Efficient 1,3-propanediol Production by Fed-Batch Culture of Klebsiella Pneumoniae: The Role of pH Fluctuation

The fermentative production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae under different fed-batch strategies was investigated. pH-stat fed-batch strategies proved to be not effective for economical 1,3-PD production for the existence of relatively high concentration of byproducts and residual glycerol at the end of the fermentation. However, in the pH-stat fed-batch strategy, an important phenomenon was observed that the yields of two main byproducts, 2,3-butanediol and lactic acid, were closely related to pH value. The dominant byproduct was 2,3-butanediol at a pH value of 5.0 to 6.5 but changed to be lactic acid at a pH value of 7.1 to 8.0. Based on the analysis of the phenomenon, a self-protection mechanism in K. pneumoniae, namely that the growing K. pneumoniae cells switch the metabolic pathways responding to environmental pH changes, was proposed. Thus a kind of feeding strategy was further applied during which the pH value was fluctuated between 6.3 and 7.3 periodically by feeding glycerol–ammonia mixture and sulphuric acid to make the metabolic pathways of 2,3-butanediol and lactic acid sub-active under the periodical low or high pH stress. At last, efficient 1,3-PD production was fulfilled under this fed-batch strategy, and the best results were achieved leading to 70 g/l 1,3-PD with a yield of 0.70 mol/mol glycerol and productivity of 0.97 g/l/h, while the two main byproducts and residual glycerol were under low concentrations.     

Application of a pH Feedback-Controlled Substrate Feeding Method in Lactic Acid Production

Substrate concentration in lactic acid fermentation broth could not be controlled well by traditional feeding methods, including constant, intermittent, and exponential feeding methods, in fed-batch experiments. A simple feedback feeding method based on pH was proposed to control pH and substrate concentration synchronously to enhance lactic acid production in fed-batch culture. As the linear relationship between the consumption amounts of alkali and that of substrate was concluded during lactic acid fermentation, the alkali and substrate in the feeding broth were mixed together proportionally. Thus, the concentration of substrate could be controlled through the adjustment of pH automatically. In the fed-batch lactic acid fermentation with Lactobacillus lactis-11 by this method, the residual glucose concentration in fermentation broth was controlled between 4.1 and 4.9 g L⁻¹, and the highest concentration of lactic acid, maximum cell dry weight, volumetric productivity of lactic acid, and yield were 96.3 g L⁻¹, 4.7 g L⁻¹, 1.9 g L⁻¹ h⁻¹, and 0.99 g lactic acid per gram of glucose, respectively, compared to 82.7 g L⁻¹, 3.31 g L⁻¹, 1.7 g L⁻¹ h⁻¹, and 0.92 g lactic acid per gram of glucose in batch culture. This feeding method was simple and easily operated and could be feasible for industrial lactic acid production in the future.     

Efficient Production of a Thermophilic 2-Deoxyribose-5-Phosphate Aldolase in Glucose-Limited Fed-Batch Cultivations of <Emphasis Type="Italic">Escherichia coli</Emphasis> by Continuous Lactose Induction Strategy

The production of a thermophilic 2-deoxyribose-5-phosphate aldolases (DERA) in Escherichia coli BL21 under continuous lactose induction strategy was investigated. The process was combined with the exponential feeding method, controlling the feeding rate to maintain the specific growth rate at 0.15 h⁻¹. The results indicate that the lactose concentration in the feed medium affected directly the expression of the target protein. The use of 50 g/L in the feed medium resulted in the biomass concentration of 39.3 g DCW/L, and an expression level of above 30%, and the maximum final DERA concentration of 16,200 U/L. Furthermore, the acetate concentration remained at a low level in the fed-batch phase, less than 0.5 g/L. In conclusion, combining glucose feeding with lactose induction is a more powerful way to achieve high cell density cultures and to efficiently produce the thermophilic DERA. The results also indicate the potential industrial utility in the scale production of other recombinant proteins.     

