Anaerobic Production of Poly(3-hydroxybutyrate) and Its Precursor 3-Hydroxybutyrate from Synthesis Gas by Autotrophic Clostridia.

Anaerobic production of the biopolymer poly(3-hydroxybutyrate) (PHB) and the monomer 3-hydroxybutyrate (3-HB) was achieved using recombinant clostridial acetogens supplied with syn(thesis) gas as the sole carbon and energy source. 3-HB production was successfully accomplished by a new synthetic pathway containing the genes thlA (encoding thiolase A), ctfA/B (encoding CoA-transferase A/B), and bdhA (encoding (R)-3-hydroxybutyrate dehydrogenase). The respective recombinant Clostridium coskatii [p83_tcb] strain produced autotrophically 0.98 ± 0.12 mM and heterotrophically 21.7 ± 0.27 mM 3-HB. As a proof of concept, production of PHB was achieved using recombinant C. coskatii and Clostridium ljungdahlii strains expressing a novel synthetic PHB pathway containing the genes thlA (encoding thiolase A), hbd (encoding 3-hydroxybutyryl-CoA dehydrogenase), crt (encoding crotonase), phaJ (encoding (R)-enoyl-CoA hydratase), and phaEC (encoding PHA synthase). The strain C. coskatii [p83_PHB_Scaceti] synthesized heterotrophically 3.4 ± 0.29% PHB per cell dry weight (CDW) and autotrophically 1.12 ± 0.12% PHB per CDW.

Tailor-made PAT platform for safe syngas fermentations in batch, fed-batch and chemostat mode with Rhodospirillum rubrum.

Recently, syngas has gained significant interest as renewable and sustainable feedstock, in particular for the biotechnological production of poly([R]-3-hydroxybutyrate) (PHB). PHB is a biodegradable, biocompatible polyester produced by some bacteria growing on the principal component of syngas, CO. However, working with syngas is challenging because of the CO toxicity and the explosion danger of H2 , another main component of syngas. In addition, the bioprocess control needs specific monitoring tools and analytical methods that differ from standard fermentations. Here, we present a syngas fermentation platform with a focus on safety installations and process analytical technology (PAT) that serves as a basis to assess the physiology of the PHB-producing bacterium Rhodospirillum rubrum. The platform includes (i) off-gas analysis with an online quadrupole mass spectrometer to measure CO consumption and production rates of H2 and CO2 , (ii) an at-line flow cytometer to determine the total cell count and the intracellular PHB content and (iii) different online sensors, notably a redox sensor that is important to confirm that the culture conditions are suitable for the CO metabolization of R. rubrum. Furthermore, we present as first applications of the platform a fed-batch and a chemostat process with R. rubrum for PHB production from syngas.

Robust at-line quantification of poly(3-hydroxyalkanoate) biosynthesis by flow cytometry using a BODIPY 493/503-SYTO 62 double-staining.

Poly(3-hydroxyalkanoates) (PHAs) are bio-based and biodegradable polyesters which have been considered as a promising alternative to petrol-based plastics. Their bacterial production is a dynamic process in which intracellular polymerization and depolymerization are closely linked and depend on the availability of carbon substrates and other nutrients. These dynamics require a fast and quantitative method to determine the optimal harvest-time of PHA containing cells or to adjust carbon supply. In principle, flow cytometry (FCM) is an ideal tool that suits these requirements and, in addition, provides data on the PHA content of different cell populations. However, FCM-based PHA quantification methods have often relied on laborious sample preparation including washing steps and long incubation times. Here, we introduce a fast method based on double-staining using BODIPY 493/503 for PHA staining and SYTO 62 for DNA that allows acquiring reliable fluorescence and cell count data in <10min. Finally, fed-batch experiments with Pseudomonas putida KT2440 and Rhodospirillum rubrum S1 revealed that the method was robust and independent of the strain and type of PHA (medium-chain-length [mcl-] and short-chain-length [scl-] PHA, respectively). Interestingly, the specific PHA fluorescence was in case of mcl-PHA larger than for scl-PHA, probably reflecting the different material properties (e.g., specific density, hydrophilicity and crystallinity).

Fruit pomace and waste frying oil as sustainable resources for the bioproduction of medium-chain-length polyhydroxyalkanoates.

