Ulva rigida Valorization into Poly(3-hydroxybutyrate), Organic Acids and Functional Ingredients.

Halomonas elongata 1H9T is a moderate halophilic strain able to produce poly(3-hydroxybutyrate) (P(3HB)), a biodegradable plastic, and gluconic acid, a valuable organic acid with wide industrial applications. In this work, the green alga Ulva rigida was used as platform to produce cultivation substrates for microbial conversion as well as functional ingredients, targeting its full valorization. The liquor obtained by autohydrolysis presented the highest concentration of oligosaccharides and protein, being an interesting feedstock to produce functional ingredients. The acid and/or enzymatic hydrolysis liquors are adequate as substrates for microbial processes. Shake flask assays with H. elongata revealed that the N-rich liquor produced after acidic treatment was the best suited for cell growth while the N-poor liquor produced by the enzymatic treatment of acid-pretreated algae residues produced the highest P(3HB) titers of 4.4 g/L. These hydrolysates were used in fed-batch cultivations as carbon and protein sources for the co-production of gluconic acid and polymer achieving titers of 123.2 g/L and 7.2 g/L, respectively. Besides gluconic acid, the Krebs cycle intermediate 2-oxoglutaric acid, also called alpha-ketoglutaric acid (KGA), was produced. Therefore, the co-production of P(3HB) and acids may be of considerable interest as an algal biorefinery valorization strategy.

Co-Production of Poly(3-hydroxybutyrate) and Gluconic Acid from Glucose by Halomonas elongata.

Polyhydroxyalkanoates (PHA) are biopolyesters regarded as an attractive alternative to petroleum-derived plastics. Nitrogen limitation and phosphate limitation in glucose cultivations were evaluated for poly(3-hydroxybutyrate) (P(3HB)) production by Halomonas elongata 1H9T, a moderate halophilic strain. Co-production of P(3HB) and gluconic acid was observed in fed-batch glucose cultivations under nitrogen limiting conditions. A maximum P(3HB) accumulation of 53.0% (w/w) and a maximum co-production of 133 g/L of gluconic acid were attained. Fed-batch glucose cultivation under phosphate limiting conditions resulted in a P(3HB) accumulation of only 33.3% (w/w) and no gluconic acid production. As gluconic acid is a valuable organic acid with extensive applications in several industries, this work presents an interesting approach for the future development of an industrial process aiming at the co-production of an intracellular biopolymer, P(3HB), and a value-added extracellular product, gluconic acid.

Ionic Liquid-Assisted Selective Extraction and Partitioning of Biomolecules from Macroalgae.

Macroalgae are a promising feedstock for several industries due to their large content of proteins and carbohydrates and the high biomass productivities. A novel extraction and fractionation concept based on ionic liquids (ILs) using Ulva lactuca as model organism is presented. Biomolecules are first extracted by means of IL-assisted mechanical shear, followed by two-phase partitioning or ultrafiltration in order to fractionate proteins and carbohydrates and to recover the IL. Ethyl methyl imidazolium dibutyl phosphate ([Emim][DBP]) is strongly selective to proteins, leading to extraction yields up to 80.4% for proteins and 30.7% for carbohydrates. The complete process, including extraction and ultrafiltration, allowed protein recovery of up to 64.6 and 15.4% of the carbohydrates in the retentate phase, while a maximum of 85.7% of the IL was recovered in the permeate phase. The native structure of the extracted proteins was preserved during extraction and fractionation as shown by gel electrophoresis. Selective extraction of proteins from macroalgae under non-denaturing conditions using ILs followed by the recovery of IL using ultrafiltration is for the first time reported. The proposed extraction-fractionation approach is simple and can be potentially applied for the biorefinery of macroalgae at the commercial scale.

Red algae industrial residues as a sustainable carbon platform for the co-production of poly-3-hydroxybutyrate and gluconic acid by Halomonas boliviensis.

Polyhydroxyalkanoate (PHA) production using halophilic bacteria has been revisited because less severe operational conditions with respect to sterility can be applied, also alleviating production costs. Halomonas boliviensis was selected because it is a moderate halophile able to grow and attain high poly-3-hydroxybutyrate (P3HB) contents under 5-45 g/L NaCl concentrations, conditions that discourage microbial contamination. Industrial residues of the red alga Gelidium corneum after agar extraction were used as sugar platform to reduce costs associated with the carbon source. These residues still comprise a high carbohydrate content (30-40% w/w) of mainly cellulose, and their hydrolysates can be used as substrates for the bioproduction of value-added products. Preliminary assays using glucose were carried out to determine the best conditions for growth and P3HB production by H. boliviensis in bioreactor fed-batch cultivations. Two strategies were addressed, namely nitrogen or phosphorus limitation, to promote polymer accumulation. Similar P3HB cell contents of 50% (gpolymer/gCDW) and yields Y P3HB/glucose of 0.11-0.15 g polymer/g glucose were attained under both conditions. However, higher specific productivities were reached under P-limitation, and thus, this strategy was adopted in the subsequent study. Two organic acids, resulting from glucose metabolism, were identified to be gluconic and 2-oxoglutaric acid. Reducing the oxygen concentration in the cultivation medium to 5% sat was found to minimize organic acid production and enhance the yield of polymer on sugar to 0.20 gP3HB/gglucose. Finally, fed-batch cultivations using G. corneum hydrolysates as the only C-source achieved an overall volumetric productivity of 0.47 g/(L.h), 40% polymer accumulation, and negligible gluconic acid production.

Economic and environmental assessment of bacterial poly(3-hydroxybutyrate) production from the organic fraction of municipal solid waste.

