Transcriptome Changes in Pseudomonas putida KT2440 during Medium-Chain-Length Polyhydroxyalkanoate Synthesis Induced by Nitrogen Limitation.

Pseudomonas putida's versatility and metabolic flexibility make it an ideal biotechnological platform for producing valuable chemicals, such as medium-chain-length polyhydroxyalkanoates (mcl-PHAs), which are considered the next generation bioplastics. This bacterium responds to environmental stimuli by rearranging its metabolism to improve its fitness and increase its chances of survival in harsh environments. Mcl-PHAs play an important role in central metabolism, serving as a reservoir of carbon and energy. Due to the complexity of mcl-PHAs' metabolism, the manner in which P. putida changes its transcriptome to favor mcl-PHA synthesis in response to environmental stimuli remains unclear. Therefore, our objective was to investigate how the P. putida KT2440 wild type and mutants adjust their transcriptomes to synthesize mcl-PHAs in response to nitrogen limitation when supplied with sodium gluconate as an external carbon source. We found that, under nitrogen limitation, mcl-PHA accumulation is significantly lower in the mutant deficient in the stringent response than in the wild type or the rpoN mutant. Transcriptome analysis revealed that, under N-limiting conditions, 24 genes were downregulated and 21 were upregulated that were common to all three strains. Additionally, potential regulators of these genes were identified: the global anaerobic regulator (Anr, consisting of FnrA, Fnrb, and FnrC), NorR, NasT, the sigma54-dependent transcriptional regulator, and the dual component NtrB/NtrC regulator all appear to play important roles in transcriptome rearrangement under N-limiting conditions. The role of these regulators in mcl-PHA synthesis is discussed.

Interactive effect of crude glycerin concentration and C:N ratio on polyhydroxyalkanoates accumulation by mixed microbial cultures modelled with Response Surface Methodology.

Response Surface Methodology (RSM) was used to investigate how the crude glycerin concentration and the carbon to nitrogen (C:N) ratio in the culture medium affect four indicators of polyhydroxyalkanoates (PHAs) accumulation by mixed microbial cultures (MMC): the observed coefficient of active-biomass yield (Yobs,BA), the observed coefficient of PHA yield (Yobs,PHA), the PHA content in biomass (XPHA) and the volumetric productivity (PrV). The C:N ratio had the largest effect on Yobs,BA and Yobs,PHA. When the C:N ratio was increased, Yobs,BA decreased and Yobs,PHA increased, regardless of the concentration of crude glycerin in the culture medium. The C:N ratio also had the largest effect on the PHA content, whereas volumetric productivity was strongly affected by both the C:N ratio and the crude glycerin concentration. The optimal conditions for PHA accumulation were a crude glycerin concentration of 8954 mg COD/L with a C:N ratio of 15.9 mg C/mg N-NH4, which gave a Yobs,BA of 0.29 mg CODBA/mg COD, a Yobs,PHA of 0.28 mg CODPHA/mg COD, a XPHA of 55.6% VSS and a PrV of 757.3 mg CODPHA/L⋅d (550.0 mg PHA/L⋅d). The accumulated PHAs consisted mainly of 3-hydroxybutyrate. By using RSM, it was possible to predict crude glycerin concentrations and C:N ratios not tested here that will allow desirable values of PHA content in biomass or PHA productivity, which can be useful for designing PHA production with MMC.

Exploring nutrient limitation for polyhydroxyalkanoates synthesis by newly isolated strains of Aeromonas sp. using biodiesel-derived glycerol as a substrate.