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are biobased and biodegradable alternatives to petrol-derived polymers, whose break-through has been prevented by high production cost. Therefore we investigated whether wastes from the food industry (nine types of fruit pomace including apricots, cherries and grapes, and waste frying oil) could replace the costly sugars and fatty acids typically used as carbon substrates for the bacterial fermentations. A selection of enzyme preparations was tested for converting the residual polysaccharides from the pomaces into fermentable monosaccharides. From the pomace of apricots, cherries and Solaris grapes, 47, 49 and 106gL(-1) glucose were recovered, respectively. Solaris grapes had the highest sugar content whereas apricots contained the fewest growth inhibitors. These two pomaces were assessed for their suitability to produce mcl-PHA in bioreactor. A 2-step fermentation was established with Pseudomonas resinovorans, hydrolyzed pomace as growth substrate and WFO as mcl-PHA precursor. Solaris grapes proved to be a very promising growth substrate, resulting in the production of 21.3gPHA(Lpomace)(-1) compared to 1.4g PHA (L pomace)(-1) for apricots. Finally, capillary zone electrophoresis analyses allowed monitoring of sugar and organic acid uptake during the fermentation on apricots, which led to the discovery of reverse diauxie in P. resinovorans.

New insights on the reorganization of gene transcription in Pseudomonas putida KT2440 at elevated pressure.

BACKGROUND: Elevated pressure, elevated oxygen tension (DOT) and elevated carbon dioxide tension (DCT) are readily encountered at the bottom of large industrial bioreactors and during bioprocesses where pressure is applied for enhancing the oxygen transfer. Yet information about their effect on bacteria and on the gene expression thereof is scarce. To shed light on the cellular functions affected by these specific environmental conditions, the transcriptome of Pseudomonas putida KT2440, a bacterium of great relevance for the production of medium-chain-length polyhydroxyalkanoates, was thoroughly investigated using DNA microarrays. RESULTS: Very well defined chemostat cultivations were carried out with P. putida to produce high quality RNA samples and ensure that differential gene expression was caused exclusively by changes of pressure, DOT and/or DCT. Cellular stress was detected at 7 bar and elevated DCT in the form of heat shock and oxidative stress-like responses, and indicators of cell envelope perturbations were identified as well.Globally, gene transcription was not considerably altered when DOT was increased from 40 ± 5 to 235 ± 20% at 7 bar and elevated DCT. Nevertheless, differential transcription was observed for a few genes linked to iron-sulfur cluster assembly, terminal oxidases, glutamate metabolism and arginine deiminase pathway, which shows their particular sensitivity to variations of DOT. CONCLUSIONS: This study provides a comprehensive overview on the changes occurring in the transcriptome of P. putida upon mild variations of pressure, DOT and DCT. Interestingly, whereas the changes of gene transcription were widespread, the cell physiology was hardly affected, which illustrates how efficient reorganization of the gene transcription is for dealing with environmental changes that may otherwise be harmful. Several particularly sensitive cellular functions were identified, which will certainly contribute to the understanding of the mechanisms involved in stress sensing/response and to finding ways of enhancing the stress tolerance of microorganisms.

Pressure to kill or pressure to boost: a review on the various effects and applications of hydrostatic pressure in bacterial biotechnology.

Much knowledge has been gained for the last 30 years about the effects of pressure on bacteria, and various pressure-based technologies have been designed. The development of modern molecular biology techniques (e.g., DNA microarrays) as well as the technological advances realized in the manufacturing of robust sampling and high-pressure devices has allowed these advances. Not only the direct effects on cell components (membranes, proteins, and nucleic acids) have been unraveled, but also the cellular response to pressure has been investigated by means of transcriptome and proteome analyses. Initially, research was performed by marine biologists who studied the microorganisms living in the deep sea at pressures of 1,000 bar. In parallel, food technologists developed pressure-based methods for inactivating microorganisms without altering the food properties as much as with temperature treatment. The preservation of specific product properties is also the rationale for pressure-based methods for the disinfection of biomaterials and for vaccine production. Therefore, attention was first focused on the "killing" potential of high pressure. On the other hand, there has been a growing interest in using elevated pressures (up to ~10 bar) for enhancing the productivity of bioprocesses. In this case, no killing effect was sought, but pressure was applied to "boost" the process by enhancing the oxygen transfer to the cell culture. This paper gives an overview on the effects of pressures in the range of 1 bar to 10 kbar on bacteria and presents the major and most recent achievements realized in the development of pressure-based biotechnological applications.

