was performed as perLaw and Slepecky (1961) using a spectrophotometer(Specord S 100, Analytic Jena, Germany). The samplecontaining the polymer in chloroform was transferred L. Sharma, N. Mallick / Bioresource Technology 96 (2005) 1304–1310  1305  to a clean test tube. The chloroform was evaporated andconcentrated H 2 SO 4  (10 ml) was added, and the solutionwas heated in a boiling water bath. After cooling andthorough mixing the absorbance of the solution wasmeasured at 235 nm against H 2 SO 4  blank. To furtherconfirm the presence of PHB, absorption spectra (200– 1000 nm) of the sample as well as the standard ( DLDL - b -hydroxybutyric acid, Sigma Chemical Company,USA), following acid digestion were taken in theSpecord S 100 Spectrophotometer. These spectra werecompared with the spectrum of crotonic acid (SigmaChemical Company, USA).  2.6. Studies on pH, light–dark cycles, nitrogen and carbon sources Fifty millilitre of the medium was taken in 150 mlErlenmeyer flasks. The pH was adjusted to differ-ent values, ranging from 5.5–10.5 (MES buffer, 4mM for pHs 5.5 and 6.5 and Tris–HCl buffer, 4mM for pHs 7.5–10.5) before introducing the cellsinto the medium, and grown for the stipulated time(21 days). PHB content was analyzed as describedabove. PHB production was also studied in cellsgrown under continuous light/light–dark cycles (14:10h). Similarly, PHB accumulation was quantified incells grown in presence of combined-nitrogen sources(nitrate and ammonium). Impact of mixotrophy onPHB accumulation was studied by supplementing thenutrient media with various concentrations (0.05– 0.4%) of glucose, fructose, maltose, ethanol, acetateand cyclohexane.  2.7. Studies on phosphorus deficiency and dark incubation To study the impact of phosphorus deficiency onPHB accumulation,  Nostoc muscorum  cells were grownin phosphate-deficient medium, where K 2 HPO 4  of themedium was replaced by equimolar concentration of KCl. The interaction of exogenous carbons (glucose,acetate and maltose) with P-deficiency was studied intwo different ways: (i) cells grown for 21 days incarbon-supplemented medium were transferred tophosphate-deficient medium, and (ii) cells grownin normal BG-11 medium were transferred to P-defi-cient medium with supplemented carbons. PHBaccumulation was analysed on 2nd, 5th, 7th and14th day.Impact of dark incubation on PHB accumulation wasstudied in cultures grown for 21 days under normallight–dark cycles followed by dark incubation with/without acetate for 3, 5 and 7 days.All the experiments were conducted thrice to checkthe reproducibility. The results were analysed by Dun-can  s new multiple range test. 3. Results 3.1. Assay of poly- b -hydroxybutyrate AssayofPHBfromthecyanobacterialsamplewasper-formed as described above and the sample as well as thestandard ( DLDL - b -hydroxybutyric acid) after acid digestionwere analysed. The absorption spectra of the samplealongwiththestandardarepresentedinFig.1,whichde-pict highest degree of similarity to crotonic acid. 3.2. Time-course of growth and PHB accumulation The time-course of growth and accumulation of PHBare presented in Fig. 2. Growth of the test organism in-creased steadily with a lag phase of 7 days followed bythe logarithmic phase and attained the stationary phaseon 18th day. Accumulation of PHB though started atthe early phase of growth, maximum accumulationwas recorded at the stationary phase i.e. on 21st day(8.6%, w/w of dry cells), after which a decline in thepolymer level was observed. 3.3. Effects of pH, light–dark cycles and nitrogensources on PHB accumulation PHB accumulation was found maximum at pH 8.5(8.9%, w/w of dry cells) followed by pHs 9.5 (7.8%)and 10.5 (7.2%) on 21st day of incubation. Acidic pHswere not found to support PHB accumulation (datanot shown). Cells grown under continuous illuminationwas found to accumulate PHB only up to 3.6% (w/w) of dry cells. Addition of combined nitrogen sources i.e.KNO 3  and NH 4 Cl to the growth medium was also Fig. 1. Comparison of absorption spectrum of crotonic acid with  N.muscorum  sample and the standard  DLDL - b -hydroxybutyric acid digestedin H 2 SO 4 .1306  L. Sharma, N. Mallick / Bioresource Technology 96 (2005) 1304–1310  found to have negative impact on PHB accumulation(Table 1). 