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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2009, p. 6222–6231 Vol. 75, No. 190099-2240/09/$08.00�0 doi:10.1128/AEM.01162-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Poly(3-Hydroxybutyrate) Production from Glycerol byZobellella denitrificans MW1 via High-Cell-Density
Fed-Batch Fermentation and SimplifiedSolvent Extraction�
Mohammad H. A. Ibrahim and Alexander Steinbuchel*Institut fur Molekulare Mikrobiologie und Biotechnologie, Westfalische Wilhelms-Universitat Munster, 48149 Munster, Germany
Received 19 May 2009/Accepted 28 July 2009
Industrial production of biodegradable polyesters such as polyhydroxyalkanoates is hampered by highproduction costs, among which the costs for substrates and for downstream processing represent the mainobstacles. Inexpensive fermentable raw materials such as crude glycerol, an abundant by-product of thebiodiesel industry, have emerged to be promising carbon sources for industrial fermentations. In this study,Zobellella denitrificans MW1, a recently isolated bacterium, was used for the production of poly(3-hydroxybu-tyrate) (PHB) from glycerol as the sole carbon source. Pilot-scale fermentations (42-liter scale) were conductedto scale up the high PHB accumulation capability of this strain. By fed-batch cultivation, at first a relativelyhigh cell density (29.9 � 1.3 g/liter) was obtained during only a short fermentation period (24 h). However, thePHB content was relatively low (31.0% � 4.2% [wt/wt]). Afterwards, much higher concentrations of PHB (upto 54.3 � 7.9 g/liter) and higher cell densities (up to 81.2 � 2.5 g/liter) were obtained by further fed-batchoptimization in the presence of 20 g/liter NaCl, with optimized feeding of glycerol and ammonia to support bothcell growth and polymer accumulation over a period of 50 h. A high specific growth rate (0.422/h) and a shortdoubling time (1.64 h) were attained. The maximum PHB content obtained was 66.9% � 7.6% of cell dry weight,and the maximum polymer productivity and substrate yield coefficient were 1.09 � 0.16 g/liter/h and 0.25 �0.04 g PHB/g glycerol, respectively. Furthermore, a simple organic solvent extraction process was employed forPHB recovery during downstream processing: self-flotation of cell debris after extraction of PHB with chlo-roform allowed a convenient separation of a clear PHB-solvent solution from the cells. Maximum PHB recovery(85.0% � 0.10% [wt/wt]) was reached after 72 h of extraction with chloroform at 30°C, with a polymer purityof 98.3% � 1.3%.
Polyhydroxybutyrate (PHB) is the best-studied example ofbiodegradable polyesters belonging to the group of polyhy-droxyalkanoates (PHAs), which are synthesized by many bac-teria and archaea as intracellular carbon and energy reserves(1, 40, 43). In the last decades, these biopolymers have re-ceived great attention due to their properties which resemblethose of conventional petrochemical-based polymers (49). Forinstance, PHB is very similar to thermoplastic polypropylene(17). Their production from renewable resources and theircomplete biodegradability give PHAs promising advantagesfrom an environmental point of view (6). In addition to theirspecial physical traits, such as the elasticity of medium-chain-length PHAs and the high crystallization rate of PHB, PHAsare biocompatible, water resistant, oxygen impermeable, andenantiomerically pure; all of these characteristics broaden thescope of their applications in industry and medicine.
So far, higher production costs than those of petrochemicalplastics have hindered the successful commercialization ofPHB (9). Many efforts have been devoted to reducing theproduction costs by developing superior microbial strains ca-
pable of utilizing cheap substrates and also by applying moreefficient fermentation strategies and economical recovery pro-cesses (10).
Fed-batch fermentation regimens are usually applied toachieve a high cell density, which is necessary for a high pro-ductivity and yield, in particular in cases of intracellular prod-ucts, by frequent or continuous feeding of nutrients whengrowth proceeds (46). Several fed-batch fermentation pro-cesses have been reported for PHA production (21, 28). Thereare two prevalent cultivation modes for PHB production thatare imposed on the microorganisms being used. The morefrequently used mode is realized by a complex two-stage cul-tivation process. In this mode, all nutrients needed for growthto a high cell density are provided during the first phase of theprocess. In the second phase, imbalanced growth conditionsare enforced by providing growth-limiting amounts of nutrientssuch as nitrogen, phosphate, or oxygen to trigger PHA biosyn-thesis and accumulation. The model organism for this mode isRalstonia eutropha (formerly known as Alcaligenes eutrophusand recently reclassified as Cupriavidus necator) (26, 27). In theother cultivation mode, PHB is accumulated concurrently withgrowth, and therefore a single-stage process is applicable. Awell-known example of this mode is PHB production by Al-caligenes latus (18, 47).
