growth ofazotobacter vinelandii uwd in fish peptone medium ... · present study sought to examine...

Vol. 59, No. 12APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1993, p. 4236-42440099-2240/93/124236-09$02.00/0Copyright X 1993, American Society for Microbiology

Growth ofAzotobacter vinelandii UWD in Fish Peptone Mediumand Simplified Extraction of Poly-,-Hydroxybutyrate

WILLIAM J. PAGE* AND ANTHONY CORNISH

Department ofMicrobiology, University ofAlberta, Edmonton, Alberta, Canada T6G 2E9

Received 6 July 1993/Accepted 30 September 1993

Azotobacter vinelandii UWD was grown in a fermentor with glucose medium with and without 0.1% fishpeptone (FP) in batch and fed-batch cultures for the production of the natural bioplastic poly-13-hydroxybu-tyrate (PHB). Strain UWD formed PHB five times faster than cell protein during growth in glucose and NH4',but PHB synthesis stopped when NH4' was depleted and nitrogen fixation started. When FP was added to thesame medium, PHB accumulated 16 times faster than cell protein, which in turn was inhibited by 40%o, andPHB synthesis was unaffected by NH4' depletion. Thus, FP appeared to be used as a nitrogen source by thesenitrogen-fixing cells, which permitted enhanced PHB synthesis, but it was not a general growth stimulator. Theaddition of FP to the medium led to the production of large, pleomorphic, osmotically sensitive cells thatdemonstrated impaired growth and partial lysis, with the leakage of DNA into the culture fluid, but these cellswere still able to synthesize PHB at elevated rates and efficiency. When FP was continuously present infed-batch culture, the yield in grams of polymer per gram of glucose consumed was calculated to range from0.43 gIg, characteristic of nongrowing cells, to an unprecedented 0.65 g/g. Separation of an FP-free growthphase from an FP-containing growth phase in fed-batch culture resulted in better growth of these pleomorphiccells and good production of PHB (yield, 0.32 g/g). The fragility of these cells was exploited in a simpleprocedure for the extraction of high-molecular-weight PHB. The cells were treated with 1 N aqueous NH3 (pH11.4) at 45°C for 10 min. This treatment removed about 10%o of the non-PHB mass from the pellet, of which60 to 77% was protein. The final product consisted of 94% PHB, 2% protein, and 4% nonprotein residualmass. The polymer molecular weight (1.7 x 106 to 2.0 x 106) and dispersity (1.0 to 1.9) were not significantlyaffected (P = 0.05) by this treatment. In addition, the NH3 extraction waste could be recycled in thefermentation as a nitrogen source, but it did not promote PHB production like FP. A scheme for improveddownstream extraction ofPHB as well as the merits of using pleomorphic cells in the production of bioplasticsis discussed.

The worldwide demand for degradable plastics is esti-mated to be 1.4 million metric tons per year by the year 2000,with much of this demand driven by legislation and environ-mental groups (20). A number of polymers are being devel-oped to meet this demand, with the majority of these beingpetrochemically based and projected to cost $6.6 kg-1 (21,26). Natural products being developed as biodegradableplastics include starch blends, polylactic acid, and bacterialpolyhydroxyalkanoates (PHA). PHA, most commonly rep-resented by poly-,-hydroxybutyrate (PHB), are naturalpolyesters that are stored as intracellular inclusions by agreat variety of bacteria (1, 2). Copolymers of poly(,B-hydroxybutyrate-co-f3-hydroxyvalerate) are currently beingproduced on a large scale by the Imperial Chemical Indus-tries in Billingham, United Kingdom (10, 22). This copoly-mer is a thermoplastic which resembles polypropylene (18)and has been successfully test marketed as shampoo bottlesin Europe (13). Although this natural product has greatpromise, it is currently priced at $17 to $22 kg-1 (26).The large-scale production of bacterial PHA poses a

number of interesting problems. Major expenses in theproduction of PHA are determined by the cost of thefermentation substrate, the extraction of the polymer frominside the cells, and the treatment of fermentation andextraction wastes. In practice, the production of 1 metric tonof polymer requires that 3 metric tons of glucose be used forPHB synthesis and the production of non-PHB biomass

* Corresponding author.

(yield in grams of polymer per gram of glucose consumed[Yp/s] = 0.33) (10, 12). The Imperial Chemical Industriesprocess usesAlcaligenes eutrophus growing in glucose saltsmedium (10). A potential cost saving is offered by the use ofAzotobacter vinelandii UWD for PHA production (34). Thisorganism will form polymer efficiently in beet molasses,which cuts the fermentation substrate cost by about one-half. However, the use of beet molasses will likely increasewaste treatment costs, unless the spent fermentation wastecan be recycled in some other application (32).At the end of the fermentation, the cells often contain 80%