3.4. Impact of carbon sources Impact of various carbon sources on PHB accumula-tion is presented in Table 2. Stimulation of PHB accu-mulation under carbon sources was as follows: 0.4%acetate + 0.4% glucose-supplemented cultures (35%,w/w of dry cells), 0.2% acetate + 0.2% glucose (32%),0.2% acetate (28%), 0.4% glucose (26%), 0.2% ethanol(20%), 0.4% maltose (18%) and 0.4% fructose (18%)on 21st day of incubation. No further rise in PHB poolwas observed at increasing concentrations of the abovecarbon compounds. Supplementation of cyclohexanewas not found to exert any significant impact on thePHB accumulation potential of the test organism. 3.5. PHB accumulation under P-deficiency Impact of phosphorus deficiency on PHB accumula-tion is presented in Fig. 3. Cultivation of   N. muscorum 0246810071421283542 Days of incubation    C   h   l  o  r  o  p   h  y   l   l       a     &   P   H   B PHB (%) w/w of dry cellsChlorophyll a (mg/L) Fig. 2. Accumulation of PHB in  N. muscorum  with reference togrowth.Table 1Accumulation of PHB in  Nostoc muscorum  cells grown in presence of combined nitrogen sourcesTreatment PHB (%) w/w of dry cellsDays of incubation14 21N 2 -fixing 7.31 ± 0.08 a 8.30 ± 0.10 a NO  3  -supplemented 3.77 ± 0.11 b 2.36 ± 0.09 b NH þ 4  -supplemented 3.80 ± 0.10 b 2.57 ± 0.08 b All values are mean ± SE. Values in the column superscripted by dif-ferent letters are significantly ( P   < 0.05) different from each other(Duncan  s new multiple range test). Separate analysis was done foreach column.Table 2Impact of exogenous carbon sources on PHB accumulation in  N.muscorum  on 21st day of incubationTreatment PHB (%) w/w of dry cellsCarbon source (%)0.2 0.4Glucose 19.74 ± 1.03 a 26.01 ± 1.31 a Acetate 28.00 ± 2.14 b 26.30 ± 2.51 a Maltose 16.80 ± 1.65 c 18.30 ± 2.01 b Fructose 16.28 ± 0.93 c 18.02 ± 0.91 b Ethanol 20.60 ± 1.64 a 16.32 ± 2.02 b Cyclohexane 10.55 ± 0.85 d 9.55 ± 0.91 c Glucose + Acetate 32.40 ± 2.26 e 34.90 ± 2.38 d Control (photoautotrophy): 8.58 ± 0.32.All values are mean ± SE. Values in the column superscripted by dif-ferent letters are significantly ( P   < 0.05) different from each other(Duncan  s new multiple range test). Separate analysis was done foreach column. 051015202507142128 Days of Incubation    P   H   B  c  o  n   t  e  n   t   (   %   )  w   /  w  o   f   d  r  y  c  e   l   l  s (a) 01020304002468101214 Days of Incubation    P   H   B  c  o  n   t  e  n   t    (   %   )  w   /  w  o   f   d  r  y  c  e   l   l  s (b) Fig. 3. Accumulation of PHB in  N. muscorum  under P-deficiency. (a)Cells subjected to P-deficiency and carbon supplementation simulta-neously, and (b) cells pre-grown in carbon-supplemented medium for21 days were subjected to P-deficiency. ( h ) P-deficient control, ( j )0.2% glucose, (  ) 0.2% acetate, ( m ) 0.2% maltose, ( n ) 0.2% glucose +0.2% acetate, and ( d ) 0.4% glucose + 0.4% acetate. L. Sharma, N. Mallick / Bioresource Technology 96 (2005) 1304–1310  1307  in P-deficient medium for 5–7 days stimulated PHBaccumulation up to 23% (w/w) of dry cells, after whichit started declining (Fig. 3a). Cells subjected to P-defi-ciency and carbon supplementation simultaneouslythough registered a rise in PHB pool; the values werenot even up to the tune of the accumulation under P-deficiency alone (Fig. 3a).Cells grown in carbon-supplemented media showedtheir highest accumulation on 21st day. When thesecells, at the peak of their accumulation, were transferredto P-deficient medium showed quite different trends(Fig. 3b). The 0.4% glucose + 0.4% acetate-, 0.2% glu-cose + 0.2% acetate- and 0.2% maltose-grown cellsshowed an immediate decline in PHB pool, whereas in0.2% acetate- and 0.2% glucose-grown cells stable condi-tions were recorded even on 14th day of P-deficiency.Therefore, the overall interaction of carbon doses andP-deficiency were not found stimulatory for PHB accu-mulation, as compared to their individual impact. 