Although several new downstream processes for the extrac-tion of PHA have been reported as being economically effec-
* Corresponding author. Mailing address: Institut fur MolekulareMikrobiologie und Biotechnologie, Westfalische Wilhelms-UniversitatMunster, Corrensstrasse 3, D-48149 Munster, Germany. Phone: 49-251-8339821. Fax: 49-251-8338388. E-mail: [email protected].
� Published ahead of print on 7 August 2009.
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tive, such as the application of surfactants and hypochlorite (9,38), dispersions of hypochlorite solution and chloroform (14,15), and the selective dissolution of cell mass by proteolyticenzymes (25) or by sulfuric acid and hypochlorite (48), solventextraction methods are still regarded as an adequate way togain intact polymers with a high purity and recovery yield (39).However, there is still a need to develop and improve theseextraction methods further to make the entire process muchsimpler and cheaper (22).
In addition to increased prices for crude oil, the abundanceof inexpensive raw materials from agriculture and industry ascheap substrates for microbial fermentations, such as crudeglycerol from the biodiesel industry, could make the produc-tion of PHA from renewable resources more competitive withcommon plastics (32). Due to increased glycerol production bythe growing biodiesel industry, the prices for glycerol becamelow enough to make this residual compound a cheap carbonsource for several industrial fermentation processes, especiallyfor the production of microbial polyesters (11, 34). However,the various amounts of actual fermentable substrates and thepresence of other nonfermentable components in feedstock,such as the various concentrations of glycerol and salts inbiodiesel coproducts, hinder their use (42). Therefore, tolerantbioprocesses and/or strains tolerant to such variable factors arerequired.
The production of PHA from glycerol has been investigatedin only a few studies (4, 12, 24, 33, 42). In a recent study (32),crude glycerol from different biodiesel manufacturers was ex-amined for suitability as a substrate for PHB production. How-ever, significant decreases in PHB productivity and productyields were recorded when NaCl-contaminated crude glycerolwas used.
Recently, a newly isolated bacterium, Zobellella denitrificansMW1, was characterized as producing large amounts of PHBfrom glycerol, with enhanced growth and polymer productivityin the presence of NaCl (20). The present study aimed atdeveloping a strategy to improve the volumetric production ofPHB by Z. denitrificans MW1, using glycerol as the sole carbonsource. For this purpose, several fed-batch cultivations wereset up to steadily improve the nutrient supply to attain a highcell density and high PHB productivity. Moreover, the conven-tional organic solvent extraction method was modified withregard to an economically more feasible large-scale PHB ex-traction, achieving a high purity and recovery of the polymer.
MATERIALS AND METHODS
Microorganism and culture conditions. Z. denitrificans strain MW1, a recentlyisolated and characterized bacterium accumulating PHB from glycerol (20), wasused in this study. Cells were grown in mineral salt medium (MSM) (41)containing the following (g/liter): Na2HPO4 � 12H2O, 9.0; KH2PO4, 1.5;MgSO4 � 7H2O, 0.2; NH4Cl, 1.0; CaCl2 � 2H2O, 0.02; and Fe(III)NH4
�-citrate,0.0012. The MSM also included 1 ml of trace element solution containing thefollowing (g/liter): EDTA, 50.0; FeCl3, 8.3; ZnCl2, 0.84; CuCl2 � 2H2O, 0.13;CoCl2 � 6H2O, 0.1; MnCl2 � 6H2O, 0.016; and H3BO3, 0.1. Glycerol (15 g/liter)was used as the sole carbon source. The pH was adjusted to 7.3 before steril-ization. MSM with 15 g/liter agar was used as a solid medium for maintenance ofthe bacterium at 4°C. Precultures were grown in 250-ml and 2-liter Erlenmeyerflasks containing 50 and 400 ml MSM, respectively. The flasks were incubated at41°C and 200 rpm for 24 h.
Cultivation at 42-liter scale. A Biostat UD-30 stainless steel reactor (B. BraunBiotech International, Melsungen, Germany) with a total volume of 42 liters(28-cm inner diameter and 71-cm height) and a d/D value relation (relation of
stirrer diameter to vessel diameter) of 0.375 was used for cultivations at the30-liter scale. This bioreactor was equipped with three stirrers, each containingsix paddles and a Funda-Foam mechanical foam destroyer (B. Braun BiotechInternational, Melsungen, Germany). In addition, sterilized probes were insertedinto ports to measure dissolved oxygen (pO2) (model 25; Mettler Toledo GmbH,Steinbach, Switzerland), pH (model Pa/25; Mettler Toledo GmbH), foam (modelL300/Rd. 28; B. Braun Biotech International), temperature (pt 100 electrode;M. K. Juchheim GmbH, Fulda, Germany), and optical density at 850 nm (OD850)(model CT6; Sentex/Monitek Technology Inc.). The operations were controlledand recorded by a digital control unit in combination with the MFCS/win soft-ware package (B. Braun Biotech International).
