Gerhart Braunegg

Polyhydroxyalkanoates, biopolyesters from renewable resources: Physiological and engineering aspects

Polyhdroxyalkanoates (PHAs), stored as bacterial reserve materials for carbon and energy, are biodegradable substitutes to fossil fuel plastics that can be produced from renewable raw materials. PHAs can be produced under controlled conditions by biotechnological processes. By varying the producing strains, substrates and cosubstrates, a number of polyesters can be synthesized which differ in monomer composition. By this means, PHAs with tailored interesting physical features can be produced. All of them are completely degradable to carbon dioxide and water through natural microbiological mineralization. Consequently, neither their production nor their use or degradation have a negative ecological impact. After a historical review, possibilities for the synthesis of novel PHAs applying different micro-organisms are discussed, and pathways of PHA synthesis and degradation are shown in detail for important PHA producers. This is followed by a discussion of the physiological role of the accumulation product in different micro-organisms. Detection, analysis, and extraction methods of PHAs from microbial biomass are shown, in addition to methods for polyester characterization. Strategies for PHA production under discontinuous and continuous regimes are discussed in detail in addition to the use of different cheap carbon sources from the point of view of different PHA producing strains. An outlook on PHA production by transgenic plants closes the review.

Sustainable Polymer Production

Abstract Sustainable biotechnological production of polyhydroxyalkanoates from renewable resources is an alternative to chemical polyster production from mineral oils. The polymers produced are biodegradable, and due to their characteristics can be used for the production of packaging materials. Many different prokaryotic microorganisms are known to accumulate polyhydroxyalkanoates intercellularly under growth limiting conditions; some of them are able to produce copolyesters from precursors added to the production medium. Production normally is done in a fed-batch process, but PHA production kinetics suggest that continuous production in a chemostat might be favorable. After biomass separation the polyester is extracted from the biomass and refined.

Archaeal Production of Polyhydroxyalkanoate (PHA) Co- and Terpolyesters from Biodiesel Industry-Derived By-Products

The archaeon Haloferax mediterranei was selected for production of PHA co- and terpolyesters using inexpensive crude glycerol phase (CGP) from biodiesel production as carbon source. CGP was assessed by comparison with the application of pure glycerol. Applying pure glycerol, a copolyester with a molar fraction of 3-hydroxybutyrate (3HB) of 0.90 mol/mol and 3-hydroxyvalerate (3HV) of 0.10 mol/mol, was produced at a volumetric productivity of 0.12 g/Lh and an intracellular PHA content of 75.4 wt.-% in the sum of biomass protein plus PHA. Application of CGP resulted in the same polyester composition and volumetric productivity, indicating the feasibility of applying CGP as feedstock. Analysis of molar mass distribution revealed a weight average molar mass M w of 150 kDa and polydispersity P i of 2.1 for pure glycerol and 253 kDa and 2.7 for CGP, respectively; melting temperatures ranged between 130 and 140°C in both setups. Supplying γ-butyrolactone as 4-hydroxybutyrate (4HB) precursor resulted in a poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate-co-4-hydroxybutyrate] (PHBHV4HB) terpolyester containing 3HV (0.12 mol/mol) and 4HB (0.05 mol/mol) in the poly[(R)-3-hydroxybutyrate] (PHB) matrix; in addition, this process runs without sterilization of the bioreactor. The terpolyester displayed reduced melting (melting endotherms at 122 and 137°C) and glass transition temperature (2.5°C), increased molar mass (391 kDa), and a polydispersity similar to the copolyesters.

Formal kinetics of poly-β-hydroxybutyric acid (PHB) production in Alcaligenes eutrophus H 16 and Mycoplana rubra R 14 with respect to the dissolved oxygen tension in ammonium-limited batch cultures

SummaryUnder chemolithoautotrophic growth conditions with the organism Alcaligenes eutrophus H16 the exponential growth phase is characterized by two different growth rates, each associated with different specific rates of ammonium consumption. On the basis of the analytical determination of Poly-β-hydroxybutyric acid (PHB), it can be conclusively shown that PHB is synthesized even during the exponential growth phase at a specific rate proportional to the specific growth rates of total biomass. After complete consumption of ammonium, the increase of biomass is exclusively due to PHB synthesis, whereas protein and rest biomass (cell dry weight minus PHB) remain constant. After an extended period of fermentation, the PHB content reaches a saturation value. The transient phase between the growth and the storage phase is very short in comparison to the duration of the whole fermentation. In the case of Alcaligenes eutrophus, strain H 16, high concentrations of dissolved oxygen strongly influence growth as well as PHB synthesis.

