Marine algal carbohydrates as carbon sources for the production of biochemicals and biomaterials.

The high content of lipids in microalgae (>60% w/w in some species) and of carbohydrates in seaweed (up to 75%) have promoted intensive research towards valorisation of algal components for the production of biofuels. However, the exploitation of the carbohydrate fraction to produce a range of chemicals and chemical intermediates with established markets is still limited. These include organic acids (e.g. succinic and lactic acid), alcohols other than bioethanol (e.g. butanol), and biomaterials (e.g. polyhydroxyalkanoates). This review highlights current and potential applications of the marine algal carbohydrate fractions as major C-source for microbial production of biomaterials and building blocks.

Feeding strategies for tuning poly (3-hydroxybutyrate-co-4-hydroxybutyrate) monomeric composition and productivity using Burkholderia sacchari.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-4HB)) co-polymers were produced at bench-scale in fed-batch cultivations by Burkholderia sacchari from glucose (main carbon-source) and gamma-butyrolactone (GBL) as co-substrate. As P(3HB-4HB) properties highly depend on the 4-hydroxybutyrate (4HB) molar fraction, it is advantageous to have a thorough knowledge of the process in order to promote the production of the targeted final product. In this work, polymers with a 4HB molar percentage ranging from 1.5 to 8.4% (mol/mol) were obtained as consequence of a fine tuning of the fed-batch operation conditions, namely regarding the co-substrate feeding rate and its addition time, as GBL is toxic to B. sacchari cells. The best results regarding both the 4HB incorporation (molar%) and the co-polymer productivity (7.1% and 1.1g/(L.h) respectively) were reached when a pulse of GBL (<10g/L) was added early in the accumulation phase followed by a constant GBL addition at a rate similar to that of consumption so that a steady co-substrate concentration in the medium was maintained.

A Burkholderia sacchari cell factory: production of poly-3-hydroxybutyrate, xylitol and xylonic acid from xylose-rich sugar mixtures.

Efficient production of poly-3-hydroxybutyrate (P(3HB)) based on glucose-xylose mixtures simulating different types of lignocellulosic hydrolysate (LCH) was addressed using Burkholderia sacchari, a wild strain capable of metabolizing both sugars and producing P(3HB). Carbon catabolite repression was avoided by maintaining glucose concentration below 10g/L. Xylose concentrations above 30g/L were inhibitory for growth and production. In fed-batch cultivations, pulse size and feed addition rate were controlled in order to reach high productivities and efficient sugar consumptions. High xylose uptake and P(3HB) productivity were attained with glucose-rich mixtures (glucose/xylose ratio in the feed=1.5w/w) using high feeding rates, while with xylose-richer feeds (glucose/xylose=0.8w/w), a lower feeding rate is a robust strategy to avoid xylose build-up in the medium. Xylitol production was observed with xylose concentrations in the medium above 30-40g/L. With sugar mixtures featuring even lower glucose/xylose ratios, i.e. xylose-richer feeds (glucose/xylose=0.5), xylonic acid (a second byproduct) was produced. This is the first report of the ability of Burkholderia sacchari to produce both xylitol and xylonic acid.

Polyhydroxyalkanoates: waste glycerol upgrade into electrospun fibrous scaffolds for stem cells culture.

This integrated study shows that waste glycerol can be bio-valorized by the fabrication of electrospun scaffolds for stem cells. Human mesenchymal stem cells (hMSC) provide an interesting model of regenerating cells because of their ability to differentiate into osteo-, chrondro-, adipo- and myogenic lineages. Moreover, hMSC have modulatory properties with potential on treatment of immunologic diseases. Electrospun fiber meshes offer tunable mechanical and physical properties that can mimic the structure of the native extracellular matrix, the natural environment where cells inhabit. Following a biorefinery approach, crude glycerol directly recovered from a biodiesel post-reaction stream was fed as major C source to Cupriavidus necator DSM 545 to produce polyhydroxyalkanoates at polymer titers of 9-25g/L. Two of the P(3HB-4HB-3HV) terpolymers produced, one containing 11.4% 4HB and 3.5% 3HV and the other containing 35.6% 4HB and 3.4% 3HV, were electrospun into fibers of average diameters of 600 and 1400nm, respectively. hMSC were cultured for 7 days in both fiber meshes, showing their ability to support stem cell growth at acceptable proliferation levels. Comparative results clearly demonstrate that scaffold topology is critical, with electrospun PHA fibers succeeding on the support of significant cell adhesion and proliferation, where planar PHA films failed.

Enhanced bioproduction of poly-3-hydroxybutyrate from wheat straw lignocellulosic hydrolysates.

