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Poly(b-hydroxybutyric acid) thermoplastic productionby Alcaligenes latus: Behavior of fed-batch cultures

E. Grothe, Y. Chisti

Abstract Fed-batch culture ofAlcaligenes latus, ATCC29713, was investigated for producing the intracellularbioplastic poly(bhydroxybutyric acid), PHB. Constantrate feeding, exponentially increasing feeding rate, andpH-stat fed batch methods were evaluated. pH-stat fedbatch culture reduced or delayed accumulation of thesubstrate in the broth and led to signicantly enhancedPHB productivity relative to the other modes of feeding.Presence of excessive substrate appeared to inhibit PHBsynthesis, but not the production of cells. In fed-batchculture, the maximum specic growth rate (0.265 h)1)greatly exceeded the value (0.075 h)1) previously observedin batch culture of the same strain. Similarly, the maxi-mum PHB production rate (up to 1.15 g l)1 h)1) wasnearly 8-fold greater than values observed in batch oper-ations. Fed-batch operation was clearly superior to batchfermentation for producing PHB. A low growth rate wasnot a prerequisite for PHB accumulation, but a reduced ordelayed accumulation of substrate appeared to enhancePHB accumulation. Under the best conditions, PHB con-stituted up to 63% of dry cell mass after 12 h of culture.

The average biomass yield coefcient on sucrose wasabout 0.35, or a little less than in batch fermentations. Thehighest PHB concentrations attained were about 18 g l)1.

List of SymbolsA parameter in Eq. (1) (g l)1)B parameter in Eq. (1) (g l)1 h)1)CNH4 concentration of ammonium ion (mg l

)1)CC mass fraction of sucrose in the feed ())CN mass fraction of ammonia in pH control solution

())CT mass fraction of trace elements in feed solution ())E element

FC mass ow rate of the sucrose solution (g

h)1

)FN mass ow rate of the ammonia solution (g h)1)

Fs feed mass ow rate (g h)1)

FT mass ow rate of the trace element solution(g h)1)

m maintenance coefcient ())N concentration of nitrogen source (g l)1)PHA polyhydroxyalkanoatePHB poly(b-hydroxybutyric acid)qS maximum specic sucrose consumption rate

(g l)1 h)1)qP specic product formation rate (g l)

1

h)1)qPmax maximum specic product formation rate

(g l)1 h)1)S total amount of substrate in fermenter at time t(g)SDS sodium dodecyl sulfateS0 mass fraction of the substrate in feed ())t time (h)t0 x-intercept in Eq. (1) (h)TES trace element solution or its amount in the medium

(ml l)1)X0 total initial biomass in fermenter (g)Xt total biomass in fermenter at time t (g)YP/S product yield coefcient on sucrose ())

YP/X specic product yield coefcient ())YR/N PHB-free biomass yield coefcient

on nitrogen source ())YR/S PHB-free biomass yield coefcient

on carbon source ())YR/T PHB-free biomass yield coefcient

on trace elements ())YX/S biomass yield coefcient on sucrose ())

Greek symbolsd mass fraction of PHB in cells ())l specied growth rate (h)1)lmax maximum specic growth rate (h

)1)

1IntroductionMicroorganisms produce a variety of thermoplastics(Poirier et al., 1995; Kim et al., 1998; Braunegg et al.,1998). Unlike petrochemical-derived polymers, microbialthermoplastics are a renewable resource with importantadvantages of biodegradability, biocompatibility, and non-toxicity. Despite their relatively high cost, bioplastics areattracting increasing commercial attention (Kim et al.,1998). Microbial poly(b-hydroxybutyric acid), PHB, isespecially attractive because its properties are broadlysimilar to those of some well-established synthetic poly-

mers such as polypropylene (Barham, 1990); hence, PHBor its blends can potentially replace traditional synthetic

Bioprocess Engineering 22 (2000) 441449 Springer-Verlag 2000

4

Received: 17 May 1999

E. GrotheDepartment of Chemical Engineering,University of Waterloo,Waterloo, Ontario, Canada N2L 3G1

Y. Chisti (&)Department of Chemical Engineering,

University of Almera,E-04071 Almera, Spain

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polymers in some applications. PHB is resistant to waterand ultraviolet radiation, and impermeable to oxygen.PHB is readily biodegraded in soil. Moreover, processingPHB into articles of commerce does not require new in-vestments in technology; existing equipment, developedoriginally for processing polyethylene and polypropylene,

can be used. Because of it's high price about 15-foldgreater than comparable synthetic plastics applicationsof PHB are limited to specialist niches. The polymer ispotentially useful in slow release drug formulations andbiodegradable surgical implants; other uses are in devel-opment (Kim et al., 1998).