PHA per total dry weight. The extraction process mustremove a relatively small percentage of impurities to give aproduct that is at least 92% pure. The extraction process alsomust be gentle enough to preserve a polymer molecularweight of at least 600,000 to make it suitable for thermoplas-tic applications (22). The Imperial Chemical Industries pro-cess uses heat disruption, a series of enzymatic digestions,peroxide treatment, and spray drying of the isolated PHAgranules (15, 24). Alternatively, the polymer can be ex-tracted from spray-dried cells with a solvent like chloroform,but this poses real problems in how to permeate the driedcell mass with the solvent and how to separate the non-PHBresidual cell mass (RM) from the very viscous PHA solution.Simple dissolution of RM with hypochlorite is commonlyused in laboratory preparations of PHA, but this treatmentwill hydrolyze the polymer (6, 19, 37, 44) and generatechlorinated wastes.When A. vinelandii UWD is grown in beet molasses

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EXTRACTION OF PHB FROM A. VINELANDII UWD 4237

medium, there is a PHA yield-promoting effect by which thefermentation substrate is directed into polymer formation toa greater extent than into cell growth (29, 30). Although theexact nature of this PHA yield promotion is unknown, thisstimulation was recently duplicated by the addition of 0.05 to0.2% fish peptone (FP), yeast extract, or certain otherpeptones to the growth medium (31, 32). It was suggestedthat peptone may act as an amino-N source, which allowsthese nitrogen-fixing, mutant cells to rapidly catabolize sugarand generate a reducing-power excess for the formation oflarge amounts of PHB (31). However, peptones and yeastextract are not commonly added to media for Azotobactersp. culture, as they usually cause pleomorphism and com-promised cell wall strength (41, 42). Even low concentra-tions of peptone or yeast extract may alter A. vinelandii cellpermeability and susceptibility to antibiotics (8). Thus, thepresent study sought to examine peptone-grown UWD cellsin more detail and to determine whether cell wall strengthwas compromised and whether PHB could be easily ex-tracted from these cells.

(Part of this research was presented at the Seventh Forumfor Applied Biotechnology, Ghent, Belgium, 30 Septemberto 1 October 1993).

MATERIALS AND METHODS

Bacterial strain and growth conditions. A. vinelandii UWD(ATCC 53799) (33) was grown in a 3% (wtlvol) glucosemedium which contained 15 mM ammonium acetate andsalts as described previously (34). The salts component ofthis medium is called Burk's buffer. The glucose mediumwas also supplemented with 0.1% (wt/vol) FP (product no.HO10OBT; Protan A/S, Drammen, Norway), as noted inResults. The cultures were inoculated with a 4% (vol/vol)inoculum, pregrown for 24 h in glucose medium or asotherwise noted. Shake flask cultures (50 ml/500-ml Erlen-meyer flask) were incubated at 28 to 30°C with shaking at 225rpm on a New Brunswick Scientific Co. model G-10 platformshaker for 24 h.

Fed-batch fermentation studies. The growth and kinetics ofPHB production by strain UWD were studied in a 2.5-literBioFloIll fermentor (New Brunswick Scientific Co.) withglucose medium as a substrate. Initially, the fermentorcontained 1.6 to 1.8 liters of medium containing 3% glucoseand 15 mM ammonium acetate. FP (0.1% final concentra-tion) was added before or after the start of the fermentation,as noted in Results. After 15 to 17 h, the fermentor was fedwith glucose for 36 h to maintain about 3% (wt/vol) glucosein the vessel. The ammonium feed used NH4HCO3, ratherthan ammonium acetate to avoid the negative effect ofacetate on PHB production (28). The feed was increasedover time to maintain about 15 mM NH4' for 15 h and wassupplied as a step gradient consisting of 266 mM NH4HCO3fed at 15 ml h- from 4 to 9 h, at 22.5 ml h-' from 9 to 14 h,and finally at 30 ml h-1 from 14 to 19 h. The medium washeld at 5% dissolved oxygen (34), and the pH remained at 7.0to 7.2 during the PHB production phase. Fed-batch fermen-tation using 5% beet molasses as a substrate was conductedas described previously (34). Other fermentation conditions,data logging, and analysis have been described (34).Chemical analysis of the cells and culture fluids. Whole-

broth fermentation samples (12 ml) were removed at hourlyintervals by an autosampler (35). Each sample was dividedinto subsamples for the determination of cell protein, totaldry weight, PHB dry weight, and glucose remaining insolution (33, 34). Ammonium and phosphate ions remaining

in solution were determined colorimetrically (7, 11). Acetateutilization was estimated indirectly by monitoring the in-crease in medium pH that was characteristic of acetate use(35). All assays were done in duplicate, and colorimetricassay results were recorded on a Hitachi U-2000 spectro-photometer. The rates of formation of products or theconsumption of nutrients were calculated from assay meansby linear regression. Non-PHB RM was calculated as thetotal cell dry weight minus PHB dry weight. The efficiency ofpolymer formation (Ypls) was calculated as the grams ofPHB formed per gram of glucose consumed. Results re-ported as significant differences were determined by Dun-can's multiple-range test (38).