Cultivations were done at 41°C and at a pO2 between 0 and 100% saturationin the medium, which was controlled by agitation rates between 100 and 800 rpmand aeration rates between 0.4 and 1.67 volume per volume per minute. Unlessstated otherwise, the pH in the medium was held between 7.0 and 7.3 bycontrolled addition of 4 N HCl or NaOH. Foam was removed by a mechanicalfoam destroyer; if this was not sufficient, the antifoam agent Silikon AntischaumEmulsion SLE (Wacker, Darwin Vertriebs GmbH, Ottobrunn, Germany) wasadded. Small samples were withdrawn from the culture fluid for analytical pur-poses.
Cell harvest from 42-liter cultivations. Cells were harvested by centrifugationin a CEPA type Z41 or type Z61 continuous centrifuge (Carl Padberg Zentri-fugenbau GmbH, Lahr, Germany). Harvested cells were frozen at �30°C andthen lyophilized (Beta 1-16; Christ, Osterode, Germany).
Determination of cell growth. Growth of Z. denitrificans MW1 was monitoredby measuring the increase of the OD600. Moreover, defined volumes of cultureswere harvested by centrifugation for 20 min at 1,200 � g and 4°C; afterwards, thecells were washed with distilled water, frozen, and lyophilized. Cell density,defined as the cell dry weight (CDW) per liter of culture broth, was determinedby weighing aliquots of lyophilized cells.
Fluorescence microscopy. The presence of cytoplasmic PHA inclusions wasevidenced by staining the biopolymer with Nile red and by observing the cellsunder a fluorescence microscope (30).
PHB extraction from Z. denitrificans MW1 whole cells. Small-scale solventextraction experiments were done to investigate the extraction efficiencies ofdifferent organic solvents for optimum PHB recovery from Z. denitrificans MW1cells. One hundred milligrams of lyophilized cells was mixed overnight at roomtemperature with 20 volumes of different solvents (chloroform, methylene chlo-ride, carbon tetrachloride, diethyl ester, ethyl acetate, and mixtures of chloro-form and acetone [1:1, 1:2, 1:3, 1:4, 2:1, 3:1, and 4:1] [vol/vol]) in 2-ml sealed glasstubes. After extraction, cells were separated by flotation or precipitation, de-pending on their behavior in the solvent being used. PHB was recovered aftersolvent evaporation. Larger-scale solvent extraction experiments were done bystirring 100 g of lyophilized cells in 1,000 ml chloroform or methylene chloride(10 volumes) at a temperature of 24, 30, or 41°C. Samples of 50 ml werewithdrawn after different incubation periods lasting up to 228 h. Before filtration,samples were left overnight in separation funnels until complete flotation of cellshad occurred, thereby yielding a clear PHB-solvent solution for easy and simplefiltration. After filtration, the PHB-solvent solutions were concentrated by dis-tillation (solvent recovery), and 2 to 4 volumes of cold methanol was used toprecipitate the PHB. Afterwards, the precipitated PHB was filtered and driedunder vacuum.
Analyses of ammonium and glycerol. The concentrations of ammonium incell-free supernatants were determined by employing a gas-sensitive type152303000 ammonium electrode (Mettler Toledo GmbH, Greifensee, Switzer-land). Analysis of residual carbon sources was carried out with a LaChrom Elitehigh-performance liquid chromatography apparatus (VWR-Hitachi Interna-tional GmbH, Darmstadt, Germany) consisting of a Metacarb 67H advanced Ccolumn (Bio-Rad Aminex equivalent; Varian, Palo Alto, CA) and a VWR-Hitachi model 22350 column oven. The column (300 mm by 6.5 mm) consistedof a sulfonated polystyrene resin in the protonated form. The primary separationmechanism included ligand exchange, ion exclusion, and adsorption. A VWR-Hitachi refractive index detector (type 2490) with an active-flow-cell temperaturecontrol and automated reference flushing eliminating temperature effects on therefractive index baseline was used for detection. Aliquots of 20 �l were injectedand eluted with 0.005 N sulfuric acid in double-distilled water at a flow rate of 0.8ml/min. Online integration and analysis of the data were done with EZ ChromeElite software (VWR International GmbH, Darmstadt, Germany).