Cultivation of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> in the Stirred Bioreactor with Different Feeding Strategies for CoQ<Subscript>10</Subscript> Production

The logistic growth model combined with the Luedeking-Piret equation was adopted in this study to model the batch production of CoQ₁₀ in the cultivation of Rhodobacter sphaeroides. The simulation results indicated that CoQ₁₀ production was a primary metabolite. As being a primary metabolite, a longer cell growing stage would tend to accumulate more biomass and lead to a higher CoQ₁₀ concentration being produced. In this context, a fed-batch operation by molasses feeding was performed to increase the biomass and subsequent CoQ₁₀ production. Three different molasses feeding strategies were operated in this study. Results suggested that the fed-batch operation with molasses controlled at 10 ± 1 g/l could increase the cell mass and CoQ₁₀ concentration to reach their maximum values of 18.6 g/l and 83.8 mg/l, respectively, nearly 2.2 times and 1.9 times their respective values obtained in the batch cultivation.     

Optimization of Fermentation Conditions for the Biosynthesis of <Emphasis Type="SmallCaps">l</Emphasis>-Threonine by <Emphasis Type="Italic">Escherichia coli</Emphasis>

In this study, the fed-batch fermentation technique was applied to improve the yield of l-threonine produced by Escherichia coli TRFC. Various fermentation substrates and conditions were investigated to identify the optimal carbon source, its concentration and C/N ratio in the production of l-threonine. Sucrose was found to be the optimal initial carbon source and its optimal concentration was determined to be 70 g/L based on the results of fermentations conducted in a 5-L jar fermentor using a series of fed-batch cultures of E. coli TRFC. The effects of glucose concentration and three different feeding methods on the production of l-threonine were also investigated in this work. Our results showed that the production of l-threonine by E. coli was enhanced when glucose concentration varied between 5 and 20 g/L with DO-control pulse fed-batch method. Furthermore, the C/N ratio was a more predominant factor than nitrogen concentration for l-threonine overproduction and the optimal ratio of ammonium sulfate to sucrose (g/g) was 30. Under the optimal conditions, a final l-threonine concentration of 118 g/L was achieved after 38 h with the productivity of 3.1 g/L/h (46% conversion ratio from glucose to threonine).     

Model-Based Nutrient Feeding Strategies for the Increased Production of Polyhydroxybutyrate (PHB) by <Emphasis Type="Italic">Alcaligenes latus</Emphasis>

Polyhydroxyalkanoates (PHAs) are biodegradable polymers which are considered as an effective alternative for conventional plastics due to their mechanical properties similar to the latter. However, the widespread use of these polymers is still hampered due to their higher cost of production as compared to plastics. The production cost could be overcome by obtaining high yields and productivity. The goal of the present research was to enhance the yield of polyhydroxybutyrate (PHB) with the help of two simple fed-batch cultivation strategies. In the present study, average batch kinetic and substrate limitation/inhibition study data of Alcaligenes latus was used for the development of PHB model which was then adopted for designing various off-line nutrient feeding strategies to enhance PHB accumulation. The predictive ability of the model was validated by experimental implementation of two fed-batch strategies. One such dynamic strategy of fed-batch cultivation under pseudo-steady state with respect to nitrogen and simultaneous carbon feeding strategy resulted in significantly high biomass and PHB concentration of 39.17 g/L and 29.64 g/L, respectively. This feeding strategy demonstrated a high PHB productivity and PHB content of 0.6 g/L h and 75%, respectively, which were remarkably high in comparison to batch cultivation. The mathematical model can also be employed for designing various other nutrient feeding strategies.     

An Enhanced Process for the Production of a Highly Purified Extracellular Lipase in the Non-conventional Yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Yarrowia lipolytica LgX64.81 is a non-genetically modified mutant that was previously identified as a promising microorganism for extracellular lipase production. In this work, the development of a fed-batch process for the production of this enzyme in this strain was described. A lipolytic activity of 2,145 U/mL was obtained after 32 h of batch culture in a defined medium supplemented with 10 g/L of tryptone, an enhancer of lipase expression. To maximize the volumetric productivity, two different fed-batch strategies had been investigated. In comparison to batch process, the intermittent fed-batch strategy had not improved the volumetric lipase productivity. In contrast, the stepwise feeding strategy combined with uncoupled cell growth and lipase production phases resulted in a 2-fold increase in the volumetric lipase productivity, namely, the lipase activity reached 10,000 U/mL after 80 h of culture. Furthermore, this lipase was purified to homogeneity by anion exchange chromatography on MonoQ resin followed by gel filtration on Sephacryl S-100. This process resulted in an overall yield of 72% and a 3.5-fold increase of the specific lipase activity. The developed process offers a great potential for an economic production of Lip2 at large scale in Y. lipolytica LgX64.81.     