Low-cost production of PHB requires improved fer-mentation strategies, inexpensive media, and easierdownstream recovery methods (Chisti, 1998; Tamer et al.,1998a, b). This work reports on production of PHB insingle-stage fed-batch cultures of the bacterium Alcali-

genes latus. In the past a different bacterium, Alcaligeneseutrophus, has been the focus of attention as a producer of

PHB, but that microorganism requires an expensive two-stage cultivation (Byrom, 1990; Marchessault et al., 1990).Unlike A. eutrophus, A. latus is a growth-associated pro-ducer of PHB; hence, a single-stage fermentation is suf-cient (Hrabak, 1992; Hanggi, 1990). Furthermore, A. latusgrows readily on sucrose which is less expensive than theglucose-based media that are typically used in A. eu-trophus fermentations. Relatively little work has been re-ported on A. latus (Yamane et al., 1996; Wang and Lee,1997; Tamer et al., 1998a, b; Grothe et al., 1999) and someof this work focused exclusively on the downstreamrecovery of PHB (Tamer et al., 1998a, b).

As for any microbial production process (Ejiofor et al.,1996a; Chisti, 1999; Chisti and Moo-Young, 1999), theperformance of A. latus culture is susceptible to manyinuences, including temperature, pH, carbon-to-nitrogenratio in the feed, concentration of substrates, concentra-tion of trace elements, ionic strength, agitation intensity,and the dissolved oxygen level. Effects of some of thesevariables have been studied (Yamane et al., 1996; Wang

and Lee, 1997; Grothe et al., 1999) and optimal growth andproduction conditions in shake ask batch culture havebeen identied in statistically designed experiments(Grothe et al., 1999). Further work is needed in morecontrolled conditions than are possible in shake asks.Many examples exist of substantial enhancement in pro-

ductivity through optimization of fermentation underconditions that are relevant to industrial culture (Chistiand Moo-Young, 1996; Chisti, 1999) and similar work isneeded for the A. latus PHB fermentation. Moreover,compared to batch culture, fed-batch operation canenhance yield and productivity by eliminating possiblesubstrate inhibition, or by otherwise modifying metabolicbehavior. This work characterizes controlled fed-batchproduction of PHB using various feeding strategies.

2Materials and methods

2.1Microorganism and culture conditionsAn intracellular PHB producer, Alcaligenes latus, ATCC29714 (or DSM 1123), was used throughout. The strain wasmaintained on agar slants and Petri dishes on the minimalMedium 1018 (Table 1) as noted in the ATCC catalog(Gharda and Rienta, 1995; Grothe et al., 1999). After suf-cient growth at 33 C, the culture was held at 4 C untilneeded. Shake asks (200 ml) containing 50 ml minimalMedium 1018 were inoculated with a loopful of cells. After34 days growth (33 C, 200 rpm shaker) the 50 mlinoculum attained cloudiness. The inoculum was useddirectly for the next preculture stage, or held at 4 C untilneeded.