Estimation of osmotic fragility and DNA release. The os-motic fragility of the cells was tested by resuspending a cellpellet (from 5 ml of culture, centrifuged at 5,000 x g for 10min and washed once with 5 ml of Burk's buffer) in 5 ml ofdistilled water. The suspension was vigorously mixed on avortex mixer for 30 s and then allowed to stand at roomtemperature for 5 min. The suspension was mixed again for30 s, and the cells were concentrated by centrifugation (5,000x g for 10 min). Lysis was also promoted by vortex mixingof duplicate samples in distilled water containing 1.5 g of 0.5-to 1.0-mm-diameter glass beads. The protein content of thecell pellet was determined (33). DNA released into theculture supernatant fluid and water extracts was quantitatedby an ethidium bromide fluorescence assay with a HitachiF-2000 spectrofluorometer (27)..PHB extraction and molecular weight determination. Strain

UWD cells were extracted with commercial bleach (contain-ing 5.25% sodium hypochlorite) at 45°C for 1 h as describedby Law and Slepecky (19). Alternatively, the bleach wasadjusted to pH 10.1, and the polymer was extracted byheating at 20°C for 10 min (44). Polymer extracted intochloroform was used as a standard for unhydrolyzed, nativepolymer (37).PHB was also extracted from the cells by use of aqueous

NH3 and heat. In most cases, cells grown in the fermentor inglucose medium containing 0.1% FP were concentrated toapproximately 50 g (dry weight) per liter by using distilledwater as the suspension medium. The cell suspension (usu-ally 5 to 10 ml) was contained in a centrifuge tube, andaqueous NH3 was added to give a final concentration of 1 Nat pH 11.4. The control for this extraction procedure con-sisted of cells suspended in distilled water only. The suspen-sions were heated at 45°C in a water bath and mixed bystirring for 10 min (Multi-stirrer model 1286; Lab-LineInstruments Inc., Melrose Park, Ill.). The suspensions werecooled to =18'C in running tap water and then concentratedby centrifugation (12,000 x g, 15 min). The NH3 extract fluidwas saved for the determination of protein and N-acetylsugar released (39). The PHB pellet was resuspended in 35ml of distilled water and homogenized by vortex mixing togive an even suspension. Triplicate subsamples of 1 to 2 mlwere removed for determination of pellet total dry weight,protein content, and PHB dry weight by using the assaysdescribed for the fermentation studies. Duplicate 5-ml sam-ples of PHB were extracted in pH 10.1 bleach (44) prior tomolecular weight determination.PHB molecular weight was determined by high-perfor-

mance liquid chromatography (HPLC) as described previ-ously (9). The weight average molecular weight (Mw), num-ber average molecular weight (Mn), and dispersity (Mw/Mn)relative to those of polystyrene standards were determinedby a gas permeation chromatography program for the Shi-madzu LC-6A HPLC (9).

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4238 PAGE AND CORNISH

3A

zm

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1 0 20

HOURS INCUBATION

4B

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Acetate-depleted

Ammonium-depleted

O_~~~~~~~~~~_0

0 5 10 15

HOURS INCUBATION

FIG. 1. Growth of strain UWD in batch culture in a fermentorwith glucose medium without FP (A) or glucose medium with FP(B). The PHB (0) and protein (-) contents of the cell pellet weredetermined at hourly intervals.

Microscopy. Cell morphology and sizes were routinelyestimated by phase-contrast microscopy with a calibratedocular.

RESULTS

Effect of FP on PHB production in batch culture. Batchculture experiments were run in the fermentor to comparethe growth and PHB production by strain UWD in glucosemedium with or without FP (Fig. 1). In glucose mediumalone, PHB was formed very slowly during the use of acetate(Fig. 1A). PHB accumulated five times faster than cellprotein (0.08 g liter-' h-1) after acetate depletion and thestart of glucose catabolism. The NH4+ in the medium wasdepleted by 12 h, after which the cells continued to synthe-size protein at the same rate during nitrogen-fixing growth.However, the increased demand of reducing power fornitrogen fixation and respiratory protection of the nitroge-nase (33) stopped PHB synthesis.When strain UWD was grown in glucose medium contain-

ing 0.1% FP, there was essentially no PHB production

TIME (h)

FIG. 2. The effect of an FP supplement to strain UWD cellsgrowing in glucose medium containing 0.1% FP. The production ofcell protein (0) was monitored for 17 h at which time glucose and FPin Burk's buffer were added as a single supplement to give 5 and0.1% final concentrations, respectively.

during growth on acetate (Fig. 1B). PHB accumulated 16times faster than protein (0.05 g liter-' h-1) during glucoseuse. When the culture became NH4' depleted, there was nosignificant decrease in the rate of protein or PHB synthesis(Fig. 1B). These results confirmed the results obtainedpreviously in shake flask cultures (28, 31, 33). They alsodemonstrated that FP was not a growth stimulator in general(31), since it caused a 40% decrease in the rate of proteinsynthesis while it increased the rate of PHB synthesistwofold.