Effect of Constant Glucose Feeding on the Production of Exopolysaccharides by <Emphasis Type="Italic">Tremella fuciformis</Emphasis> Spores

A fed-batch culture system with constant feeding (glucose 80 g L⁻¹, 0.25 ml min⁻¹) was used to study the influence of glucose on cell dry weight and exopolysaccharides production from submerged Tremella fuciformis spores in a 5-L stirred-tank bioreactor. The results showed that high levels of cell mass (9.80 g L⁻¹) and exopolysaccharides production (3.12 g L⁻¹) in fed-batch fermentation were obtained after 1 h of feeding, where the specific growth rate (μ) and exopolysaccharides yield on substrate consumed (YP/S) were 0.267 d⁻¹ and 0.14 g g⁻¹. Unlike batch fermentation, maximal cell mass and exopolysaccharides production merely reached 7.11 and 2.08 g L⁻¹; the specific growth rate (μ) and exopolysaccharides yield on substrate consumed (YP/S) were 0.194 d⁻¹ and 0.093 g g⁻¹, respectively. It is concluded that the synthesis of exopolysaccharides can be promoted effectively when feeding glucose at a late exponential phase.     

A Simple Substrate Feeding Strategy using a pH Control Trigger in Fed-Batch Fermentation

A simple automated glucose feeding strategy based on pH control was developed to produce high-cell-density fed-batch fermentation. In this strategy, the pH control scheme utilized an acidified concentrated glucose solution to lower the pH. The frequency of glucose addition to the fermentor is determined by the culture’s growth kinetics. To demonstrate the effectiveness of the coupled pH and glucose control strategy in biomass and/or secondary metabolite production, several fed-batch fermentations of indigenous Escherichia coli and recombinant E. coli were carried out. Both strains produced biomass with optical density of greater than 40 at 600 nm. We also tested the glucose control strategy using two types of pH controller: a less sophisticated portable pH controller and a more sophisticated online proportional-integral-derivative (PID) controller. Our control strategy was successfully applied with both controllers, although better control was observed using the PID controller. We have successfully demonstrated that a glucose feeding strategy based on a simple pH control scheme to indirectly control the glucose concentration can be easily achieved and adapted to conventional bioreactors in the absence of online glucose measurement and control.     

The Fed-Batch Production of a Thermophilic 2-Deoxyribose-5-Phosphate Aldolase (DERA) in <Emphasis Type="Italic">Escherichia coli</Emphasis> by Exponential Feeding Strategy Control

2-Deoxyribose-5-phosphate aldolase (DERA) catalyzes a sequential aldol reaction useful in synthetic chemistry. In this work, the effect of a feeding strategy on the production of a thermophilic DERA was investigated in fed-batch cultures of recombinant Escherichia coli BL21 (pET303-DERA008). The predetermined specific growth rate (μ _set) was evaluated at 0.20, 0.15, and 0.10 h⁻¹, respectively. The DERA concentration and volumetric productivity were associated with μ _set. The cells synthesized the enzyme most efficiently at μ _set = 0.15 h⁻¹. The maximum enzyme concentration (5.12 g/L) and total volumetric productivity (0.256 g L⁻¹ h⁻¹) obtained were over 10 and five times higher than that from traditional batch cultures. Furthermore, the acetate concentration remained at a relatively low level, less than 0.4 g/L, under this condition which would not inhibit cell growth and target protein expression. Thus, a specific growth rate control strategy has been successfully applied to induce fed-batch cultures for the maximal production of the thermophilic 2-deoxyribose-5-phosphate aldolase.     

Production of Butyric Acid from Glucose and Xylose with Immobilized Cells of <Emphasis Type="Italic">Clostridium tyrobutyricum</Emphasis> in a Fibrous-bed Bioreactor

Butyric acid has many applications in chemical, food, and pharmaceutical industries. In the present study, Clostridium tyrobutyricum ATCC 25755 was immobilized in a fibrous-bed bioreactor to evaluate the performance of butyrate production from glucose and xylose. The results showed that the final concentration and yield of butyric acid were 13.70 and 0.46 g g⁻¹, respectively, in batch fermentation when 30 g L⁻¹ glucose was introduced into the bioreactor. Furthermore, high concentration 10.10 g L⁻¹ and yield 0.40 g g⁻¹ of butyric acid were obtained with 25 g L⁻¹ xylose as the carbon source. The immobilized cells of C. tyrobutyricum ensured similar productivity and yield from repeated batch fermentation. In the fed-batch fermentation, the final concentration of butyric acid was further improved to 24.88 g L⁻¹ with one suitable glucose feeding in the fibrous-bed bioreactor. C. tyrobutyricum immobilized in the fibrous-bed bioreactor would provide an economically viable fermentation process to convert the reducing sugars derived from plant biomass into the final bulk chemical (butyric acid).     