A further preculture step was necessary for inoculationof bioreactor. Thus, 5 ml of above described inoculum wastransferred to a 2 l shake ask containing Medium 1(Table 1) with 0.5 ml l)1 of trace element solution TES 1instead of the normal 1 ml l)1 noted in Table 1 (Laffertyand Braunegg, 1990; Ramsay et al., 1990a; Yamane et al.,

Table 1. Composition of cul-ture media Component Composition (g l

)1)

Medium 1018 Medium 1 Medium 2 Medium 3

Sucrose 20 20 20 20(NH4)2SO4 1 2 5.16 1.4

KH2PO4 4.4 1.5 0.14 1.5Na2HPO4 4.8 3.6 0.68 1.8MgSO47H2O 0.5 0.2 0.3 0.2Trace element solution (ml l)1) 1 1 1 1

Composition of trace element solution (g l)1)

TES 0 TES 1 TES 2 TES 3

Ammonium Fe(III)citrate 50 60 60 6CaCl22H2O 5 10 6.2 10H3BO3 0.3 2.8 0.3CoCl26H2O 0.2 3 0.2ZnSO47H2O 0.1 1 0.1MnCl24H2O 0.03 2 0.03Na2MoO42H2O 0.03 0.5 0.03NiSO47H2O 0.02 1 0.02CuSO45H2O 0.01 0.4 0.01

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1996). Phosphates were dissolved separately to preventprecipitation, and the trace element solution was ltersterilized. The measured pH was 7.2. After 4-days (33 C,200 rpm), the entire contents of the ask were used toinoculate 4.5 l of fermentation medium in the bioreactor.Unless otherwise noted, the fermentation medium was

identical to that used in preculture. All media were ster-ilized at 121 C, 20 min, and cooled to 33 C culture tem-perature that had been earlier established as optimal(Grothe et al., 1999). The fermenter was a 15 l (nominal)bottom-stirred device (MBR Bioreactor AG, Switzerland)equipped with pH, temperature, and dissolved oxygencontrollers. The 0.2 m diameter vessel was fully bafed (4-bafes). The working volume was 8 l. The starting volumeof each fermentation was 5 l. Two six-bladed impellers,both 0.1 m in diameter, were used on the same shaft. Thelower impeller was a downward pumping hydrofoil type,whereas the upper impeller was a Rushton turbine (Chisti,1999; Chisti and Moo-Young, 1999). One of the impellers

always remained above the culture level, serving to dis-perse foam. In addition, Sigma 298 silicon antifoam (SigmaChemical Co., St. Louis, MO, Catalog no. A 8436) wasautomatically dispensed in response to a foam sensor. Theagitation speed varied over 300800 rpm in response to adissolved oxygen controller. The dissolved oxygen con-centration generally remained above 15% of air saturationvalue. The aeration rate was held constant at 1 vvm. Theinoculation pH was 7.0. Once the pH declined to a previ-ously identied optimum of pH 6.5 (Grothe et al., 1999),it was controlled at that value by addition of aqueousammonia (20% w/v) solution.

Feeding commenced after an initial batch phase thatlasted up to $35 h. Three different feeding strategies weretested: (i) continuous constant rate feeding; (ii) exponen-tially increasing feed rate (Ejiofor et al., 1996); and (iii)feeding in response to a pH control signal (pH-stat) asdetailed later. Medium 1 and Medium 3 (Table 1) wereused for feeding in separate experiments.

2.2Cell dry massBiomass content was determined by gravimetry. Culturesamples (10 ml) were centrifuged (15,000 g, 4 min, 4 C),the supernatant was refrigerated for further analysis, andthe cell pellet was washed in deionized water, recovered(15,000 g, 4 min, 4 C), dried to constant weight (90 C,

24 h), cooled in a desiccator, and weighed. The biomassyield coefcient on sucrose (YX/S) was calculated as the celldry weight produced per unit mass of sucrose consumed.All measurements were in duplicate.

2.3SucroseThe supernatant (1 ml) from biomass determinations wasused in quantifying sucrose by refractive index measure-ments in an Abbe refractometer (Atago 3T, Japan). Therefractometer had been calibrated using dilutions offreshly prepared sucrose-containing uninoculated culturemedium (Grothe et al., 1999). In addition, a few of the

measurements were veried by HPLC (Millipore, Milford,USA) on a lead sulfate column. Automatic sample injec-

tion (Waters 700 Saterlite WISP, 20 ll injection volume)was employed. An external differential refractometer(model R401) was used as the detector. Chromatogramswere acquired and integrated with a Baseline 810 Chro-matography Workstation. Double deionized water was theeluent (85 C, 0.6 ml min)1). The HPLC calibration

standards were identical to those used for the refracto-meter (Grothe et al., 1999).