Quantitative analyses. To determine the PHB contents of the cells, sampleswere subjected to methanolysis in the presence of 15% (vol/vol) sulfuric acid.The resulting 3-hydroxybutyric acid methyl esters were analyzed by gas chroma-tography (GC) (19). PHB content (% [wt/wt]) was defined as a percentage ofCDW. The purity of extracted PHB was also determined by GC from a known
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mass of solvent-extracted PHB. The percentage of PHB recovered was calculatedfrom a known amount of PHB in the biomass, based on the purity of the totalmass of samples recovered. The substrate conversion factor (g PHB/g glycerol)was calculated as grams of PHB produced per grams of glycerol utilized. Allresults are from duplicate or triplicate measurements, and mean values andstandard deviations are presented.
RESULTS
Production of PHB by fed-batch cultivation of Z. denitrifi-cans MW1. Four fed-batch fermentations at the 42-liter scalewere carried out to obtain high cell densities and high polymercontents of cells of the newly isolated bacterium Z. denitrificansMW1, using glycerol as the sole carbon source. In accordancewith the results of a previous study done with small Erlenmeyerflasks (20), cultivation temperature and pH were controlled at41°C and 7.3, respectively. In the first fermentation (Fig. 1), anovernight seed culture was used to inoculate 24 liters of MSM(4% [vol/vol] inoculum size). Feeding was started after 6 h ofbatch culture, using a 50% (vol/vol) solution of glycerol. Thedissolved oxygen (pO2) level in the medium was controlled auto-matically at 30% by increasing the agitation and aeration rates to800 rpm and 1.67 volume per volume per minute, respectively.
The feeding regimen was designed to provide sufficientamounts of glycerol (10 to 20 g/liter) to support both cell growthand polymer accumulation during growth of Z. denitrificansMW1. Ammonium chloride solution (25% [wt/vol]) was addedif the ammonium concentration in the culture fell below 1.0g/liter. The specific growth rate (�) during the batch phase was0.09/h, and the maximum specific growth rate recorded was0.351/h, with a doubling time of 1.97 h. At the end of thisfermentation (24 h), a high cell density and also high volumet-ric polymer productivity were obtained (29.9 � 1.3 g CDW/liter and 9.28 � 1.7 g PHB/liter, respectively). However, the
PHB content was relatively low (31.0% � 4.2% [wt/wt]). Thetwo main problems which prevented the extension of this fer-mentation to reach higher cell densities and polymer contentswere the excessively added NaOH solution to control the sharpdecrease in pH during the last 6 hours of this fermentation,which should cause significant cell lysis, and the increasedmedium volume because of nutrient feeding and base addition.The total amount of 4 kg glycerol used yielded a total of 0.33kg PHB, thereby indicating a low substrate conversion factor of0.10 � 0.02 g PHB/g glycerol.
Fed-batch fermentation in the presence of NaCl. In the nextfed-batch fermentations (batches 2, 3, and 4) (Table 1), thereported enhancing effect of sodium chloride on growth andpolymer accumulation (20) was investigated on a large scale(42 liters). In addition, some modifications in the feeding strat-egy were conducted. A 24-h-old preculture (800 ml) with ap-proximately 3 g CDW/liter was used to inoculate 20 liter MSMcontaining 15 g/liter glycerol. The total amount of sodiumchloride for 30 liters (600 g) was added at the beginning of thefermentation. The feeding solution used contained 1% (wt/vol)MgSO4 and 50% (vol/vol) glycerol. The pO2 was kept at 20%saturation. Culture pH was controlled at 7.3 during the first20 h of growth and then at 6.8 until the end of fermentation,using sodium hydroxide (4 N) and ammonia water (25% [wt/vol]); the latter was also used as a nitrogen source, as well asNH4Cl (25% [wt/vol]). In fed-batch 2, a similar cell density(32.8 � 0.6 g CDW/liter) to that of fed-batch 1 was reached, inspite of using only half the amount of glycerol (Table 2). Therelatively optimized cultivation conditions resulted in a higherpolymer content of 52% � 1.4% (wt/wt), producing 17.1 �0.8 g PHB/liter after 36 h of cultivation. The product yield wasimproved to 0.26 � 0.01 g PHB/g glycerol.
FIG. 1. Fed-batch cultivation of Z. denitrificans for PHB production from glycerol in the absence of NaCl (fed-batch 1). Z. denitrificans MW1was cultivated in a Biostat UD-30 stirred-tank reactor containing 24 liters of MSM with glycerol as the sole carbon source. The bioreactor wasinoculated with 4% (vol/vol) 18-h preculture. The pO2 was controlled automatically at 30% saturation by adjusting aeration and agitation rates.Cells were grown for 24 h at 41°C and pH 7.3. During the time course of cultivation, samples were withdrawn, and the concentrations of glyceroland ammonium, as well as the cell density and the PHB content of the cells, were determined as described in Materials and Methods.