The management of municipal solid waste is a major logistic and environmental problem worldwide. Nonetheless, the organic fraction of municipal solid waste (OFMSW) is a valuable source of nutrients which can be used for a variety of purposes, according to the Circular Economy paradigm. Among the possible applications, the bioproduction of a biodegradable polyester, poly(3-hydroxybutyrate) [P(3HB)], using OFMSW as carbon platform is a promising strategy. Here, an economic and environmental assessment of bacterial P(3HB) production from OFMSW is presented based on previously published results. The SuperPro Designer® software was used to simulate P(3HB) production under our experimental parameters. Two scenarios were proposed depending on the fermentation medium: (1) enzymatic hydrolysate of OFMSW supplemented with glucose and plum waste juice; and (2) basal medium supplemented with glucose and plum waste juice. According to our results, both scenarios are not economically feasible under our experimental parameters. In Scenario 1, the low fermentation yield, the cost of the enzymes, the labour cost and the energy consumption are the factors that most contribute to that result. In Scenario 2, the cost of the extraction solvent and the low fermentation yield are the most limiting factors. The possibility of using process waste as raw material for the generation of other products must be investigated to enhance economic feasibility. From an environmental viewpoint, the photochemical oxidation potential (derived from the use of anisole as extraction solvent) and the generation of acid rain and global warming effect (caused by the burning of fuels for power generation) are the most relevant impacts associated to P(3HB) production under our experimental parameters.

Giving credit to residual bioresources: From municipal solid waste hydrolysate and waste plum juice to poly (3-hydroxybutyrate).

Municipal solid waste (MSW) is massively generated all over the world. Its organic fraction (OFMSW), which represents a high percentage of MSW, mainly contains biodegradable materials, namely food waste, paper and garden waste. The social cost of OFMSW treatment and/or disposal is a serious and widespread problem, particularly in highly populated areas. Thus, effective and innovative solutions, which include the upgrading of OFMSW, are being currently sought. In fact, the OFMSW abundance, availability and average composition suggest its considerable potential within the circular economy desideratum, paving the way to valorisation approaches. In this context, an OFMSW sugar-rich hydrolysate and its validation as a substrate for the production of the polyester poly(3-hydroxybutyrate) (P(3HB)), to date the only bioplastic easily biodegradable in marine environment, were successfully obtained in a previous study. Based on those results, this work addresses the upscaling of the fermentative production, in fed-batch mode, of P(3HB) by Burkholderia sacchari. The OFMSW hydrolysate was used as cultivation medium due to its balanced nutrient composition, while a plum waste juice, also rich in sugars, was applied as feed to the bioreactor. By implementing this strategy, a maximum P(3HB) production of 30 g·L-1 with an accumulation of 43% g (P(3HB))/g cell dry weight (CDW) after 51 h, was achieved. The use of the hydrolysate as initial medium resulted in higher CDW (71 g·L-1) than that of the simulated hydrolysate (62 g·L-1 in average), probably because the OFMSW hydrolysate favours biomass growth in detriment of P(3HB) production.

Upgrading the organic fraction of municipal solid waste to poly(3-hydroxybutyrate).

The organic fraction of municipal solid waste was studied as feedstock for the production of poly(3-hydroxybutyrate) (P(3HB)). To release the monosaccharides, a diluted acid pre-treatment followed by an enzymatic hydrolysis was applied. A sugar yield of 49% was achieved using a pre-treated waste and an enzyme cocktail of Pentopan 500 BG and Celluclast BG. The addition of Glucoamylase NS 22035 helped to hydrolyze the starch fraction, improving the hydrolysis yield to 56%. The hydrolysate was used as culture medium to produce P(3HB) by Burkholderia sacchari DSM 17165. Assays at shaking flask scale showed that when the hydrolysate was used as substrate, the attained cell concentration was slightly higher than in the control medium. It was necessary to supplement the hydrolysate with extra glucose to increase the C/N ratio and with a mineral solution to overcome the nutritional deficiencies. The P(3HB) accumulation using the supplemented hydrolysate was 58% (g polymer/g biomass).

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.

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Burkholderia sacchari using wheat straw hydrolysates and gamma-butyrolactone.

Burkholderia sacchari DSM 17165 is able to grow and produce poly(3-hydroxybutyrate) both on hexoses and pentoses. In a previous study, wheat straw lignocellulosic hydrolysates (WSH) containing high C6 and C5 sugar concentrations were shown to be excellent carbon sources for P(3HB) production. Using a similar feeding strategy developed for P(3HB) production based on WSH, fed-batch cultures were developed aiming at the production of the copolymer P(3HB-co-4HB) (poly(3-hydroxybutyrate-co-4-hydroxybutyrate)) by B. sacchari. The ability of this strain to synthesize P(3HB-co-4HB) was first shown in shake flasks using gamma-butyrolactone (GBL) as precursor of the 4HB units. Fed-batch cultures using glucose as carbon source (control) and GBL were developed to achieve high copolymer productivities and 4HB incorporations. The attained P(3HB-co-4HB) productivity and 4HB molar% were 0.7g/(Lh) and 4.7molar%, respectively. The 4HB incorporation was improved to 6.3 and 11.8molar% by addition of 2g/L propionic and acetic acid, respectively. When WSH were used as carbon source under the same feeding conditions, the values achieved were 0.5g/(Lh) and 5.0molar%, respectively. Burkholderia sacchari, a strain able to produce biopolymers based on xylose-rich lignocellulosic hydrolysates, is for the first time reported to produce P(3HB-co-4HB) using gamma butyrolactone as precursor.

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.