Variable Volume Fed-Batch Fermentation for Nisin Production by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp<Emphasis Type="Italic">. lactis W28</Emphasis>

A feeding technology that was suitable for improving the nisin production by Lactococcus lactis subsp. lactis W28 was established. The effects of initial sucrose concentration (ISC) in the fermentation broth, feeding time, and feeding rate on the fermentation were studied. It was observed that a fed-batch culture (ISC = 10 g l⁻¹) with 100 ml sucrose solution (190 g l⁻¹) being evenly fed (9–10 ml h⁻¹) into the fermenter after 3-h fermentation gave the best performance in terms of biomass and nisin yield. Under these conditions, the total biomass and the total nisin yield were approximately 23% and 51% higher than those in batch fermentation, respectively. When the sucrose concentration was controlled at 5–10 g l⁻¹ in variable volume intermittent fed-batch fermentation (VVIF) with ISC = 10 g l⁻¹, the total biomass and the total nisin yield were 29% and 60% above those in batch fermentation, respectively. The VVIF proved to be effective to eliminate the substrate inhibition by maintaining sucrose at appropriate levels. It is also easy to be scaled up, since various parameters involved in industrial production were taken into account.     

Fed-Batch Strategies for Production of PHA Using a Native Isolate of <Emphasis Type="Italic">Halomonas venusta</Emphasis> KT832796 Strain

In this study, polyhydroxyalkanoates (PHA) accumulation by Halomonas venusta KT832796, a moderate halophilic bacteria isolated from marine source was studied. Both nutritional requirements and process parameters for submerged cultivation of the organism in bioreactor have been standardized. From the shake flask studies, glucose and ammonium citrate as carbon and nitrogen source produced maximum PHA at a ratio 20 with 3.52 g/L of dry cell weight and 70.56% of PHA content. However, ammonium sulfate as the nitrogen source was found to be more suitable for fed-batch cultivation. Several feeding strategies including pH-based fed-batch and variants of pulse feeding were studied to improve the PHA levels. pH-based feeding, although improved PHA level to 26 g/L, most of the carbon flux was diverted towards biomass formation; hence, the percent PHA was only 39.15% of the dry cell weight. Maximum PHA of 33.4 g/L, which corresponded to 88.12% of the dry cell, was obtained from high concentration single pulse method. There was a net 8.65-fold increase in PHA using this feeding strategy when compared to batch studies. According to our knowledge, this is the highest amount of PHA reported for a Halomonas venusta strain.     

(Liquid + liquid) equilibrium data for the separation of 2,3-butanediol from aqueous streams using tetraoctyl ammonium 2-methyl-1-naphthoate

Microbiological production of 2,3-butanediol (2,3-BD) through fermentation using renewable feedstock is a promising option for the production of bio based chemicals. Liquid–liquid extraction could be a more efficient process, if a proper solvent is used. Tetraoctyl ammonium 2-methyl-1-naphthoate [TOA MNaph] is a tailor made hydrophobic ionic liquid that may be applied for the recovery of 2,3-BD from aqueous streams. In this work, the (liquid + liquid) equilibrium data for {2,3-BD + water + [TOA MNaph]} at (313.2, 333.2, and 353.2) K have been obtained and correlated to the NRTL and UNIQUAC activity coefficient models. Root square mean deviations (RMSD) values of 1.54% (NRTL) and 1.88% (UNIQUAC) were obtained, showing that both models can properly describe the experimental data. Compared to conventional solvents [TOA MNaph] presents a good balance between distribution coefficient (D_2,3BD = 1.08) and selectivity (S = 11.47).     