Effect of FP on PHB production in fed-batch culture. Whenadditional glucose and FP (5 and 0.1% final concentrations,respectively) were fed to strain UWD growing in glucosemedium containing 0.1% FP, there was a doubling in theprotein content of the culture (Fig. 2). The protein increasewas complete after about one cell doubling time (=3 h), andthen there was a decrease in cell protein after a lag of about3 h. This decrease was not due to dilution, as the glucose andFP supplement was added as a single addition, and a plateaushould have been seen if growth had only stopped. Thedecrease was most likely caused by cell lysis and a gradualloss of protein from the cell pellet. This fermentation runterminated with massive foam generation as a result ofwhichthe entire contents of the fermentor were blown into a trapon the exhaust gas line. PHB synthesis (0.6 g liter-' h-') andglucose consumption (2.7 + 0.1 g liter- h-') continuedunabated after the FP feed. Therefore, despite the apparentinstability of the cells after the FP feed, they appeared toremain metabolically active and able to produce PHB.To analyze further the production of PHB in glucose

medium containing FP, the fermentor cultures were fedglucose and FP in a continuous feed (Table 1). When glucoseand FP were fed to cells pregrown in glucose mediumcontaining FP, there was very little protein synthesis, ex-actly as noted in the batch culture experiment. However,RM increased 11-fold during the course of the fermentation,from 0.35 to about 4.0 g/liter, while PHB increased 167-foldat a constant rate, from 0.15 to 25 g/liter. Total cell massincreased from 0 to 20 h (1.2 g liter-' h-'), and the furtherincrease in cell mass from 20 to 38 h (1.0 g liter-' h-') wasdue to PHB synthesis (Table 1). The Ypls increased from0.43 g/g (0 to 20 h) to 0.65 g/g (20 to 38 h). Therefore, itappeared that the cells were growing slowly during incuba-tion in glucose medium containing FP, but the cells were

rapidly and very efficiently filling with PHB.In an attempt to increase the rate of protein synthesis,

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TABLE 1. Effect of FP on the growth and PHB production of UWD in fed-batch culture in a fermentor

MaximumInitial growth Nutrient feedb PHB dry wt % PHB PHB Cell protein PHB/cellmedium'aH r t %PB PBCl rti H/el Y/ 99c Tze()(g/liter) (dry wt) (g liter-' h-1) (g liter-' h-1) protein (g/g) Y (g/g) Time (h)C

Glucose-FP 5% G-FP 25 85 1.0 0.03 15 0.65 373% G-FP-NH4+ 14 74 0.8 0.13 6.5 0.36 37

Glucosed 3% G-FP 25 79 1.0 0.10 10 0.32 475% molasses' 5% molasses 22 66 1.1 0.25 5.3 0.29 36

a Three percent glucose medium, with 0.1% FP added as noted, or 5% beet molasses medium (see Materials and Methods).b Nutrient feed to maintain the concentration of glucose (G) noted plus 0.1% FP was started after 14 to 17 h of incubation in the initial growth medium. The

NH4' feed (from 4 to 19 h) is described in Materials and Methods. The beet molasses feed continued from 12 to 32 h, and a single addition of 6 mM potassiumphosphate (final concentration) was made at 18 h.

c Time when maximum PHB yield (in grams per liter) was obtained.d Mean values from four fermentations.e Mean values from three fermentations.

cells growing in glucose medium containing FP were fedglucose, FP, and NH4( (Table 1). This resulted in a 14-foldincrease in RM production, from 0.35 to 4.9 g/liter, andincreased protein synthesis during the NH4' feed. At theend of the NH4' feed, protein production returned to therate (0.04 g liter-' h-1) found in NH4'-depleted glucosemedium containing FP. The synthesis of PHB occurred at aconstant rate during the glucose, FP, and NH4( feed, andPHB increased 93-fold from 0.15 to 14 g/liter in 32 h. The rateof phosphate use (0.32 mmol liter-1 h-1) was much higherwith NH4( feeding than without (0.093 mmol liter-1 h-'),and phosphate limitation likely stopped PHB synthesis at 32h. Thus, NH4+ feeding did promote greater growth, but thiswas at the expense of PHB formation.The best conditions for PHB production were obtained by

starting the fermentation in glucose medium and then feedingglucose and FP (Table 1). This was an attempt to separatethe growth phase in glucose-NH4' from the PHB productionphase in glucose-FP (Fig. 3). In this case, protein synthesiswas initiated without FP and continued during the glu-cose-FP feed. Total cell dry weight also increased (1.2 gliter-' h-') during the sugar-FP feed. There was an initialslow rate of PHB formation during growth in glucose andNH4' without FP (0.4 g liter-' h-'; YPlS = 0.23 g/g) and thena lag during NH4+-depleted growth (13 to 17 h). During theglucose-FP feed (17 to 38 h), the consumption of glucose

40

30

i2010 mmonium-depleted

-i10

0 5 10 15 20 25 30 35 40 45 50

FIG. 3. Growth of strain UWD in a fed-batch fermentor. Theculture was grown for 17 h in glucose medium and then fed withglucose and FP (3 and 0.1% final concentrations, respectively) for 20h (bar on figure). The glucose content in the medium (-), the PHBcontent (E), and the cell protein content (0) were determined athourly intervals.