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Using these improved results, further optimizations weredone using the same fed-batch technique, but with increasedamounts of glycerol and ammonia. Aiming to reach a highercell density despite the lack of information available for therecently isolated Z. denitrificans strain MW1, nutrient require-ments for cell growth were estimated according to the generalcalculation method for bacteria (13). In the third fed-batchfermentation (Fig. 2), the same feeding solution used in fed-batch 2 was supplemented with trace elements, iron, and cal-cium, providing two times their original concentrations inMSM during the entire time course of the fermentation. Thetotal estimated amounts of glycerol and ammonia were fed tokeep concentrations of 10 to 20 g/liter of glycerol and 1 to 2g/liter of ammonium. In this fed-batch fermentation, signifi-cant increases in cell density (195%) and volumetric PHBproductivity (201%) compared to those in fed-batch 2 wereobtained using a semicontinuous feeding regimen, which pro-vided adequate carbon and nitrogen for growth and polymeraccumulation over a period of 63 h. In addition, the polymercontent increased slightly, to 56% � 0.7% (wt/wt), and thePHB productivity increased substantially, to 0.57 � 0.02 g/liter/h. The substrate conversion factor was kept higher than0.2 g PHB/g glycerol.
In the last fed-batch fermentation (fed-batch 4) (Fig. 3), ahigher cell density and polymer content were targeted whilekeeping the satisfying product yield obtained during the pre-vious fed-batch cultivations. The concentration of glycerol inthe feeding solution was increased to 80% (vol/vol). NH4OH(25% [wt/vol]) was used in parallel with NaOH (4 N), insteadof ammonium chloride solution, for improved control of thepH of the medium and also to provide enough nitrogen forgrowth. On the basis of offline analytical data (high-perfor-mance liquid chromatography analysis of the glycerol concen-tration in samples withdrawn from the bioreactor), the feedingrate was increased or decreased if necessary during continuousfeeding to keep the glycerol concentration between 10 and 20g/liter. The maximum growth rate and doubling time were0.422/h and 1.64 h, respectively. The highest cell densityachieved was 81.2 � 2.5 g CDW/liter, and the maximum OD600
obtained was 351 � 5.3. In addition, a high PHB content(66.9% � 7.6% [wt/wt]) was reached after a relatively shortfermentation time (50 h), thereby enhancing the polymer vol-umetric productivity up to 54.2 � 7.9 g PHB/liter, with animproved product yield (0.25 � 0.04 g PHB/g glycerol). Insummary, during this fed-batch fermentation (30-liter final vol-ume), 2.44 kg CDW containing 1.63 kg PHB was producedfrom 6.52 kg glycerol. Growth-associated PHB accumulation
was monitored microscopically. Figure 3C shows the increasedprevalence of PHB granules inside Z. denitrificans MW1 cellsduring fed-batch fermentation 4.
Extraction of PHB from Z. denitrificans MW1 cells. In thisstudy, extraction of PHB from cells of Z. denitrificans MW1was investigated by different methods, such as the use ofdetergents or decolorizing agents, dispersion of cells in hy-pochlorite solution and chloroform, and the selective disso-lution of cell mass by sulfuric acid and hypochlorite. How-ever, the degree of purity obtained by these methods wasvery low in comparison to the purity of PHB prepared bysolvent extraction (data not shown). Many problems werefaced during separation of non-PHB cell materials or duringresidual hypochlorite wash-off steps, in addition to an exces-sive need for high-speed centrifugation. These problems wouldbe magnified in the large-scale extraction process for PHB andwould require special precautions, especially if highly viscousPHB-solvent solutions were employed.
During the search for a simple but perfect recovery of PHBfrom Z. denitrificans MW1 cells, different organic solvents wereinvestigated in small-scale experiments (100 mg cells in 2 mlorganic solvent) to determine their efficiency in recoveringPHB and how easy the separation of PHB from cell debrisafter extraction could be. Only chloroform and methylenechloride showed remarkable efficiencies in PHB recovery. Nodetectable PHB was extracted from the cells with carbon tet-rachloride, diethyl ester, ethyl acetate, or mixtures of chloro-form and acetone under the conditions studied (data notshown). Self-flocculation and flotation of Z. denitrificans MW1cells were recorded for carbon tetrachloride, chloroform, andmethylene chloride, whereas in diethyl ester and ethyl acetatethe cells settled down. On the other hand, floating/settlingpriority in chloroform-acetone mixtures depended on the prev-alence of chloroform, i.e., if the percentage of chloroform wasless than 75% (vol/vol), then the cells would sediment.
Because of their PHB extraction efficiencies, chloroform andmethylene chloride were selected for large-scale experiments(100 g cells in 10 volumes solvent) to optimize PHB extractionfrom Z. denitrificans MW1 cells with a high recovery and effi-cient floating. The data in Table 3 show the effects of incuba-tion temperature and time on the efficiency of solvent recoveryof PHB. At room temperature (24°C), chloroform recovered54.5% � 0.06% (wt/wt) of the PHB from the cells after 24 h ofextraction. Extending the extraction time to up to 228 h wasnot correlated with an increase in the amount of recoveredpolymer. However, the chloroform extraction efficiency in-creased significantly when incubation was done at a higher
TABLE 1. PHB production by Zobellella denitrificans MW1 in fed-batch fermentationsa
Fed-batchfermentation no.