Aeromonas spp. strains isolated from activated sludge in a municipal wastewater treatment plant were found to be able to synthesize polyhydroxyalkanoates (PHA) utilizing pure and crude glycerol. The 16S rRNA gene sequence of the isolates exhibited similarity to Aeromonas hydrophila, A. aquatica, and A. salmonicida. Our results confirmed that the adequate supply of nitrogen and phosphorus during culture in 250-ml shake flasks did not stimulate the synthesis of PHAs. The results indicate that the PHA content of cells was higher under a phosphorus-limiting environment compared to nitrogen starvation. In the two-stage cultivation using glucose (in the first step) and crude glycerol from biodiesel industry (in the second step) as a component of the growth medium, the analyzed strains grew to 3.06 g/l of cell dry weight containing up to 22% of PHAs. Furthermore, during the same culture strategy up to 42% of PHAs were extracted, when in the second step of the process, Aeromonas sp. AC_03 was grown on pure glycerol under phosphorus limitation. The purified biopolymer was confirmed to be polyhydroxybutyrate. Aeromonas sp. AC_02 was also capable to accumulate the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer when pure glycerol was added as a substrate under nitrogen-deficiency one-step bioprocess. Our results confirm that due to the biopolymer productivity, newly isolated strains could be exploited for obtaining valuable biopolymers using wastes generated from biodiesel industry.

Transcriptome remodeling of Pseudomonas putida KT2440 during mcl-PHAs synthesis: effect of different carbon sources and response to nitrogen stress.

Bacterial response to environmental stimuli is essential for survival. In response to fluctuating environmental conditions, the physiological status of bacteria can change due to the actions of transcriptional regulatory machinery. The synthesis and accumulation of polyhydroxyalkanoates (PHAs) are one of the survival strategies in harsh environments. In this study, we used transcriptome analysis of Pseudomonas putida KT2440 to gain a genome-wide view of the mechanisms of environmental-friendly biopolymers accumulation under nitrogen-limiting conditions during conversion of metabolically different carbon sources (sodium gluconate and oleic acid). Transcriptomic data revealed that phaG expression is associated with medium-chain-length-PHAs' synthesis not only on sodium gluconate but also on oleic acid, suggesting that PhaG may play a role in this process, as well. Moreover, genes involved in the ß-oxidation pathway were induced in the PHAs production phase when sodium gluconate was supplied as the only carbon and energy source. The transition from exponential growth to stationary phase caused a significant expression of genes involved in nitrogen metabolism, energy supply, and transport system. In this study, several molecular mechanisms, which drive mcl-PHAs synthesis, have been investigated. The identified genes may provide valuable information to improve the efficiency of this bioprocess and make it more economically feasible.

Molecular identification of polyhydroxyalkanoates-producing bacteria isolated from enriched microbial community.

Polyhydroxyalkanoates (PHAs) are especially interesting because of their similar properties to synthetic plastics and their potential use as biodegradable polymers. Many strategies have been employed to effectively and economically produce PHAs, among them a production process based on mixed microbial populations, enriched from activated sludge could be one of the alternative technologies. Defining the bacterial species creating these anonymous populations is crucial for the improvement of cultivation strategy. Moreover, enriched bacterial populations could be a promising source for microbes, useful in many biotechnological projects. The main object of this study was to characterize the microorganisms creating the microbial consortium cultured towards PHAs production. After cultivation, bacteria were identified using the 16S rRNA gene sequencing approach. The presence of genes engaged in PHAs synthesis was detected using PCR. The performed analysis revealed that among eleven isolated bacterial strains, four possessed the ability of polyhydroxybutyrate synthesis.

Theoretical and observed biogas production from plant biomass of different fibre contents.

The methane productivity of silage of four crop species -Zeamays L., Sorghumsaccharatum, Miscanthusxgiganteus and Miscanthussacchariflorus - was investigated. The experiments revealed that at a hydraulic retention time of 60days the volumetric methane yields from the Z.mays L. or S.saccharatum silages were higher than those from the Miscanthusxgiganteus or M.sacchariflorus silages because of the higher crude fibre content in Miscanthus spp. However, at comparable lignin concentrations in the feedstock, methane productivity for M.sacchariflorus (0.19+/-0.08L/g volatile solids) was twice that of Miscanthusxgiganteus (0.10+/-0.03L/g volatile solids). The efficiency of cellulose conversion varied from 83.6% (S. saccharatum) to 52.1% (Miscanthusxgiganteus), and hemicellulose from 88.9% (Z. mays L.) to 59.7% (Miscanthusxgiganteus). Conversion of cellulose and hemicellulose depended on the ratio of these polysaccharides to the lignin concentration of the feedstock.