increased 1.7-fold, while PHB synthesis increased to 1.0 gliter-' h-1 and became more efficient (Ypls = 0.34 g/g).Phosphate limitation did not occur in this culture. RMincreased 13.5-fold from 0.6 to 8.1 g/liter, while PHB in-creased 139-fold from 0.23 to 32 g/liter in 47 h. Thus, growthwas stimulated by this feeding regimen and the cells filledwith PHB at a fast and efficient rate.By comparison, UWD cells grown in fed-batch culture

with beet molasses as the substrate formed PHB at the samerate as that observed in glucose medium containing FP butformed cell protein >2.5-fold faster (Table 1). During thecourse of this fermentation, the RM increased 44-fold from0.25 to 11 g/liter, which was much greater than that observedin glucose medium containing FP. The PHB content of thecells increased 244-fold from 0.09 to 22 g/liter, but PHBrarely accounted for more than 70% of the total dry weight.Consistent with better cell growth, these fermentationsalways became phosphate limited and were fed phosphateduring the molasses feeding stage. Thus, FP addition toglucose medium appeared to reproduce the PHB yield-promoting activity but not the growth-promoting activity ofbeet molasses.Appearance of the cells grown in glucose medium with FP.

Phase-contrast light microscopy showed that most cellsgrown in glucose medium with FP were 2- to 3-,um-diameterspheres after 24 h of incubation. Dividing, motile cells filledwith phase-bright PHB inclusions were visible at 18 to 24 h,but the majority were phase-bright, often motile cells thatlooked like soccer balls. This appearance was from thenumerous phase-bright PHB inclusions in the cell that wereclearly outlined under the cell envelope. Much smaller,.0.5- to 1-,um-diameter, phase-bright PHB granules fre-quently appeared in the culture fluid or associated with thecell surface, especially in older cultures. Larger 4- to 5-,um-diameter cells and very large 7- to 8-,um-diameter "monster"cells, filled with phase-bright PHB inclusions, were abun-dant at .24 h. These monster cells appeared to be morefragile than the other cells, and a small amount of pressureon a coverslip over a wet mount was sufficient to rupturethem.

Strain UWD grown in glucose medium without FP con-tained very little PHB, and the cells were motile and dividingafter 24 h of incubation. The cells appeared phase dark, withfewer phase-bright PHB inclusions than when FP waspresent. As the culture aged (.24 h), the cells becamespherical, but the average diameter was 2 to 3 ,um. Monstercells were rarely observed without FP addition.

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TABLE 2. Effect of FP on the osmotic sensitivity of UWD cellsgrown in glucose medium

DNA released

PHB/cell (,Jg/mg of cell protein)'Pregrowth Growth rt'mediuma medium' protei In growth In distilled With(g/g)c medium water after vortex

after 24 h 5 mine shearing,Glucose Glucose 1.1 2.6 4.6 59

Glucose-FP 6.0 3.6 8.1 60Glucose-FP Glucose 0.5 15 18 69

Glucose-FP 3.5 10 7.0 85

a Cells were incubated for 24 h in 3% glucose medium, with 0.1% FP addedas noted." Cultures (50 ml/500-ml flask) were inoculated with cells from the pre-

growth medium and incubated in 3% glucose medium, with 0.1% FP (asnoted), for 24 h.

c Means from triplicate cultures.d Mean values from duplicate assays.e Cells were washed with vigorous vortex mixing in distilled water (see

Materials and Methods).f Cells were washed with vigorous vortex mixing in distilled water contain-

ing glass beads (see Materials and Methods).

Stability of cells grown with FP. Cell fragility was measuredby observing the amount of DNA released into the culturefluid after 24 h of growth or by observing the amount ofDNAreleased into a distilled water wash of the cells (Table 2). Thepresence of FP in the medium used to prepare the inoculum,or FP added to the growth medium, made the cells morefragile. The most-fragile cells were from cultures wherethere was a shift down to medium without FP or where FPwas continuously present. In the latter case, the cells ap-peared to be relatively stable in the distilled water wash,possibly because the most fragile cells had ruptured duringthe growth phase. The amounts of DNA released into thegrowth medium and into the distilled water wash fluidindicated partial lysis of the culture, and extensive lysis wasnot obvious from direct microscopic observation. When thecells were subjected to additional shearing by mixing withglass beads (Table 2), much greater amounts of DNA werereleased and definite lysis was visible by light microscopy.The best yield of PHB was found in cells that had been

pregrown without FP and used to inoculate medium contain-ing FP (Table 2), as shown in the fermentor experiments(Table 1). Cells that had been exposed to FP only in thegrowth medium had decreased cell fragility and a better rateof cell protein formation in the fermentor, whereas cellscontinuously exposed to FP were unstable and had thelowest cell protein formation rate in the fermentor (Tables 1and 2).

Extraction of PHB with hypochlorite. Polymer extractedwith alkaline bleach (20°C for 10 min) had a molecular weightof 2.62 x 10' (dispersity, 2.4). When more severe extractionin common bleach (45°C for 60 min) was used to remove RM,there was definite polymer hydrolysis, especially when theratio of hypochlorite to cell mass was high (Fig. 4). How-ever, the =50% loss in molecular weight caused by the mostsevere treatment still allowed the recovery of a product witha molecular weight of 1.8 x 106. In comparison, PHBextracted from strain UWD with chloroform had an Mw of2.83 x 106 to 3.4 x 106 (dispersity, 1.6 to 2.2). The finalproduct from the most severe hypochlorite treatment was97% PHB by weight and contained only 0.27% protein.