3.6. Impact of dark incubation on acetate supplemented culture Cells grown for 21 days under normal light–darkcycles when subjected to dark incubation depicted anincrease in PHB pool up to 14% (w/w) of dry cells after5 days (Table 3). However, a declining trend was noticedafter 7 days. Interestingly, supplementation of 0.2% ace-tate at the initiation of dark incubation boosted theaccumulation of PHB up to 35% (w/w) of dry cells. 4. Discussion Since Carr  s publication (Carr, 1966) on the occur-rence of PHB in a cyanobacterium,  Chlorogloea fritschii  ,a thin flow of reports containing the presence of PHB invarious cyanobacterial strains followed (Vincenzini andPhilippis, 1999). Our study with  Nostoc muscorum  underphotoautotrophic condition (Fig. 2) is in well agreementwith Stal (1992) where he detected accumulation of PHA up to 6–9% (w/w) of dry cells in  Oscillatoria limosa and  Gloeothece  sp. This is however, in contrast to Carr(1966) where PHB accumulation in  Chlorogloea fritschii  was observed only in presence of acetate.In  Nostoc muscorum  PHB accumulated during thelinear growth phase, reached a maxima when the cul-tures become stationary (Fig. 2). A significant decreasein PHB content during the lag phase, following the inoc-ulation, is very similar to that of   Oscillatoria limosa  and Gloeothece  sp. (Stal, 1992). Therefore, it is tempting toadopt the same explanation that PHB may be used asa specific carbon reserve, which allows the test organismto synthesize components that are required in order toregain the full capacity of photoautotrophic growth.The chloroform extraction is now the most widely ac-cepted method for extraction of PHA (Lee, 1996). Theprecipitation of the polymer with diethyl ether and ace-tone in this method rules out the possibilities of any con-taminating lipids, and therefore yields the purified PHA.Our results also give testimony to the above view, wherethe absorption spectrum of acid-digested sample did notshow presence of any impurities, and when comparedwith the standard ( DLDL - b -hydroxybutyric acid) depictedhighest degree of similarity (Fig. 1). These spectra of acid-digested sample as well as the standard  DLDL - b -hydroxybutyric acid also showed complete matchingwith the spectrum of crotonic acid, thus confirmingthe occurrence of PHB in the test cyanobacterium.The absence of any other peak in the spectrum of theacid-digested sample indicates that PHA of   Nostoc mus-corum  presumably contains polyhydroxybutyrate, only.However, further analysis with GC/HPLC is necessaryto confirm this view, as the crotonic acid method wouldnot detect other hydroxyalkanoates.As observed in this study, alkaline pHs supports PHBaccumulation. This is in well agreement with Khatipovet al. (1998) where PHB accumulation in the bacterium, Rhodobacter sphaeroides , was significantly enhancedwhen pH of the culture medium was increased to 7.5from 6.8. Under photoautotrophic conditions, the N 2 -fixing cultures contained higher levels of PHB than thenitrate-/ammonium-grown  Nostoc  (Table 1). This isquite in tune with the observations of  Rippka et al.(1971) and Stal (1992) for  Gloeothece  sp., but contra-dicts with  Oscillatoria limosa  (Stal, 1992). Further,  Nos-toc  cultures grown under normal light–dark cyclesaccumulated more PHB than cultures grown under con-tinuous illumination (data not shown), thus agreeingwith the view of  Asada et al. (1999) that dark periodsare inevitable for PHB accumulation in photoautotro-phic cultures.Cultures incubated with various carbon sources de-picted a significant rise in PHB pool in acetate-, glu-cose-, fructose-, maltose- and ethanol-supplemented Table 3Impact of dark incubation on PHB accumulation of   Nostoc muscorum grown for 21 days under light–dark cyclesTreatment Durationof darkincubation (days)PHB(%) w/wof dry cellsControl Light–dark cycles 8.5 ± 0.10 a Dark 3 12.19 ± 0.19 b 5 13.85 ± 0.28 b 7 6.06 ± 0.12 a Dark + acetate (0.2%) 3 13.97 ± 0.23 b 5 20.35 ± 0.32 c 7 35.07 ± 0.24 d All values are mean ± SE. Values in the column superscripted by dif-ferent letters are significantly ( P   < 0.05) different from each other(Duncan  s new multiple range test).1308  L. Sharma, N. Mallick / Bioresource Technology 96 (2005) 1304–1310


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