Cultivation-dependent and -independent characterization of microbial community producing polyhydroxyalkanoates from raw-glycerol.

High substrate costs decrease the profitability of polyhydroxyalkanoates (PHAs) production, and thus low-cost carbon substrates coming from agricultural and industrial residuals are tested for the production of these biopolymers. Among them crude glycerol, formed as a byproduct during biodiesel production, seems to be the most promising source of carbon. The object of this study was to characterize the mixed population responsible for the conversion of crude glycerol into PHAs by the cultivation-dependent and -independent methods. Enrichment of the microbial community was monitored by applying the Ribosomal Intergenic Spacer Analysis (RISA) and the identification of community members was based on 16S rRNA gene sequencing of cultivable species. Molecular analysis revealed that mixed populations consist of microorganisms affiliated with four bacterial lineages: alpha, gamma- Proteobacteria, Actinobacteria and Bacteroides. Among them, three Pseudomonas strains and Rhodobacter sp. possessed genes coding for polyhydroxyalkanoates synthase. Comparative analysis revealed that most of the microorganisms detected by direct molecular analysis were obtained by the traditional culturing method.

Changes in microbial community structure during adaptation towards polyhydroxyalkanoates production.

Polyhydroxyalkanoates (PHAs) are interesting as material for bioplastic production because they are recognized as biodegradable and could be produced from renewable resources. The industrial production of PHAs has already been used in practice by pure cultures. In recent years, many studies have been addressed of PHA production by mixed cultures. Nevertheless, while fermentation strategy to improve the PHA content of biomass, yield and productivity in pure cultures are well defined, knowledge about the operational condition for PHA synthesis by mixed culture is still very limited. The ecology of the microbial community of activated sludge remains largely unknown, primarily because of the difficulty of making detailed observation. Recently, developed molecular techniques allow determination of community composition from DNA extracted directly from biomass samples. This study examined the changes of bacterial communities in activated sludge through application of the molecular technique, ribosomal intergenic spacer analysis (RISA). Microbial communities from anaerobic-aerobic and ammonia limited fermentations were ascertained. The applied operational conditions were shown to select for a restricted microbial population, which were different in term of structure with respect to the initial microbial consortia in the activated sludge used as inoculum.

Molecular insight into activated sludge producing polyhydroxyalkanoates under aerobic-anaerobic conditions.

One of the options enabling more economic production of polyhydroxyalkanoates compared to pure cultures is the application of mixed cultures. The use of a microbial community in a sequencing batch reactor has a few advantages: a simple process control, no necessity for sterile processing, and possibilities of using cheap substrates as a source of carbon. Nevertheless, while cultivation methods to achieve high PHAs biomass concentration and high productivity in wild and recombinant strains are defined, knowledge about the cultivation strategy for PHAs production by mixed culture and species composition of bacterial communities is still very limited. The main object of this study was to characterize on the molecular level the composition and activity of PHAs producing microorganism in activated sludge cultivated under oxygen limitation conditions. PHAs producers were detected using a PCR technique and the created PHA synthase gene library was analyzed by DNA sequencing. The obtained results indicate that PHAs-producers belonged to Pseudomonas sp., and possessed genes coding for mcl-PHA synthase. The kinetics of mcl-PHA synthase expression was relatively estimated using real-time PCR technology at several timepoints. Performed quantitative and qualitative analysis of total bacterial activity showed that there were differences in total activity during the process but differential expression of various groups of microorganisms examined by using DGGE was not observed.