Extraction of PHB in aqueous NH3. An alternative PHBextraction procedure using aqueous NH3 and heat was tried,

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Cell mass/hypochlorite (g/g)FIG. 4. Extraction of PHB from strain UWD by using hypochlo-

rite. The molecular weight (O) and dispersity (-) of the polymerwere determined after 60 min of treatment at 450C at the hypochlo-rite-to-cell mass (cell mass/hypochlorite) ratios shown.

with the hope that RM would be extracted by the alkaline pHor by ammonolysis and that the waste stream could berecycled in the fermentation process.

Initially, cells from the fermentor were suspended indistilled water to give a dense slurry (50 g/liter) typical of thatgenerated in a full-scale industrial process (24). When thecells were boiled in distilled water for 10 min, there was anincrease in PHB dry weight in the pellet from the original 80to 86% in the treated sample. However, the same increasewas obtained by mixing the cells in distilled water for 10 minat 22°C, and boiling did not significantly (P = 0.05) decreasethe protein content of the cell pellet. This suggested that thecells were lysing in the distilled water during the preparationof the cell slurry. Lysis was evident by light microscopy,which showed that the suspension was filled with 4- to5-,um-diameter individual or aggregated cells and many freePHB granules. At least 1 N NH3 (pH 11.4) was needed toeffect some extraction of protein and other RM from thepellet, but the use of 5 N NH3 (pH 12.0) or 10 N NH3 (pH12.7) was not more effective (data not shown). Treatmentwith 1 N NH3 (pH 11.4) for 10 min at 45°C, without an initialboiling step, was considered optimal for the scale used here(Fig. 5). Protein, which accounted for about 10% of the dryweight of the original pellet, was reduced about 2% by thedistilled water treatment (Fig. 5, zero time point) and anadditional 6% by NH3 treatment for 10 min. Longer treat-ment did not extract more protein from the PHB pellet.Protein was released into the NH3 extract as well as N-acetyl sugar. The decline in released protein and N-acetylsugar values over time probably was caused by hydrolysisby ammonolysis.

Regardless of the density of the cell suspension (7 to 50g/liter), NH3 treatment removed about 10% of the originalRM from the pellet (Table 3). Of this RM, about 60 to 77% ofthe protein originally present was removed. Thus, the bestpurification of PHB occurred when the initial PHB contentof the cells was high. An analysis of the RM originallypresent in these dense (50-g/liter) cell suspensions showedthat protein was more readily removed by NH3 treatmentthan nonprotein RM (Fig. 6).

After treatment of the cells with aqueous NH3, there wasthe complete rupture of at least 90% of the cells and therelease of free, nonaggregated PHB granules. This was

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EXTRACTION TIME (min)

FIG. 5. Extraction of protein and N-acetyl sugar from strainUWD by using aqueous NH3. The cells were treated with 1 N NH3at 45°C for up to 120 min. The release of protein (0) and N-acetylsugar (L1) into the NH3 extract and the protein remaining in thepellet (-) were determined.

difficult to estimate accurately because of cell aggregation inthe untreated cell slurry. Increasing the temperature of theNH3 treatment to 100°C removed slightly r'ore RM (notsignificant at P = 0.05), removed significantly more protein(Table 3), and resulted in the complete rupture of the cells.Treatment of a very dense suspension (200 g/liter) with NH3also removed about the same proportion of protein as wasreleased from the 50-g/liter suspension, but overall there wasless RM removed (Table 3).Treatment of strain UWD (50 g/liter) with 1 N NH3 at 45°C

for 10 to 120 min, 1 N NH3 at 20 to 100°C for 10 min, or 5 NNH3 at 20 to 80°C for 10 min did not result in a significant (P= 0.05) change in PHB Mw compared with that of thedistilled water control. The Mw of the control PHB (n = 7)was 1.7 x 106 to 2.0 x 106 (dispersity, 1.3 to 1.5), while theMw from all extraction trials (n = 15) was 1.7 x 106 to 2.7 x106 (dispersity, 1.0 to 1.9). The only treatment that had asignificant negative effect on the polymer was 5 N NH3 at100°C for 10 min, which decreased the M, to 1.4 x 106 andincreased the dispersity to 2.3.

TABLE 3. Extraction of PHB from strain UWD withaqueous NH3

Initial cell % of initial dry wt % of final dry wt Proteinmass PHB RM PHB Protein extracted

(gfliter)a content extracted content content

7 74 11 85 3.5 7620 80 9 89 3.3 6050C 82 1 83 8.5 050d 84 10 94 1.5 77SOC 83 11 92 0.3 96200 86 6 92 1.7 70

a All samples were treated with 1 N NH3 at 45'C for 10 min, except asnoted.

b Final grams of protein divided by initial grams of protein in PHB pellettimes 100%.

c Control treated in distilled water at 45'C for 10 min (mean values from fourseparate trials).

d Mean values from three separate trials.e This sample was treated with 1 N NH3 at 100'C for 10 min.

84%FIG. 6. Analysis of the extraction of protein and nonprotein RM

(NP-RM) from strain UWD by using aqueous NH3. The pelletconstituents are shown as percentages of PHB, protein, and NP-RM. Of the latter two fractions, an amount was extracted with NH3(shaded sectors), and an amount remained with the pellet (blacksectors).