2.4AmmoniumA calibrated ammonia sensor electrode (model 8002-8,Electrical Instruments Ltd, Chertsey, U.K.) was used tomeasure the NH4 concentration. The measurement rangewas 502,000 mg l)1 as NH4 . Just prior to measurement,the supernatant (5 ml) from cell dry mass determinationswas made alkaline with concentrated sodium hydroxide(1 ml) to convert the dissolved ammonium ion toammonia (Grothe et al., 1999).

2.5Poly(b-hydroxybutyric acid)A gravimetric method similar to those employed previ-ously (Marchessault et al., l990; Ramsay et al., l990; Grotheet al., 1999) was used. A sodium dodecyl sulfate (SDS)solution (1% w/v SDS, 10 ml, pH 10) was added to thebiomass pellet obtained as described for cell dry massmeasurements. The mixture was incubated on an orbitalshaker (60 min, 200 rpm, 37 C). The solids were recov-ered by centrifugation (4 min, 7,000 g) and washed withcommercial sodium hypochlorite solution (Javex-5, Col-gate-Palmolive Canada Inc., Toronto; 1 ml, 5.64% w/vsodium hypochlorite) that had been diluted to 20 ml. The

pellet was centrifuged (4 min, 7,000 g), washed with de-ionized water (20 ml), and centrifuged again. The nalpellet was dried (90 C, 24 h) to constant weight in pre-weighed aluminum dishes (Grothe et al., 1999). The PHByield coefcient relative to biomass (YP/X) was calculatedas the mass of PHB obtained per unit cell dry weight.Measurements were in duplicate.

3Results and discussion

3.1Constant rate and exponentially increasingfeed rate cultures

In batch culture, Medium 1018 (Table 1) is known to yieldbiomass with $62% PHB content, but Medium 3 providesa higher total amount of PHB because it supports a sub-stantially greater quantity of biomass than does Medium1018 (Grothe et al., 1999). Similarly, Medium 3 signi-cantly outperforms Medium 1 and Medium 2 (Table 1) inproviding a greater nal amount of biomass and PHB forotherwise identical conditions (Grothe et al., 1999). Me-dium 2 had been developed (Grothe et al., 1999) using apublished elemental analysis of A. latus (Table 2) as thebasis. Medium 2 is richer in nitrogen compared to theother media listed (Table 1) and it is quite low in phos-phates. With respect to trace elements, Medium 2 is gen-

erally richer than the other media. Media 1 and 3 wereused in fed-batch experiments.

E. Grothe, Y. Chisti: Fed-batch production of poly(b-hydroxybutyric acid)

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The rst experimental fed-batch culture was fed at aconstant rate of 50 ml h)1 commencing after 33 h ofbatch operation. The feed was concentrated Medium 1(Table 1) that contained 50% sucrose and 10 ml TES 1 perliter, so that the sucrose feed rate was 5 g l)1 h)1. Theselection of the feed rate was based on an expected nalcell concentration and broth volume, an expected growthyield on sucrose YX/S of 0.37, and known initial liquidvolume in the bioreactor after inoculation (5 l). The rapidpickup in growth upon commencement of feeding (Fig. 1)is apparently due to alleviation of nitrogen limitation: by

20 h the nitrogen had declined to quite low levels, butmore than 10 g l)1 sucrose remained (Fig. 1). At 68 hwhen feeding ceased, the cell concentration was$27 g l)1 and PHB constituted 31% of the cell dryweight. The maximum specic growth rate was 0.105 h)1.At 68 h the culture entered a decline phase although suf-cient carbon and nitrogen were available. The constantfeed rate strategy was unsatisfactory as the carbon- andnitrogen substrates accumulated in the broth (Fig. 1).Accumulation of the carbon feed, or more likely the ni-trogen source, apparently contributed to the onset of thedecline phase. Throughout, the biomass yield coefcientYX/S was fairly stable at 0.20.4, but the PHB yield coef-

cient on biomass declined signicantly during rapidgrowth (Fig. 1).After 20 h, the initial pH of 7.0 declined to 6.3 (Fig. 1)

and automatic pH regulation with concentrated ammoniasolution commenced in order to maintain pH at a setpointof 6.5. The latter value had been identied as optimal forgrowth in batch culture (Grothe et al., 1999). The dy-namics of pH regulation were reected in the measuredconcentration of the ammonium ion (Fig. 1, lower part).During pH regulation, the ammonium concentration atany time t closely followed the empirical equation:

CNH4 Aett0aB Y 1

where the best-t values of the various parameters were:A (the y-intercept) = 12.52 g l)1; B (slope) = 16.53

g

l)1

h)1; and t0 (x-intercept) = 4.22 h. The exponentialprole of the ammonium concentration directly reectedthe exponential growth pattern of the biomass (Fig. 1).

In batch culture without pH control, Medium 3 (Ta-ble 1) had earlier been shown to be the best medium forobtaining an overall high production of PHB in A. latuscultures (Grothe et al., 1999). A second fermentation(Fig. 2) utilized Medium 3 at a lower constant feed ratethan earlier, in attempts to reduce possible substrate in-hibition. Note that carbon and nitrogen associated inhi-bition has been reported with A. eutrophus (Belfares et al.,1995), a microorganism that is related to A. latus usedhere. The feed rate was 44 ml h)1, or 4.4 g l)1 h)1 in

terms of sucrose. The culture (Fig. 2) employed lowerinitial sucrose and nitrogen levels than the earlier de-scribed fermentation (Fig. 1). The sucrose concentrationnever exceeded $15 g l)1. Ammonium sulfate was thenitrogen source in the batch phase. The initial C:N ratiowas 28.3. Both the nitrogen source and the C:N ratio usedhad been established as optimal in earlier work (Grotheet al., 1999). The cell concentration attained after 12 h was14.9 g l)1; the cells had 63% PHB by weight, or more thantwice the PHB content in the earlier noted fermentation(Fig. 1). The specic growth rate was also higher at0.265 h)1. The fermentation had to be stopped after 12 hbecause of leakage from the impeller shaft seal. The yield

coefcients YX/S and YP/X were fairly stable at 0.51 and0.63, respectively, all through the culture duration (Fig. 2).

Table 2. Composition of A. latus (percent of dry weight)

Element Percent C:E ratio

Carbon 47.90 1.0Oxygen 34.78 1.4Hydrogen 6.92 6.9

Nitrogen 6.22 7.7Sodium 1.25 38.3Phosphorus 1.045 45.8Potassium 0.225 212.9Sulfur 0.175 273.7Magnesium 0.163 293.9Iron 2.23 21.5Calcium 0.0095 5042Cobalt 0.0042 11405Manganese 0.0030 15967Boron 0.0027 17741Zinc 0.0013 36846Nickel 0.0013 36846Copper 0.0005 95800Molybdenum 0.0001 479000

Source: Yamane et al. (1996)

Fig. 1. Fed-batch fermentation prole ofA. latus. Time coursesare shown for biomass, PHB, sucrose, pH, ammonium, and thecalculated values of the yield coefcients YP/X and YX/S. Duringthe fed-batch phase, the culture was fed at a constant rate of50 ml h)1 using Medium 1


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As opposed to the constant feed rates of Figs. 1 and 2,the fermentation shown in Fig. 3 employed an exponentialfeeding method using Medium 3. Thus, based on the dataobtained in earlier fermentations, the following parametervalues were assumed: total initial biomass content X0 of35 g equivalent to 7 g l)1 biomass in the broth, a maxi-mum specic growth rate (lmax) of 0.27 h

)1, and a biomassyield coefcient on sucrose (YX/S) of 0.37. The theoreticalfeeding prole was calculated using the equation:

Fs X0

YXaSS0 lelt Y 2

where l is the specied growth rate, t is the fermentation

time, X0 is the total amount of biomass in the fermenter attime zero, Fs is the feed mass ow rate, and S0 is the massfraction of the substrate in the feed. The S0 value was 0.5.Equation (2) assumes rst order growth kinetics, i.e.:

Xt X0elt Y 3

where Xt is the total amount of biomass in the fermenter attime t. Equation (2) is derived from a mass balance on thesubstrate in the fermenter; thus, the substrate consump-tion rate is expressed as:

dS

dt FsS0

l

YXaSXt

qPYPaS

Xt mXt Y 4

where S is the total substrate in the fermenter at time t,Fs S0 is the substrate supply term, and the other terms on

the right-hand-side of the equation represent, respectively,the substrate consumption due to microbial growth,product formation, and maintenance metabolism. Here,qp is the specic product formation rate, and YP/S is theproduct yield based on substrate. Generally, under con-ditions of exponential growth, the maintenance coefcientm is quite small and the maintenance term may be ne-glected. Furthermore, because PHB is an intracellularproduct, the third term on the right-hand-side of Eq. (4)may be lumped with the second term; hence, Eq. (4)becomes:

dS

dt FsS0

lXt

YXaS X 5

When the substrate feed rate exactly matches the con-sumption rate, dS/dt = 0; hence, from Eq. (5) follows:

Fs lXt

YXaSS0X 6

Substitution of Eq. (3) into Eq. (6) yields the theoreticalfeed prole, Eq. (2).

Apparently because of a high assumed value ofl in Eq.(2), the calculated substrate feed rate exceeded the con-sumption rate and the sucrose concentration in the fer-menter rose rapidly (Fig. 3); thus, exponential feeding was

terminated at 9.5 h. After 21 h of culture, feeding was re-sumed, but at a constant rate of 4.4 g l h)1 until 45 h.

Fig. 2. Fed-batch culture prole of A. latus. Time courses areshown for biomass, PHB, sucrose, pH, ammonium, and the cal-culated values of the yield coefcients YP/X and YX/S. During thefed-batch phase (Medium 3), the feed rate was constant at44.4 ml h)1. The C:N ratio was 28.3

Fig. 3. Fed-batch culture prole of A. latus. Time courses areshown for biomass, PHB, sucrose, pH, ammonium, and the cal-culated values of the yield coefcients YP/X and YX/S. The feedrate increased exponentially for the rst 3 h then stopped; feedingcommenced again at a constant reduced rate (4.4 g l)1 h)1)after 21 h and continued until 45 h


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Around 10 h, most of the nitrogen had been depleted(Fig. 3) and the culture may have been nitrogen limited;hence, a reduced growth rate between 720 h (Fig. 3).With continuing nitrogen feed in response to the pHcontrol signal, the nitrogen level picked up beyond 20 hand this may have contributed to increased growth rate

after that time (Fig. 3). The sucrose level was always abovethe limiting value beyond 10 h of culture. At 7 h, thecalculated maximum specic growth rate was 0.254 h)1, orlower than the value initially assumed for calculating thefeed prole. After 48 h, the maximum biomass concen-tration was 16.3 g l)1, corresponding to a product yieldof$50%. The maximum PHB productivity was estimatedat 0.544 g l)1 h)1. The maximum sucrose consumptionrate was 3.9 g l)1 h)1, or $89% of the assumed value.

The pH regulation commenced at 7 h with the fed-batchphase (Fig. 3). Again, during pH regulation, the ammo-nium ion concentration increased exponentially with timeaccording to the equation:

CNH4 11X2 et14X8a7X9 Y 7

that conrmed CNH4 as a direct feed-back indicator of thebiomass growth. The product yield coefcient on biomassincreased from 0.4 to 0.5 during culture (Fig. 3); however,the biomass yield on sucrose declined from a high value of0.6 to 0.4. This meant that a greater percentage of sucrosewas converted to PHB in the earlier half of the fermenta-tion than in the later half. Toward the end, the PHB yieldon sucrose was 16%.