Constitutive Expression of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Lipase LIP2 in <Emphasis Type="Italic">Pichia pastoris</Emphasis> Using GAP as Promoter

A gene encoding Yarrowia lipolytica lipase LIP2 (YlLIP2) was cloned into a constitutive expression vector pGAPZαA and electrotransformed into the Pichia pastoris X-33 strain. The high-yield clones obtained by high copy and enzyme activity screening were chosen as the host strains for shaking flask and fermentor culture. The results showed that glucose was the optimum carbon source for YlLIP2 production, and the maximum hydrolytic activity of recombinant YlLIP2 reached 1,315 U/ml under the flask culture at 28 °C, pH 7.0, for 48 h. The fed-batch fermentation was carried out in 3- and 10-l bioreactors by continuously feeding glucose into the growing medium for achieving high cell density and YlLIP2 yields. The maximum hydrolytic activity of YlLIP2 and cell density obtained in the 3-l bioreactor were 10,300 U/ml and 116 g dry cell weight (DCW)/l, respectively. The peak hydrolytic activity of YlLIP2 and cell density were further improved in the 10-l fermentor where the values respectively attained were 13,500 U/ml and 120 g DCW/l. The total protein concentration in the supernatant reached 3.3 g/l and the cell viability remained approximately 99% after 80 h of culture. Furthermore, the recombinant YlLIP2 produced in P. pastoris pGAP and pAOX1 systems have similar content of sugar (about 12%) and biochemical characteristics. The above results suggest that the GAP promoter-derived expression system of P. pastoris is effective for the expression of YlLIP2 by high cell density culture and is probably an alternative to the conventional AOX1 promoter expression system in large-scale production of industrial lipases.     

High-cell-density cultivation of Pseudomonas putida IPT 046 and medium-chain-length polyhydroxyalkanoate production from sugarcane carbohydrates

We studied high-density cultures of Pseudomonas putida IPT 046 for the production of medium-chain-length polyhydroxyalkanoates (PHAMCL) using an equimolar mixture of glucose and fructose as carbon sources. Kinetics studies of P. putida growth resulted in a maximum specific growth rate of 0.65 h(-1). Limitation and inhibition owing to NH4+ ions were observed, respectively, at 400 and 3500 mg of NH4+/L. The minimum concentration of dissolved oxygen in the broth must be 15% of saturation. Fed-batch strategies for high-cell-density cultivation were proposed. Pulse feed followed by constant feed produced a cell concentration of 32 g/L in 18 h of fermentation and low PHAMCL content. Constant feed produced a cell concentration of 35 g/L, obtained in 27 h of fermentation, with up to 15% PHAMCL. Exponential feed produced a cell concentration of 30 g/L in 20 h of fermentation and low PHAMCL content. Using the last strategy, 21% PHAMCL was produced during a period of 34 h of fed-batch operation, with a final cell concentration of 40 g/L and NH4+ limitation. Using phosphate limitation, 50 g/L cell concentration, 63% PHAMCL and a productivity of 0.8 g/(L x h) were obtained in 42 h of fed-batch operation. The PHAMCL yield factors from consumed carbohydrate for N-limited and P-limited experiments were, respectively, 0.15 and 0.19 g/g.     

Kinetic Analysis and pH-Shift Control Strategy for Propionic Acid Production with <Emphasis Type="Italic">Propionibacterium Freudenreichii</Emphasis> CCTCC M207015

The production of propionic acid by Propionibacterium freudenreichii CCTCC M207015 was investigated in a 7.5-l stirred-tank fermentor. Batch fermentations by P. freudenreichii CCTCC M207015 at various pH values ranging from 5.5 to 7.0 were studied. Based on the analysis of the time course of specific cell growth rate (μ _x) and specific propionic acid formation rate (μ _p), a two-stage pH-shift control strategy was proposed. At first 48 h, pH was controlled at 6.5 to obtain the maximal μ _x, subsequently pH 6.0 was used to maintain high μ _p to enhance the production of propionic acid. By applying this pH-shift control strategy in propionic acid fermentation, the maximal propionic acid and glucose conversion efficiency had a significant improvement and reached 19.21 g/l and 48.03%, respectively, compared with those of constant pH operation (14.58 g/l and 36.45%). Fed-batch fermentation with pH-shift control strategy was also applied to produce propionic acid; the maximal propionic acid yield and glucose conversion efficiency reached 25.23 g/l and 47.76%, respectively.     

