Growth independent rhamnolipid production from glucose using the non-pathogenic Pseudomonas putida KT2440

<p>Abstract</p> <p>Background</p> <p>Rhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen <it>Pseudomonas aeruginosa </it>during growth on hydrophobic substrates such as plant oils. The heterologous production of rhamnolipids entails two essential advantages: Disconnecting the rhamnolipid biosynthesis from the complex quorum sensing regulation and the opportunity of avoiding pathogenic production strains, in particular <it>P. aeruginosa</it>. In addition, separation of rhamnolipids from fatty acids is difficult and hence costly.</p> <p>Results</p> <p>Here, the metabolic engineering of a rhamnolipid producing <it>Pseudomonas putida </it>KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, <it>P. putida </it>KT2440 features almost no changes in growth rate and lag-phase in the presence of high concentrations of rhamnolipids (> 90 g/L) in contrast to the industrially important bacteria <it>Bacillus subtilis, Corynebacterium glutamicum</it>, and <it>Escherichia coli. P. putida </it>KT2440 expressing the <it>rhlAB</it>-genes from <it>P. aeruginosa </it>PAO1 produces mono-rhamnolipids of <it>P. aeruginosa </it>PAO1 type (mainly C₁₀:C₁₀). The metabolic network was optimized in silico for rhamnolipid synthesis from glucose. In addition, a first genetic optimization, the removal of polyhydroxyalkanoate formation as competing pathway, was implemented. The final strain had production rates in the range of <it>P. aeruginosa </it>PAO1 at yields of about 0.15 g/g_glucosecorresponding to 32% of the theoretical optimum. What's more, rhamnolipid production was independent from biomass formation, a trait that can be exploited for high rhamnolipid production without high biomass formation.</p> <p>Conclusions</p> <p>A functional alternative to the pathogenic rhamnolipid producer <it>P. aeruginosa </it>was constructed and characterized. <it>P. putida </it>KT24C1 pVLT31_<it>rhlAB </it>featured the highest yield and titer reported from heterologous rhamnolipid producers with glucose as carbon source. Notably, rhamnolipid production was uncoupled from biomass formation, which allows optimal distribution of resources towards rhamnolipid synthesis. The results are discussed in the context of rational strain engineering by using the concepts of synthetic biology like chassis cells and orthogonality, thereby avoiding the complex regulatory programs of rhamnolipid production existing in the natural producer <it>P. aeruginosa</it>.</p

Modeling and optimization of extracellular polysaccharides production by Enterobacter A47

Polysaccharides are gaining increasing attention as potential environmental friendly and sustainable building blocks in many fields of the (bio)chemical industry. The microbial production of polysaccharides is envisioned as a promising path, since higher biomass growth rates are possible and therefore higher productivities may be achieved compared to vegetable or animal polysaccharides sources. This Ph.D. thesis focuses on the modeling and optimization of a particular microbial polysaccharide, namely the production of extracellular polysaccharides (EPS) by the bacterial strain Enterobacter A47. Enterobacter A47 was found to be a metabolically versatile organism in terms of its adaptability to complex media, notably capable of achieving high growth rates in media containing glycerol byproduct from the biodiesel industry. However, the industrial implementation of this production process is still hampered due to a largely unoptimized process. Kinetic rates from the bioreactor operation are heavily dependent on operational parameters such as temperature, pH, stirring and aeration rate. The increase of culture broth viscosity is a common feature of this culture and has a major impact on the overall performance. This fact complicates the mathematical modeling of the process, limiting the possibility to understand, control and optimize productivity. In order to tackle this difficulty, data-driven mathematical methodologies such as Artificial Neural Networks can be employed to incorporate additional process data to complement the known mathematical description of the fermentation kinetics. In this Ph.D. thesis, we have adopted such an hybrid modeling framework that enabled the incorporation of temperature, pH and viscosity effects on the fermentation kinetics in order to improve the dynamical modeling and optimization of the process. A model-based optimization method was implemented that enabled to design bioreactor optimal control strategies in the sense of EPS productivity maximization. It is also critical to understand EPS synthesis at the level of the bacterial metabolism, since the production of EPS is a tightly regulated process. Methods of pathway analysis provide a means to unravel the fundamental pathways and their controls in bioprocesses. In the present Ph.D. thesis, a novel methodology called Principal Elementary Mode Analysis (PEMA) was developed and implemented that enabled to identify which cellular fluxes are activated under different conditions of temperature and pH. It is shown that differences in these two parameters affect the chemical composition of EPS, hence they are critical for the regulation of the product synthesis. In future studies, the knowledge provided by PEMA could foster the development of metabolically meaningful control strategies that target the EPS sugar content and oder product quality parameters

Evaluation of Effects of Methods Used for Forage Analysis and Dietary Inclusion of Buffer on Fiber Digestibility and Rumen Fermentation in Diets High in Distillers Grains