Microbial PHA Production from Waste Raw Materials

The application of biotechnological processes for industrial production can be regarded as promising for sustainable development, although for a range of products, biotechnological production strategies have not yet passed the test of economic viability. This is often caused by the cost of the raw materials. Here, a viable solution strategy is identified by the utilization of a broad range of waste and surplus materials that can be upgraded to the role of feedstocks for the biomediated production of desired end products such as polyhydroxyalkanoate biopolymers. The selection of the appropriate waste stream as a feedstock for biotechnological purposes mainly depends on the global region where the production plant will be constructed. To save costs for transportation, facilities for the production of biopolymers, biofuels and biochemicals should be integrated into existing production lines, where the feedstocks directly accrue as waste streams. In Europe and North America, surplus whey from the dairy industry is available in large quantities, whereas huge amounts of non-wood lignocellulosic materials from rice, corn and sugar cane plants are found in many different countries worldwide. The enormously increasing production of biofuels provides a range of by-products such as glycerol and low-quality fatty acid esters from biodiesel production or distillery residues from bioethanol factories. The utilization of waste streams for production of value-added products not only enhances the economics of such products, but also provides industry with a strategy to overcome disposal problems.

Strategies for recovery and purification of poly[(R)-3-hydroxyalkanoates] (PHA) biopolyesters from surrounding biomass

To provide competitive alternatives to classical plastics, production of poly[(R)‐3‐hydroxyalkanoate] (PHA) biopolyesters has to become more economical. Downstream processing for PHA recovery from biomass plays a vital role in the PHA manufacturing process with respect to cost performance, material quality, and eco‐balance. Several factors impact the selection of the adequate PHA recovery method: the microbial production strain, type and composition of PHA, PHA load in biomass, required product purity, availability of chemicals for PHA recovery, and impact on physical properties of PHA. In this review, we compare classical and novel strategies for PHA recovery from microbial biomass. Approaches for reducing solvent and energy inputs, focusing on obtaining endotoxin‐poor PHA for medical application, are presented, as well as recent developments in efficient disruption of PHA‐rich biomass. In addition, particularities of extremophiles and genetically modified microorganisms with properties facilitating the release and separation of PHA granules are discussed.

Biotechnological production of poly(3-hydroxybutyrate) with Wautersia eutropha by application of green grass juice and silage juice as additional complex substrates

Alternative inexpensive complex nitrogen- and phosphate sources from agriculture, green grass juice (GGJ) and silage juice (SJ), were added to cultivation medium in order to investigate their impact on growth of the well-known polyhydroxyalkanoate (PHA) accumulating strain Wautersia eutropha. The influence of these additives was directly compared with cultivations on defined minimal mineral medium (M) as well as on the same medium supplemented with more expensive complex additives: corn steep liquor (CSL) and casamino acids (CA). It turned out that the supplementation with most complex additives results in shortening of lag-phases of bacterial growth and in higher end-concentrations of residual biomass compared with M-medium. Finally, higher volumetric productivities for poly(3-hydroxybutyrate) (3-PHB) were achieved. The effect of the inexpensive additive SJ on volumetric productivity was similar to the result for the expensive CA (0.653 vs. 0.619 g L−1 h−1). The same was found for the biomass concentration (7.00 vs. 7.44 g L−1 respectively). Together with an economic appraisal presented in this study, the results suggest it is possible to make the sustainable process of microbial PHA-production more economically feasible. A survey of the thermal characteristics and molecular mass properties of the isolated polymers completes this work.

High production of poly(3-hydroxybutyrate) from a wild Bacillus megaterium Bolivian strain

Taking into account that a novel strain of Bacillus megaterium was isolated from Uyuni salt lake (Bolivia) in a previous work, the objectives of this new study were to determine the maximal Poly‐3‐hydroxybutyrate production potential of B. megaterium strain uyuni S29 in an industrial conventional media, the possibility that the strain accumulates different types of polyhydroxyalkanoates, the cellular morphology during the biosynthesis process and the characterization of the produced biopolymers.

Bacterial epoxide hydrolases of opposite enantiopreference

Epoxide hydrolases of matching opposite enantiopreference were found among various Actinomyces spp. While (S)-2,2-disubstituted oxiranes were hydrolyzed by Rhodococcus and Nocardia spp., several strains of methylotrophic bacteria, such as Mycoplana rubra and Methylobacterium spp., exhibited a preference for the (R)-enantiomers. Thus, the stereochemical course of the reaction can be controlled by a simple choice of the appropriate enzyme source.

Novel precursors for production of 3-hydroxyvalerate-containing poly[(R)-hydroxyalkanoate]s

Abstract Polyhydroxyalkanoates (PHAs) with tailored properties are needed to meet consumer demands regarding the use of eco-compatible biobased polymeric materials and relevant plastic items. Inserting 3-hydroxvalerate (3HV) monomeric units in PHA biopolyesters results in poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyvalerate] (PHBHV) copolyesters aimed at their conversion into production of biodegradable eco-friendly plastic items. As inexpensive novel precursor substrate structurally related to 3HV, a mixture of odd-numbered carboxylic acids with 9–17 carbon atoms was produced by oxidative ozonolysis of alkenes. This mixture was successfully applied for biomediated PHBHV production by Cupriavidus necator. Applying this mixture as carbon substrate, a molar 3HV fraction exceeding 0.12 was obtained. The isolated copolyesters featured a low degree of crystallinity, narrow molar mass distribution, and low melting temperatures. These properties should make application of the novel 3HV-precursors interesting for large-scale production of easily processable copolyesters.