Polyhydroxyalkanoates (PHAs) are bioplastics that can replace conventional petroleum-derived products in various applications. One of the major barriers for their widespread introduction in the market is the higher production costs compared with their petrochemical counterparts. In this work, a process was successfully implemented with high productivity based on wheat straw, a cheap and readily available agricultural residue, as raw material. The strain Burkholderia sacchari DSM 17165 which is able to metabolise glucose, xylose and arabinose, the main sugars present in wheat straw hydrolysates (WSHs), was used. Results in shake flask showed that B. sacchari cells accumulated about 70%gpoly(3-hydroxybutyrate)(P(3HB))/g cell dry weight (CDW) with a yield of polymer on sugars (YP/S) of 0.18g/g when grown on a mixture of commercial C6 and C5 sugars (control), while these values reached about 60%gP(3HB)/g CDW and 0.19g/g, respectively, when WSHs were used as carbon source. In fed-batch cultures carried out in 2L stirred-tank reactors (STRs) on WSH, a maximum polymer concentration of 105 g/L was reached after 61 hours of cultivation corresponding to an accumulation of 72% of CDW. Polymer yield and productivity were 0.22 gP(3HB)/g total sugar consumed and 1.6g/L hour, respectively. The selected feeding strategy successfully overcame the carbon catabolite repression (CCR) phenomenon observed with sugar mixtures containing hexoses and pentoses. This is the first work describing fed-batch cultivations aiming at PHA production using real lignocellulosic hydrolysates. Additionally, the P(3HB) volumetric productivities attained are by far the highest ever achieved on agricultural waste hydrolysates.

On the heterogeneous composition of bacterial polyhydroxyalkanoate terpolymers.

Poly(3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxyvalerate) (P(3HB-4HB-3HV)) terpolymers of low 3-hydroxyvalerate (3HV) content (1.7-6.4%) with 4-hydroxybutyrate (4HB) molar fractions from 1.8% to 35.6% were produced by fed-batch cultivation of Cupriavidus necator DSM545. Waste glycerol, γ-butyrolactone and propionic acid were used as main carbon source, 4HB and 3HV precursors, respectively. Uniaxial tensile tests were performed on the corresponding biopolymers. The Young's modulus and tensile strength of P(3HB-4HB-3HV) decreased, whereas the elongation at break increased with the 4HB molar%, following the general trend described for poly(3-hydroxybutyrate-4-hydroxybutyrate) (P(3HB-4HB)) but with pronounced lower elasticity. Differential scanning calorimetry results indicate that the temperature of crystallization and enthalpy of melting decreased as the 4HB% increased. No crystallization was observed in terpolymers containing more than 30% of heteromonomers (4HB and 3HV) even though multiple melting events were detected. Terpolymer fractions of different composition were obtained by solvent-fractionation of the original bacterial terpolymers.

Effect of cultivation parameters on the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and poly(3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxyvalerate) by Cupriavidus necator using waste glycerol.

Short-chain polyhydroxyalkanoate co-polymers (poly(3-hydroxybutyrate-co-4-hydroxybutyrate)) (P(3HB-co-4HB)) and terpolymers (poly(3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxyvalerate)) (P(3HB-4HB-3HV)) were produced using high-cell density fed-batch cultures of Cupriavidus necator DSM 545. C-source for growth and 3HB synthesis was waste glycerol (GRP) from a biodiesel plant. Incorporation of 4HB monomers was promoted by γ-butyrolactone (GBL). Propionic acid (PA), a stimulator of 4HB accumulation, increased the 4HB molar ratio 2-fold, but also acted as 3HV precursor, yielding P(3HB-4HB-3HV). Dissolved oxygen (DOC) was a key parameter for % PHA accumulation and volumetric productivity (Prod(vol)). 4HB molar ratio increased in the presence of PA and with extended accumulation time. By manipulating DOC and cultivation time, P(3HB-4HB) with between 11.4 and 21.5 molar% of 4HB were attained. Similarly, P(3HB-4HB-3HV) was obtained with 4HB molar% between 24.8% and 43.6% and 3HV% from 5.6% to 9.8%. Mw varied between 5.5 × 10(5) and 1.37 × 10(6)Da. PHA production from GRP helps reducing production costs with concomitant GRP valorization.

Adaptation of Cupriavidus necator to conditions favoring polyhydroxyalkanoate production.

The fatty acid (FA) composition of the bacterial membrane of Cupriavidus necator DSM 545 was assessed during the time course of two-stage fed-batch cultivations for the production of short-chain polyhydroxyalkanoates (PHA). Changes in the relative proportion of straight, methyl and cyclopropyl saturated, unsaturated, hydroxy substituted and polyunsaturated FA were observed, depending on the C sources and cultivation conditions used to favor the synthesis of poly(3-hydroxybutyrate) (P(3HB)), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) or poly(3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxyvalerate) (P(3HB-4HB-3HV)), under N limiting conditions. The relative percentage of each FA class was studied using glucose or waste glycerol (GRP), as main C source for P(3HB) production. The FA profile was also assessed when GRP was used together with i) γ-butyrolactone (GBL) (precursor of 4HB monomers) for P(3HB-4HB) synthesis and ii) GBL and propionic acid (PA) (3HV precursor) to yield P(3HB-4HB-3HV). The effect of GBL and PA utilization as PHA monomer precursors on the FA profile of the cell membrane was studied under two different dissolved oxygen concentrations (DOC).