Forage quality is an important factor affecting intake and utilization of forage, thus making it imperative to evaluate the methods used to determine the nutritive values of forage in order to predict animal performance. Additionally, fibrous feeds for ruminants are less subject to competitive demand. The plant cell wall is the largest hindrance to complete digestion of feeds, particularly forages and by-products and to the utilization of the nutrients and energy they contain, necessitating effective strategies for increasing the rate and efficiency of utilization of forage fiber and the energy therein. It is critically important to increase fiber digestion for productivity and environmental reasons. Two studies were conducted in an attempt to make recommendations based on the methods used for forage analysis and rumen fermentation of dietary inclusion of buffer in diets high in distillers grains. The first study compared five forages: Hay (Hay), Conventional Corn Silage (CCS), Conventional Haylage (CHL), Hybrid Corn Silage (HCS), and Hybrid Haylage (HHL) in an in situ (dry/wet) and in vitro trials for differences in dry matter and fiber degradability. Results showed different methods vary in digestibility values, but difference among forage followed similar patterns among method. Further research will be warranted to standardize procedures to be used for methods to evaluate forage quality. The second study evaluated the effects of High buffer (HiBuffer) and Low buffer (LoBuffer) inclusion on nutrient digestibility, rumen parameters, and blood metabolites in steers limit-fed diets high in distillers grains. Five dairy cannulated steers (Brown Swiss and Holstein) 303.4± 45 d of age were used in a cross over design experiment within a 2-week period. Two treatment diets containing 40% DDGS with High (HiBuffer) or Low (LoBuffer) buffer inclusion concentrations were fed. Results show differences in DMI and G:F, while BW and ADG were similar among treatments. The rumen total VFA, acetate: propionate, and pH were similar among treatments. For blood metabolites there were treatment effects for glucose and cholesterol, while plasma urea nitrogen concentrations were similar among treatments. Total tract digestion of nutrients was similar among treatments. Result demonstrates that buffer inclusion had limited impact on utilization of DDGS. However, future research is warranted to determine the precise amount of buffer inclusion and DDGS feeding rate

Mechanistic understanding of mixed-culture fermentations by metabolic modelling

Biorefineries are set to become an important agent in the shift towards a circular economy due to their potential to valorise organic wastes into marketable products. Anaerobic fermentations yielding volatile fatty acids are a key process in this production scheme as their products act as intermediates between the organic wastes and the final biorefinery products. However, their product selectivity is highly influenced by the environmental conditions and the mechanisms governing the process remain unknown. In this thesis, predictive tools were developed with the objective of understanding the mechanisms governing anaerobic fermentations and of designing processes targeting specific volatile fatty acids with high productivity

Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3

Nowadays, we are witnessing highly dynamic research activities related to the intriguing field of biodegradable materials with plastic-like properties. These activities are currently intensified by a strengthened public awareness of prevailing ecological issues connected to growing piles of plastic waste, microplastic formation, and increasing greenhouse gas emissions; this goes hand-in-hand with the ongoing depletion of fossil feedstocks, which are traditionally used to produce full carbon backbone polymers. To a steadily increasing extend, polyhydroxyalkanoate (PHA) biopolyesters, a family of plastic-like materials with versatile material properties, are considered a future-oriented solution for diminishing these concerns. PHA production is based on renewable resources, and occurs in a bio-mediated fashion by the action of living organisms. If accomplished in an optimized way, PHA production and the entire PHA lifecycle are embedded into nature´s closed cycles of carbon. Holistic improvement of PHA production, applicable on an industrially relevant scale, calls for inter alia: consolidated knowledge about the enzymatic and genetic particularities of PHA accumulating organisms, in-depth understanding of the kinetics of the bioprocess, the selection of appropriate inexpensive fermentation feedstocks, tailoring the composition of PHA on the level of the monomeric constituents, optimized biotechnological engineering, and novel strategies for PHA recovery from biomass characterized by minor energy and chemical requirement

THE KINETICS OF TWO HETEROTROPHIC TETRACHLOROETHENE-RESPIRING POPULATIONS AND THEIR EFFECTS ON THE SUBSTRATE INTERACTIONS WITH DEHALOCOCCOIDES STRAINS

This study focused on evaluating how interactions between the hydrogenotroph Dehalococcoides ethenogenes strain 195, which is able to completely dechlorinate tetrachloroethene (PCE) to ethene, and the two heterotrophs Desulfuromonas michiganensis strain BB1 and Desulfitobacterium sp. strain PCE1, which dechlorinate PCE to either cis-dichloroethene (cis-DCE) or trichloroethene (TCE), on the fate of PCE under common in situ bioremediation scenarios. Meaningful kinetic parameter estimates were obtained for the heterotrophic dehalorespirers under a wide range of conditions. Batch culture assays and numerical experiments were conducted with Desulfuromonas michiganensis to evaluate the effect of the initial conditions including the ratio of the initial substrate concentration (S0) to the initial biomass concentration (X0) and the ratio of S0 to the half-saturation constant (KS) on parameter correlation. Most importantly, S0/KS, but not S0/X0, strongly influenced parameter correlation. Correlation between the Monod kinetic parameters could be minimized by maximizing S0/KS. In the present study, dechlorination of high PCE concentrations by Desulfuromonas michiganensis and Desulfitobacterium sp. strain PCE1 was monitored. The maximum level of PCE that could be dechlorinated by each strain was not constant, and varied with X0. This phenomenon could not be described using conventional Monod kinetics; therefore, a new model that incorporated an inactivation term into the biomass growth equation was developed to describe dechlorination at high PCE concentrations. The interactions among Dehalococcoides ethenogenes and heterotrophic dehalorespirer in continuous-flow stirred tank reactors (CSTRs) were performed under two conditions that reflect either a natural attenuation or engineered bioremediation treatment scenario. Extant kinetic estimates accurately predicted the steady-state chlorinated ethene concentrations in the CSTRs. However, intrinsic kinetic parameter estimates better described the CSTR start-up phase. The modeling and experimental results suggested that the ability of Dehalococcoides ethenogenes to utilize PCE and TCE is limited by the presence of a PCE-to-TCE/cis-DCE dehalorespirer, which forces Dehalococcoides ethenogenes to function primarily as a cis-DCE-respiring population. This study provides insight into how the activities of different dehalorespiring cultures are interrelated and will aid in the design of engineered bioremediation approaches that optimize the potential benefits associated with different dehalorespiring populations to achieve efficient and effective clean-up of PCE- and TCE-contaminated sites