Optimized Fed-Batch Fermentation of Scheffersomyces stipitis for Efficient Production of Ethanol from Hexoses and Pentoses

Scheffersomyces stipitis was cultivated in an optimized, controlled fed-batch fermentation for production of ethanol from glucose–xylose mixture. Effect of feed medium composition was investigated on sugar utilization and ethanol production. Studying influence of specific cell growth rate on ethanol fermentation performance showed the carbon flow towards ethanol synthesis decreased with increasing cell growth rate. The optimum specific growth rate to achieve efficient ethanol production performance from a glucose-xylose mixture existed at 0.1 h^?1. With these optimized feed medium and cell growth rate, a kinetic model has been utilized to avoid overflow metabolism as well as to ensure a balanced feeding of nutrient substrate in fed-batch system. Fed-batch culture with feeding profile designed based on the model resulted in high titer, yield, and productivity of ethanol compared with batch cultures. The maximal ethanol concentration was 40.7 g/L. The yield and productivity of ethanol production in the optimized fed-batch culture was 1.3 and 2 times higher than those in batch culture. Thus, higher efficiency ethanol production was achieved in this study through fed-batch process optimization. This strategy may contribute to an improvement of ethanol fermentation from lignocellulosic biomass by S. stipitis on the industrial scale.     

The pH Shift and Precursor Feeding Strategy in a Low-Toxicity FR-008/Candicidin Derivative CS103 Fermentation Bioprocess by a Mutant of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. FR-008

CS103, the novel derivative of polyene macrolides antibiotic FR-008/candicidin with lower toxicity has been isolated from the culture mycelia of the mutant of Streptomyces sp. FR-008, with targeted deletions of the fscP cytochrome P450 gene from its chromosome. To enhance biosynthesis of CS103, pH shift and precursor feeding strategy for fermentation process by the mutant of Streptomyces sp. FR-008 in a stirred tank bioreactor was developed. According to the process parameters analysis, the effectiveness of the strategy was examined and confirmed by experiments. A maximal CS103 concentration of 139.98 μg/mL was obtained, 2.05-fold higher than that in the pH-uncontrolled fermentation. Compared to other three cases as pH-uncontrolled, pH-controlled, and two-stage pH-controlled batch cultures, the proposed “pH shift and precursor feeding strategy” effectively avoided the scarcity of the antibiotic precursor, increased the CS103 yield from biomass (Y _P/X) and substrate (Y _P/S) by 110.61% and 48.52%, respectively, and at the time the fermentation time was shortened from 120 to 96 h. The highest CS103 production rate (1.46 μg mL⁻¹ h⁻¹) of the pH shift and precursor feeding strategy was 284.21%, 97.30%, and 58.70% higher than that of pH-uncontrolled, pH-controlled, and two-stage pH-controlled batch culture cases, respectively.     

High-Cell-Density cultivation of pseudomonas putida IPT 046 and medium-chain-length polyhydroxyalkanoate production from sugarcane carbohydrates

We studied high-density cultures of Pseudomonas putida IPT 046 for the production of medium-chain-length polyhydroxyalkanoates (PHA_MCL) using an equimolar mixture of glucose and fructose as carbon sources. Kinetics studies of P. putida growth resulted in a maximum specific growth rate of 0.65h⁻¹. Limitation and inhibition owing to NH₄ ⁺ ions were observed, respectively, at 400 and 3500 mg of NH₄ ⁺/L. The minimum concentration of dissolved oxygen in the broth must be 15% of saturation. Fed-batch strategies for high-cell-density cultivation were proposed. Pulse feed followed by constant feed produced a cell concentration of 32 g/L in 18 h of fermentation and low PHA_MCL content. Constant feed produced a cell concentration of 35 g/L, obtained in 27 h of fermentation, with up to 15% PHA_MCL. Exponential feed produced a cell concentration of 30 g/L in 20 h of fermentation and low PHA_MCL content. Using the last strategy, 21% PHA_MCL was produced during a period of 34 h of fed-batch operation, with a final cell concentration of 40 g/L and NH₄ ⁺ limitation. Using phosphate limitation, 50 g/L cell concentration, 63% PHA_MCL and a productivity of 0.8 g/(L·h) were obtained in 42 h of fed-batch operation. The PHA_MCL yield factors from consumed carbohydrate for N-limited and P-limited experiments were, respectively, 0.15 and 0.19 g/g.