3.2pH-stat fed batch cultureOne way of controlling pH in pH-stat fed-batch culture isto couple the feeding rate to the consumption of the pHcontrol additive. For example, for an acid producing fer-mentation, the quantity of acid produced is a function ofthe growth rate and the amount of biomass (i.e., the rateof consumption of the carbon source); thus, the amount ofalkali needed to neutralize the acid is proportional to thesubstrate consumption rate. In principle, feeding in re-sponse to demand of alkali for pH control should assurethat the nutrients are fed in proportion to their rate ofconsumption; hence, the level of nutrients in the fermentermay be held nearly constant (Yamane et al., 1996). In thefed-batch phase, when the added ammonium is the solenitrogen source, and at steady state, a biomass balance

may be written as:

CCFCYXaS CNFNYRaN CTFTYRaT X 8

Here CC, CN, and CT are the mass fractions of the carbonsource, the nitrogen source, and trace elements in the re-spective feed solutions. The ow rates of the carbon, ni-trogen, and the trace element feed streams are FC, FN, andFT, respectively. The yield coefcient of the biomass (cellsand intracellular PHB) on the carbon source is YX/S; andthe yield coefcients of PHB-free biomass (or `residual'biomass) on ammonium and trace elements are YR/N andYR/T, respectively. Note that the carbon source is con-sumed both in producing PHB and the PHB-free biomass,

whereas PHB does not contain nitrogen and traceelements. From Eq. (8), the ratios of carbon-to-nitrogen

and trace-elements-to-nitrogen feeds are readily shownto be:

FCFN

CNCC

YRaN

YXaSY 9

and

FTFN

CNCT

YRaNYRaT

Y 10

respectively. Because

YXaS YRaS

1 dY 11

Equation (9) may be written as:

FCFN

CNCC

YRaN

YRaS1 d Y 12

where d is the mass fraction of PHB in the cells.A pH-stat method was used for the fed-batch phase of

the fermentation proled in Fig. 4. Approximately 1.5 l of50% w/v sucrose solution and 160 ml of 20% w/v ammoniasolution were fed from separate reservoirs held on bal-ances (Mettler, PE11 or PM3000) and continuously stirredwith a magnetic stirrer. The feeds were supplied by using asingle peristaltic pump drive with two pump headsequipped with different diameter tubing to obtain thedesired ratio of carbon-to-nitrogen feeds. The trace ele-ments were fed with the sucrose medium that contained10 ml l)1 trace element solution. The pump was con-trolled in response to a signal from a pH controller.

Fig. 4. Time prole of A. latus fed-batch culture. A pH-stat fed-batch method was used with FC/FN = 10


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The fed-batch phase commenced as soon as the pH ofbatch culture dropped to below the pH set point of 6.5(Fig. 4). Published values of YX/S = 0.37 and YR/N = 3.92(Yamane et al., 1996) were used in Eq. (9) to estimate anexpected value of FC/FN of 9.64; however, an experimentalFC/FN value of 10 was used for the fermentation proled in

Fig. 4. Control of pH commenced at 17 h and feedingcommenced at 27 h of culture when the pH had droppedto below the set point value of 6.5. As shown in Fig. 4, theconcentration of sucrose did not remain constant althoughthe pH was controlled effectively. Apparently, the esti-mated value ofFC/FN was high because of discrepancies inthe YX/S and YR/N values reported in the literature (Ya-mane et al., 1996) and those actually observed. Also, d andYX/S varied during the fermentation (Fig. 4) whereas theyhad been assumed constant in calculating the FC/FN ratio.The maximum nal PHB content in the biomass was 53%.The maximum specic growth rate was estimated to be0.149 h)1; the maximum rates of PHB synthesis and

sucrose consumption were 0.782 and 3.57 g

l)1

h)1

,respectively.The fermentation proled in Fig. 5 also used a pH-stat

feeding mode as explained previously, except that thevalue of the FC/FN ratio was reduced from 10 (Fig. 4) to 7(Fig. 5). Consequently, a desired low sucrose concentra-tion was maintained over a longer period (Fig. 5). Thefeeding and pH regulation commenced together at 24 hand ended at 40 h. For a short period near the end of thebatch phase, insufciency of nitrogen (Fig. 5, lower part)may have limited cell growth. The maximum specicgrowth rate was 0.154 h)1 and the nal PHB content inbiomass reached 52%. The PHB productivity qP was0.99 g l)1 h)1 for a sucrose consumption rate of3.42 g

l)1

h)1. The biomass yield on sucrose was about

40%. Both the constant rate fed cultures (Figs. 1, 2) ac-cumulated sucrose, suggesting a greater than needed feedrate value.