Recycling the NH3 extraction waste fluid. The NH3 extractfluid was neutralized by titration with acetic acid, and thefinal NH4' content was determined colorimetrically. Thisneutralized extract and chemical ammonium acetate wereused to make glucose medium containing 15 to 25 mM NH4'and 0.1% FP (each in duplicate shake flasks). There was nosignificant difference (P = 0.05) in the yield ofPHB (in gramsper liter or percent dry weight) or the Ypls between thesesources of ammonium acetate at 15 mM (Table 4) or 25 mM(data not shown).The neutralized extract also was added to glucose medium

without FP to test whether the cell materials released intothe NH3 extract could substitute for FP (Table 4). The yieldof PHB (in grams per liter or percent dry weight) and Yplswas increased significantly (P = 0.05) in medium lacking FPby the presence of extract, but this was not equal to thatobtained with 0.1% FP. Therefore, the extract could berecycled as a source of NH4+ in the fermentation, but itcould not substitute fully for FP as a PHB yield promoter.

DISCUSSIONPeptones are not usually added toAzotobacter sp. culture

media, and this is for a good reason. The peptone- and yeastextract-mediated generation of enlarged, osmotically fragile,

TABLE 4. Reuse of the NH3 extract fluid as a nitrogen andpeptone sourcea

Addition to basal mediuma PHB PHB YP/sNH4' source (15 mM) FP (0.1%) (g/liter) (% dry wt) (g/g)

Ammonium acetate + 5.0 76 0.28- 1.1 46 0.10

Extract" + 4.6 70 0.29- 3.2 64 0.20

a Basal medium was 3% glucose in Burk's buffer salts. Shake flask cultures(in duplicate) were harvested after 22 h.

b The NH3 extract fluid was neutralized with acetic acid and diluted indistilled water.

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fungoid and pleomorphic cells has been documented in theseorganisms (41, 42). It has been suggested that the activeingredient in these extracts that affects A. vinelandii orAzotobacter chroococcum is glycine, possibly in combina-tion with other amino acids such as L-methionine, L-trypto-phan, or L-phenylalanine (5, 42). The inhibitory effect ofglycine on bacterial cell growth and the generation of en-larged, osmotically fragile cells has been known since atleast 1948 (23). It has been shown in a variety of gram-positive and gram-negative bacteria that glycine interfereswith peptidoglycan synthesis and results in the accumulationof UDP-glycopeptide precursors in the cell cytoplasm (14,36, 40). During the course of this study, we looked for theaccumulation of these UDP-glycopeptide precursors (39) inthe cytoplasm of strain UWD during growth in glucosemedium containing FP but observed no definite trends(hence, data are not shown). The concentration of glycinerequired to promote pleomorphism of A. vinelandii is 38 to50 mg/liter (42). We have confirmed these values by usingstrain UWD, where pleomorphism is first noticeable withglycine at 50 mg/liter and very definite at 100 mg/liter (datanot shown). The 0.1% FP used in these experiments adds 66mg of glycine per liter of glucose medium (calculated frommanufacturer's data sheets). This in combination with otheramino acids should be sufficient to promote pleomorphism.The UWD cells continue to replicate in glucose medium

containing FP, but the growth rate is slow compared withthat in beet molasses. Pregrowth in medium containing FPdoes not prepare the cells for better growth in the samemedium. In fact, these cells grow poorly and are more fragilethan cells given a shift up into medium containing FP. Thissuggests that the cells do not adapt to being pleomorphic butremain impaired in the ability to replicate. The shock ofbeing shifted into FP is demonstrated in Fig. 2. There is animmediate doubling of cell protein in response to the nitro-gen component of the added FP, and then the cells enter intocycles of slow cell growth and lysis. Continuous feeding ofFP does not reveal these cycles of cell lysis, but theypresumably are still occurring. The FP-grown cell populationis heterologous, with smaller motile cells that are likelyinvolved in replication and enlarged, osmotically fragilemonster cells that are likely terminal forms stuffed withPHB. Simply filling these monster cells with PHB cannotaccount for the increased yield of PHB obtained with FPaddition, but increased cell volume (e.g., 4.9 ,um3 of 2- to3-,um-diameter cells increasing to 28 to 50 ,um3 of 6- to8-,um-diameter monster cells) coupled to a modest increasein cell numbers (e.g., 10-fold, based on RM) increases thecell capacity for PHB accumulation 57- to 100-fold.The Ypls values of FP-grown cells were calculated to be

0.36, 0.43, and 0.65 g/g. The lowest value is typical ofgrowing cells metabolizing sugar by the Entner-Doudoroffpathway and producing PHB by an A. eutrophus-type bio-synthetic pathway (10, 12). The middle value is typical ofnongrowing cells using the same pathways for PHB, NAD+,and ATP production and NADPH recycling (45). Findingthis Ypls in cells cultivated continuously on FP is notunexpected as their growth was severely impaired. The Yplsvalue of 0.65 g/g is extremely high and unprecedented, but itis reproducible (this and unpublished work). The maximumtheoretical Ypls from glucose (based on chemical stoichiom-etry) is 0.72 g/g, but this assumes no loss of carbon as CO2(45). Since the very high Ypls occurred late in the culture ofFP-grown cells, it might be safe to assume that biochemicalpathways were also impaired. A minimum alteration wouldrequire reduced respiratory activity and efficient CO2 recy-

