Poly(3-Hydroxybutyrate) Production from Glycerol by Zobellella denitrificans MW1 via High-Cell-Density Fed-Batch Fermentation and Simplified Solvent Extraction

Industrial production of biodegradable polyesters such as polyhydroxyalkanoates is hampered by high production costs, among which the costs for substrates and for downstream processing represent the main obstacles. Inexpensive fermentable raw materials such as crude glycerol, an abundant by-product of the biodiesel 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-hydroxybutyrate) (PHB) from glycerol as the sole carbon source. Pilot-scale fermentations (42-liter scale) were conducted to scale up the high PHB accumulation capability of this strain. By fed-batch cultivation, at first a relatively high cell density (29.9 ± 1.3 g/liter) was obtained during only a short fermentation period (24 h). However, the PHB content was relatively low (31.0% ± 4.2% wt/wt]). Afterwards, much higher concentrations of PHB (up to 54.3 ± 7.9 g/liter) and higher cell densities (up to 81.2 ± 2.5 g/liter) were obtained by further fed-batch optimization in the presence of 20 g/liter NaCl, with optimized feeding of glycerol and ammonia to support both cell growth and polymer accumulation over a period of 50 h. A high specific growth rate (0.422/h) and a short doubling 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 for PHB recovery during downstream processing: self-flotation of cell debris after extraction of PHB with chloroform 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 purity of 98.3% ± 1.3%.Polyhydroxybutyrate (PHB) is the best-studied example of biodegradable polyesters belonging to the group of polyhydroxyalkanoates (PHAs), which are synthesized by many bacteria and archaea as intracellular carbon and energy reserves (1, 40, 43). In the last decades, these biopolymers have received great attention due to their properties which resemble those of conventional petrochemical-based polymers (49). For instance, PHB is very similar to thermoplastic polypropylene (17). Their production from renewable resources and their complete biodegradability give PHAs promising advantages from an environmental point of view (6). In addition to their special physical traits, such as the elasticity of medium-chain-length PHAs and the high crystallization rate of PHB, PHAs are biocompatible, water resistant, oxygen impermeable, and enantiomerically pure; all of these characteristics broaden the scope of their applications in industry and medicine.So far, higher production costs than those of petrochemical plastics have hindered the successful commercialization of PHB (9). Many efforts have been devoted to reducing the production costs by developing superior microbial strains capable of utilizing cheap substrates and also by applying more efficient fermentation strategies and economical recovery processes (10).Fed-batch fermentation regimens are usually applied to achieve a high cell density, which is necessary for a high productivity and yield, in particular in cases of intracellular products, by frequent or continuous feeding of nutrients when growth proceeds (46). Several fed-batch fermentation processes have been reported for PHA production (21, 28). There are two prevalent cultivation modes for PHB production that are imposed on the microorganisms being used. The more frequently used mode is realized by a complex two-stage cultivation process. In this mode, all nutrients needed for growth to a high cell density are provided during the first phase of the process. In the second phase, imbalanced growth conditions are enforced by providing growth-limiting amounts of nutrients such as nitrogen, phosphate, or oxygen to trigger PHA biosynthesis and accumulation. The model organism for this mode is Ralstonia eutropha (formerly known as Alcaligenes eutrophus and recently reclassified as Cupriavidus necator) (26, 27). In the other cultivation mode, PHB is accumulated concurrently with growth, and therefore a single-stage process is applicable. A well-known example of this mode is PHB production by Alcaligenes latus (18, 47).Although several new downstream processes for the extraction of PHA have been reported as being economically effective, 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 proteolytic enzymes (25) or by sulfuric acid and hypochlorite (48), solvent extraction methods are still regarded as an adequate way to gain intact polymers with a high purity and recovery yield (39). However, there is still a need to develop and improve these extraction methods further to make the entire process much simpler and cheaper (22).In addition to increased prices for crude oil, the abundance of inexpensive raw materials from agriculture and industry as cheap substrates for microbial fermentations, such as crude glycerol from the biodiesel industry, could make the production of PHA from renewable resources more competitive with common plastics (32). Due to increased glycerol production by the growing biodiesel industry, the prices for glycerol became low enough to make this residual compound a cheap carbon source for several industrial fermentation processes, especially for the production of microbial polyesters (11, 34). However, the various amounts of actual fermentable substrates and the presence of other nonfermentable components in feedstock, such as the various concentrations of glycerol and salts in biodiesel coproducts, hinder their use (42). Therefore, tolerant bioprocesses and/or strains tolerant to such variable factors are required.The production of PHA from glycerol has been investigated in only a few studies (4, 12, 24, 33, 42). In a recent study (32), crude glycerol from different biodiesel manufacturers was examined for suitability as a substrate for PHB production. However, significant decreases in PHB productivity and product yields were recorded when NaCl-contaminated crude glycerol was used.Recently, a newly isolated bacterium, Zobellella denitrificans MW1, was characterized as producing large amounts of PHB from glycerol, with enhanced growth and polymer productivity in the presence of NaCl (20). The present study aimed at developing a strategy to improve the volumetric production of PHB by Z. denitrificans MW1, using glycerol as the sole carbon source. For this purpose, several fed-batch cultivations were set up to steadily improve the nutrient supply to attain a high cell density and high PHB productivity. Moreover, the conventional organic solvent extraction method was modified with regard to an economically more feasible large-scale PHB extraction, achieving a high purity and recovery of the polymer.