Initialvol
(liters)
Finalvol
(liters)
Cultivationtime (h)
Amt ofglycerol (g)
Residual amt ofglycerol (g)
CDW(g/liter)
% PHA(wt/wt)
PHA concn(g/liter)
PHB productivity(g/liter/h)
Yield(g PHB/g glycerol)
1 24 36 24 4,059 768 29.9 � 1.3 31.0 � 4.2 9.3 � 1.7 0.39 � 0.07 0.10 � 0.022 (NaCl) 20 26 36 2,100 360 32.8 � 0.6 52.0 � 1.4 17.1 � 0.8 0.47 � 0.02 0.26 � 0.013 (NaCl) 20 33 63 5,725 250 63.9 � 2.0 56.0 � 0.7 35.8 � 1.6 0.57 � 0.02 0.22 � 0.014 (NaCl) 20 30 50 8,400 1,884 81.2 � 2.5 66.9 � 7.6 54.3 � 7.9 1.09 � 0.16 0.25 � 0.04
a Fed-batch fermentations were done by cultivating Z. denitrificans MW1 in a 42-liter Biostat UD-30 stainless steel reactor. Medium components, feeding solutions,and cultivation conditions were as described in the text. All fermentations were operated at 41°C. Fed-batch fermentations 2, 3, and 4 were supplied with 20 g/liter NaCl.Analyses were done in triplicate, and averages and standard deviations are presented.
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temperature. At 30°C, 74.2% � 0.09% (wt/wt) of the PHB wasrecovered from the cells after 24 h. The maximum recoveryrate, 85.0% � 0.10% (wt/wt) of PHB, with a purity of 98.3% �1.3%, was achieved after 72 h by use of 10 volumes of CHCl3at 30°C. Figure 4 shows the complete flotation of cells in aseparation funnel after 16 h of incubation at room tempera-ture. A slightly lower recovery rate (81.7% � 0.09% [wt/wt])and purity (97.9% � 0.8%) were detected after 120 h. Thelatter may be due to excessive cell lysis during a prolongedincubation time, which may hinder flotation of cell debris.
Increasing the temperature during chloroform extraction to41°C did not enhance the recovery efficiency, since low PHBrecovery (65.9% � 0.08%) and low purity (94.2% � 2.5%)were observed in comparison to those reached at 30°C. Also, athigh temperature, increased cell lysis resulted in a very lowfloating velocity of cell debris, and after 48 h of extraction at41°C, cells could not be separated by flotation.
Extraction with methylene chloride yielded a lower recoveryrate than that attained with chloroform. For instance, after24 h, only 32.6% � 0.04% (wt/wt) of the PHB could be recov-ered from the cells at 24°C; this is only about 60% of the PHBrecovered by chloroform for the same temperature and incu-bation period. PHB recovery by methylene chloride was onlymarginally enhanced by increasing the extraction time to 72 h(Table 3).
DISCUSSION
Production of PHB from glycerol by the recently isolatedbacterium Z. denitrificans MW1 was scaled up by using thefed-batch fermentation mode at the 42-liter scale. In the first
fed-batch fermentation, a relatively high cell density and highpolymer productivity were achieved after a short incubationtime (24 h). Both were higher, by factors of 8.5 and 13.4,respectively, than those recently recorded in flask-scale exper-iments after 96 h (20). In spite of the low PHB content and thelow product yield reached in this fed-batch cultivation experi-ment, the cell density and also the PHB content and productyield were comparable to the data published for the productionof PHB from glycerol (Table 2). A slightly lower product yield(0.080 g PHB/g glycerol) was recorded for recombinant E. colistrains in 5.6-liter batch fermentations (12). A similar cell den-sity was also reported for a recombinant arcA mutant of E. coliin a 5.6-liter fed-batch culture (33). A higher PHB content(76% [wt/wt]) was achieved by an unidentified osmophilic or-ganism in a 42-liter fed-batch fermentation using glycerol li-quor phase with yeast extract and peptone. However, the pro-ductivity was very low (0.09 g/liter/h) because of the longcultivation time (182 h) (24). Several flask-scale cultivationswere carried out with a variety of bacterial strains, such as arecombinant E. coli strain harboring PHB synthesis genes fromStreptomyces aureofaciens (31, 37), different Pseudomonasstrains (2, 3), and several Vibrio sp. isolates (7), but only com-parably low PHA productivities were attained. Batch culture ofR. eutropha DSM 11348 in a 2.5-liter fermenter with glyceroland casein peptone or Casamino Acids resulted in the maxi-mum productivity (0.254 g/liter/h) reported for PHB produc-tion from glycerol (4).