F-07~~~~~~~~~~~~~~~~~~~13 7 7

26 6~~~~~~~~~~~

10

3 7FIG. 7. Downstream extraction of PHB by an enzyme digestion

process (A) or an aqueous NH3 process (B). The units in thedownstream process include centrifuge (units 1), water input (units2), fermentation culture waste fluid (streams 3), holding tank (unit4), heat disruption cell (unit 5), blending tank (units 6), extractionwaste stream (streams 7), heat denaturation cell (unit 8), peroxidetreatment tank (unit 9), and polymer slurry to final spray drying (unit10). Further details are found in the text.

cling (fixation). In cases where there is no loss of CO2 fromthe growth substrate (e.g., by using butyrate or butanol), theYP/S can reach values of 0.65 to 0.77 g/g (45). It also ispossible that extensive lysis made available other carbonsources for PHB production, which are not accounted for inthe calculation of YP/fiucose, An analysis of enzyme activitiesin FP-grown cells is in progress.

Extraction of PHB from these pleomorphic cells was quitesimple. The use of aqueous NH3 as the extraction solvent isnovel and has not been reported previously. However, in ourexperience, NH3 extraction was only successful with strainUWD grown in FP medium (data not shown). Thus, it islikely that the compromised cell wall strength of thesepleomorphic cells is crucial to the success of the NH3extraction procedure. Analysis of the extraction processshows that protein is easily removed from the cell slurry andthere is some release of N-acetyl sugars, likely by thealkaline pH and ammonolysis of the weakened cell wallesters, respectively. The final preparation was "94% PHB,2% protein, and 4% nonprotein RM. These values comparefavorably with those obtained by using the heat disruptionand enzyme treatment process described by Holmes andLim (15).A comparison of the process described by Holmes and

Lim and summarized by Marchessault et al. (15, 24) and anNH3 extraction scheme is shown in Fig. 7. Basically, theenzyme extraction process (Fig. 7A) requires the following:(i) a tank equal to the volume of the fermentor (unit 4), wherethe culture is held as it is diluted from 80 to 100 g/liter to "50g/liter with process water; (ii) continuous heat disruption ofthe diluted suspension to lyse the cells and denature DNA(unit 5); (iii) concentration of the polymer and the generationof a large-volume waste stream (centrifuge 1 and stream 3);(iv) enzyme treatment in two blending tanks (units 6) sepa-rated by concentration (centrifuge 1 and stream 7) anddilution and heat treatment (unit 8) to protect the secondproteolytic enzyme from the first proteolytic enzyme; (v)concentration of the polymer (centrifuge 1 and stream 7) andthen bleaching in H202 (unit 9). The NH3 extraction proce-dure (Fig. 7B) would start with (i) concentration of the cellsand removal of the spent beet molasses waste stream (cen-trifuge 1 and stream 3), (ii) dilution of the pellet to 50 to 200g/liter and treatment with aqueous NH3 (unit 6) followed byextract removal (centrifuge 1 and stream 7), and (iii) a

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second treatment in NH3 or H202 added for good measure.Both downstream processes would end with concentrationof the polymer and spray drying (unit 10).The NH3 extraction procedure offers a number of real

savings in the downstream extraction of PHB. There is anobvious reduction in the amount of equipment in the processwhich should reduce the capital costs significantly. There isalso a considerable reduction in the waste stream volumes.In the enzymatic approach, the culture volume must at leastbe doubled and all wastes are directed to waste treatment. Inthe NH3 treatment scheme, the spent culture fluid is re-moved at the beginning, with the hope that this waste streamwill be used for something in the future (e.g., as an animalfeed). In any event, the results show that the culture at 50 to200 g/liter can be effectively treated with NH3, provided thePHB content of the cells is high. The NH3 extraction wastesalso can be recycled in the process as a nitrogen source in atwo-stage process (Table 4 and work in progress).

Glycine addition (23, 43) is a simple and effective treat-ment to weaken the cell wall and promote the release of cellconstituents. This old procedure is receiving renewed inter-est as a means of promoting periplasmic or cytoplasmicenzyme production and release from a variety of gram-negative and gram-positive bacteria (3, 4, 16, 17, 25, 46). Theintentional growth of pleomorphic cells, however, requires afine balance to be struck between cell wall softening and cellstabilization. How to stabilize these enlarged cells andpromote their growth is an interesting and potentially usefulsubject for study.

ACKNOWLEDGMENTS

We thank Luis D'Elia, Brent Rudy, and Yvonne Davidson forexcellent technical assistance.

This study was supported by grants from the Natural Sciences andEngineering Research Council of Canada, Strategic (biotechnology)and Cooperative Research and Development programs, and a con-tract from the Nova Corporation of Alberta.

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growth ofazotobacter vinelandii uwd in fish peptone medium ... · present study sought to examine...

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