3.3Comparison of feeding methodsThe ve fermentations and the three feeding strategiesused are compared in Table 3 in terms of several measuresof process performance. In general, feeding methods thatreduced or delayed accumulation of substrates led tohigher productivity of PHB. Thus, at 0.97 g l)1 h)1 themean PHB productivity of fermentations 2, 4 and 5

(Table 3) was more than twice a mean value of 0.43g l)1 h)1 for fermentations 1 and 3. Also, from Table 3,while accumulation of substrate had a strong negativeinuence on PHB productivity, substrate accumulationhad little effect on biomass productivity. The latter waswithin 22% of a mean value of 3.49 g l)1 h)1 for the vefermentations. Note from Table 3, that a low specicgrowth rate (e.g., in fermentation 1) is not a requirementfor high accumulation of PHB, but a reduced substrateinhibition is. High specic growth rates combined withcontrolled feeding to reduce substrate accumulation en-hance PHB productivity as in fermentations 2, 4, and 5.Consequently, a workable method of feeding, such thatsubstrate supply does not limit growth yet there is noaccumulation of substrate, is indicated for improvingproductivity and yield of PHB. Of the feeding methods

Fig. 5. Time prole of A. latus fed-batch culture. A pH-stat fed-batch method was used with FC/FN = 7

Table 3. Comparison offeeding strategies Parameter Feed strategy

Continuous constant rate Exponential pH-stat

Fermentation no. 1 2 3 4 5Culture time (h) 68 12 51 48 46.5Biomass (g l)1) 24.7 14.9 32.9 27.3 35.4PHB (g l)1) 8.5 9.4 16.3 14.5 18.2l (h)1) 0.105 0.265 0.254 0.149 0.154qPmax (g l

)1 h)1) 0.31 1.15 0.54 0.78 0.99

qSmax (g l)1

h)1) 3.15 3.40 3.92 3.57 3.42YP/X 0.31 0.63 0.49 0.53 0.51YX/S 0.24 0.51 0.36 0.29 0.39YP/S 0.15 0.32 0.16 0.15 0.21


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examined, pH-stat strategy was useful but not entirelysatisfactory in controlling substrate accumulation. Themethods used, invariably required a priori knowledge ofparameters such as specic growth rates, yield coefcients,and the biomass concentration at the end of the batchphase. Because these parameters vary, the fermentations

generally tended to accumulate the substrate at variousrates.

One fed-batch culture method that potentially canfeed to match the maximum substrate demand and doesnot cause substrate accumulation, is that of Oh et al.(1998). That method, based on on-line monitored sub-strate uptake rate, does not require assumptions or apriori data on specic growth rates, yield coefcients, orthe biomass concentration. The method needs on-linemeasurements of dissolved oxygen, but does not requiremonitoring of the substrate concentration. In view ofthese advantages, the feeding method of Oh et al. (1998)is recommend as having the potential to enhance PHB

productivity.Compared to published data for the same strain inbatch culture (Grothe et al., 1999), the highest observedmaximum specic growth rate in fed-batch operation was3.5 times more. Similarly, the maximum PHB productionrate was nearly 8-fold greater than values observed inbatch culture. Clearly, relatively controlled fed-batch cul-tivation of A. latus is superior to batch fermentation forproducing PHB.

4ConclusionsFed-batch culture is superior to batch fermentation of

A. latus in producing PHB. Among the various feedingoptions, those that reduce or delay accumulation of sub-strate substantially improve PHB productivity relative tocases when the substrate is allowed to build up in thebroth. Thus, the maximum PHB production rate (up to1.15 g l)1 h)1) was nearly 8-fold greater than valuesobserved in batch operations. Under the best conditions,PHB constituted up to 63% of dry cell mass after 12 h ofculture. The maximum PHB concentration attained wasabout 18 g l)1, but this value may be further enhanced byimproved control of feeding. Feeding needs to be con-trolled so that microbial growth is not limited, yet theconcentration of substrate in the broth remains low orincreases slowly to a low upper limit so that the substrate

inhibition of PHB synthesis is minimal.

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