Significant decreases in PHB content (from 70 to 48% [wt/wt]) and also in product yield (from 0.37 to 0.14 g/g) wererecorded for PHB production by Cupriavidus necator JMP 134using NaCl-contaminated crude glycerol (5.5% [wt/vol] NaCl)
FIG. 2. Fed-batch cultivation of Z. denitrificans for PHB production from glycerol in the presence of NaCl (fed-batch 3). Z. denitrificans MW1was cultivated in the presence of NaCl in a Biostat UD-30 stirred-tank reactor containing 20 liters of MSM with glycerol as the sole carbon source.A total of 2% NaCl was added at the beginning of the fermentation. The fermenter was inoculated with 4% (vol/vol) 24-h preculture. The pH ofthe medium was controlled at 7.3 during the first 20 h of growth and then at 6.8 until the end of fermentation, using NaOH (4 N) or NH4OH (25%[wt/vol]). The glycerol feeding solution (50% [vol/vol]) was supplemented with 1% MgSO4 and two times the normal concentrations of traceelements, iron, and calcium in MSM. The pO2 was controlled automatically at 20% saturation by adjusting aeration and agitation rates. Cells weregrown for 63 h at 41°C. During the time course of cultivation, samples were withdrawn, and the concentrations of glycerol and ammonium, as wellas the CDW and PHB content of the cells, were determined as described in Materials and Methods.
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(32), which might be related to the increased osmotic pressurecaused by NaCl accumulation during glycerol feeding. Thisproblem can be ignored if halophilic bacteria or moderatehalophiles are used under such conditions. On the other hand,some of these bacteria have been studied for PHB productivity(16, 36), but the need for additional salts for strain require-ments elevated the production costs (29, 40) and could furtheraccelerate the corrosion of commonly used stainless steel fer-menters (8, 35).
In this respect, further optimizations of fed-batch fermenta-tions by Z. denitrificans MW1 were made by utilizing the re-ported enhanced growth and polymer content in the presenceof sodium chloride (20). First, the modified feeding and pHcontrol improved the product yield to 260%, since half theamount of glycerol (1.74 kg) was utilized, producing 445.4 gPHB with a higher polymer productivity (0.47 � 0.02 g/liter/h).Further optimizations of fed-batch fermentation with in-creased feeding rates of glycerol and ammonia, together with
the precise control of pH, maximized PHB productivity, to1.09 � 0.16 g/liter/h, with a higher polymer content (66.9% �7.6% [wt/wt]) and a high product yield (0.25 � 0.04 g PHB/gglycerol), which is the highest yield reported so far for theproduction of PHA from glycerol (Table 2).
Although Z. denitrificans MW1 accumulates PHB duringgrowth, slightly increased PHB contents were detected at a lowconcentration of ammonium chloride (20). Therefore, a shortfinale of limited nitrogen conditions was provided in all fed-batch fermentations (batches 1, 2, 3, and 4) for enhancedpolymer accumulation. An increased PHB content after apply-ing the nitrogen limitation condition was also reported forgrowth-associated PHB accumulation by A. latus during batchand fed-batch cultures (45).
As shown in Fig. 3A, the offline direct control feeding reg-imen used has some critical points, such as the drop in glycerolconcentration to 5 g/liter at 12 and 29 h, and also the am-monia concentration decreased to 0.17 g/liter at 43 h. At thesame time that glycerol was depleted (12 and 29 h), an increasein pO2 was detected (Fig. 3B), and this could be a basis for theapplication of indirect control of glycerol concentration in fur-ther optimization. However, precise direct control of glycerolis also anticipated to maximize cell density and polymer pro-ductivity. High PHB productivity from glucose (2.42 g/liter/h)was achieved by a glucose-utilizing mutant of A. eutrophus H16when a direct online glucose control was applied compared tothe productivity reached with pH state for a fed-batch (0.25g/liter/h) of the same strain (23). Also, for higher-cell-densitycultivation, a pure oxygen supply is a significant concern, sincethe maximum stirring and airflow used in fed-batch 4 could notprovide enough dissolved oxygen in the exponential growthphase (up to 36 h) (Fig. 3B). Due to its physicochemical char-acteristics, glycerol as a substrate will pose a challenge in ox-ygen transfer rates as well.