Putting cells under pressure: a simple and efficient way to enhance the productivity of medium-chain-length polyhydroxyalkanoate in processes with Pseudomonas putida KT2440.

The success of bioprocess implementation relies on the ability to achieve high volumetric productivities and requires working with high-cell-density cultivations. Elevated atmospheric pressure might constitute a promising tool for enhancing the oxygen transfer rate (OTR), the major growth-limiting factor for such cultivations. However, elevated pressure and its effects on the cellular environment also represent a potential source of stress for bacteria and may have negative effects on product formation. In order to determine whether elevated pressure can be applied for enhancing productivity in the case of medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida KT2440, the impact of a pressure of 7 bar on the cell physiology was assessed. It was established that cell growth was not inhibited by this pressure if dissolved oxygen tension (DOT) and dissolved carbon dioxide tension (DCT) were kept below ∼30 and ∼90 mg L(-1), respectively. Remarkably, a little increase of mcl-PHA volumetric productivity was observed under elevated pressure. Furthermore, the effect of DCT, which can reach substantial levels during high-cell-density processes run under elevated pressure, was investigated on cell physiology. A negative effect on product formation could be dismissed since no significant reduction of mcl-PHA content occurred up to a DCT of ∼540 mg L(-1). However, specific growth rate exhibited a significant decrease, indicating that successful high-cell-density processes under elevated pressure would be restricted to chemostats with low dilution rates and fed-batches with a small growth rate imposed during the final part. This study revealed that elevated pressure is an adequate and efficient way to enhance OTR and mcl-PHA productivity. We estimate that the oxygen provided to the culture broth under elevated pressure would be sufficient to triple mcl-PHA productivity in our chemostat system from 3.4 (at 1 bar) to 11 g L(-1)h(-1) (at 3.2 bar).

A reduction in growth rate of Pseudomonas putida KT2442 counteracts productivity advances in medium-chain-length polyhydroxyalkanoate production from gluconate.

BACKGROUND: The substitution of plastics based on fossil raw material by biodegradable plastics produced from renewable resources is of crucial importance in a context of oil scarcity and overflowing plastic landfills. One of the most promising organisms for the manufacturing of medium-chain-length polyhydroxyalkanoates (mcl-PHA) is Pseudomonas putida KT2440 which can accumulate large amounts of polymer from cheap substrates such as glucose. Current research focuses on enhancing the strain production capacity and synthesizing polymers with novel material properties. Many of the corresponding protocols for strain engineering rely on the rifampicin-resistant variant, P. putida KT2442. However, it remains unclear whether these two strains can be treated as equivalent in terms of mcl-PHA production, as the underlying antibiotic resistance mechanism involves a modification in the RNA polymerase and thus has ample potential for interfering with global transcription. RESULTS: To assess PHA production in P. putida KT2440 and KT2442, we characterized the growth and PHA accumulation on three categories of substrate: PHA-related (octanoate), PHA-unrelated (gluconate) and poor PHA substrate (citrate). The strains showed clear differences of growth rate on gluconate and citrate (reduction for KT2442 > 3-fold and > 1.5-fold, respectively) but not on octanoate. In addition, P. putida KT2442 PHA-free biomass significantly decreased after nitrogen depletion on gluconate. In an attempt to narrow down the range of possible reasons for this different behavior, the uptake of gluconate and extracellular release of the oxidized product 2-ketogluconate were measured. The results suggested that the reason has to be an inefficient transport or metabolization of 2-ketogluconate while an alteration of gluconate uptake and conversion to 2-ketogluconate could be excluded. CONCLUSIONS: The study illustrates that the recruitment of a pleiotropic mutation, whose effects might reach deep into physiological regulation, effectively makes P. putida KT2440 and KT2442 two different strains in terms of mcl-PHA production. The differences include the onset of mcl-PHA production (nitrogen limitation) and the resulting strain performance (growth rate). It remains difficult to predict a priori where such major changes might occur, as illustrated by the comparable behavior on octanoate. Consequently, experimental data on mcl-PHA production acquired for P. putida KT2442 cannot always be extrapolated to KT2440 and vice versa, which potentially reduces the body of available knowledge for each of these two model strains for mcl-PHA production substantially.