Eventually, ease of recovery of PHA is a very importantparameter for economically feasible production of the polyes-ter (22). In this respect, flotation of Z. denitrificans MW1 cellsafter solvent extraction is considered the easiest way to achievea cell-free PHB-solvent solution by an in situ separation step,avoiding additional costs for centrifugation and waste of poly-mer during recovery steps. The highest recovery rate wasreached with chloroform at 30°C after 72 h. Recently, anothermethod of selective dissolved-air flotation for PHA recoveryfrom cell debris of Pseudomonas putida was also investigated(44). A slightly lower recovery and purity were reported for theextraction of PHB from A. eutrophus by chlorinated solvent
FIG. 3. PHB production by Z. denitrificans MW1 during continuous feeding of glycerol (fed-batch 4). Cultivation was done in a Biostat UD-30stirred-tank reactor containing 20 liters of MSM with glycerol as the sole carbon source. A total of 2% NaCl was added at the beginning of thefermentation. The bioreactor was inoculated with 4% (vol/vol) 24-h preculture. (A) Glycerol was fed continuously to keep its concentration higherthan 10 g/liter. The glycerol feeding solution (80% [vol/vol]) was supplemented with MgSO4 (1% [wt/vol]) and two times the normal concentrationsof trace elements, iron, and calcium in MSM. During the time course of cultivation, samples were withdrawn, and the concentrations of glyceroland ammonium, as well as the CDW and PHB content of cells, were determined as described in Materials and Methods. (B) The followingparameters were obtained by online monitoring: OD850, pO2 (controlled automatically at 20% saturation by adjusting aeration and agitation rates),and the pH of the medium (controlled at 7.3 during the first 20 h of growth and then at 6.8 to the end of fermentation, using NaOH [4 N] andNH4OH [25% {wt/vol}] in parallel). Fermentation was operated for 50 h at 41°C. (C) Microscopic photographs showing the accumulation of PHBgranules in cells during the entire time course of fermentation (9, 14, 24, 32, 45, and 50 h). The left panels (with size bars) show pictures obtainedby phase-contrast light microscopy of cells possessing bright cytoplasmic inclusions. The right panels (without size bars) show fluorescencemicrographs of PHB inclusions stained with Nile red.
TABLE 3. Solvent extraction of PHB from Z. denitrificansMW1 cellsa
Solvent (temp) Time(h)
Amt of extractedPHB (g/50 ml
solvent)
Recovery(%) Purity (%)
Chloroform (24°C) 24 1.82 � 0.04 54.5 � 0.06 ND48 1.84 � 0.04 55.4 � 0.06 ND72 1.85 � 0.06 55.6 � 0.06 97.6 � 2.1
228 1.81 � 0.03 54.4 � 0.06 96.8 � 1.8
Chloroform (30°C) 24 2.47 � 0.10 74.2 � 0.09 ND48 2.81 � 0.03 84.3 � 0.10 ND72 2.83 � 0.13 85.0 � 0.10 98.3 � 1.396 2.82 � 0.12 84.7 � 0.10 ND
120 2.72 � 0.21 81.7 � 0.09 97.9 � 0.8
Chloroform (41°C) 24 2.19 � 0.05 65.9 � 0.08 94.2 � 2.5
Methylene chloride 24 1.09 � 0.03 32.6 � 0.04 ND(24°C) 48 1.13 � 0.03 34.1 � 0.04 ND
72 1.21 � 0.10 36.2 � 0.04 98.8 � 1.0228 1.05 � 0.03 31.5 � 0.04 96.4 � 2.7
a Lyophilized cells of Z. denitrificans MW1 (100-g portion) from fed-batchfermentation 4 (66.9% � 7.6% PHB �wt/wt�) were stirred into 10 volumes ofsolvent at different extraction temperatures. Fifty-milliliter sample portions werewithdrawn at the indicated times. Samples were separated in separation funnelsovernight at room temperature. Clear PHB-solvent solutions were withdrawnand concentrated. Afterwards, 4 volumes of methanol was added to precipitatePHB, which was then filtered and dried under vacuum. Recovery and purity ofPHB were analyzed by GC. Analyses were done in triplicate, and averages andstandard deviations for three different analyses are presented. ND, not deter-mined.
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reflux (39). In spite of the short extraction time recorded (15min), the high temperatures (61°C for CHCl3 and 40°C forCH2Cl2) at which reflux was done and the need for 24 h of cellpretreatment with acetone should hinder the application ofthis recovery method at the industrial scale.
Compared to all other strains used for PHB productionfrom glycerol (Table 2), this study recommends Z. denitrifi-cans MW1 as a new and attractive option for large-scaleproduction of PHB by use of residual crude glycerol fromthe biodiesel industry.
ACKNOWLEDGMENTS
Financial support of this study by BASF AG (Ludwigshafen, Ger-many) is gratefully acknowledged.
We thank Yasser Elbahloul, Ahmad Sallam, Kaichien Lin, Kay Frey,and Herbert Ahlers for their assistance during fermentation.
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