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.

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.

Bioaugmentation and biostimulation strategies to improve the effectiveness of bioremediation processes.

Bioremediation, involving bioaugmentation and/or biostimulation, being an economical and eco-friendly approach, has emerged as the most advantageous soil and water clean-up technique for contaminated sites containing heavy metals and/or organic pollutants. Addition of pre-grown microbial cultures to enhance the degradation of unwanted compounds (bioaugmentation) and/or injection of nutrients and other supplementary components to the native microbial population to induce propagation at a hastened rate (biostimulation), are the most common approaches for in situ bioremediation of accidental spills and chronically contaminated sites worldwide. However, many factors like strain selection, microbial ecology, type of contaminant, environmental constraints, as well as procedures of culture introduction, may lead to their failure. These drawbacks, along with fragmented literature, have opened a gap between laboratory trials and on-field application. The present review discusses the effectiveness as well as the limitations of bioaugmentation and biostimulation processes. A summary of experimental studies both in confined systems under controlled conditions and of real case studies in the field is presented. A comparative account between the two techniques and also the current scenario worldwide for in situ biotreatment using bioaugmentation and biostimulation, are addressed.

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.

Assessment of three-dimensional biofilm structure using an optical microscope.

A method allowing the evaluation of the structure and the calculation of the volume of a biofilm, using an optical microscope, is proposed based on the linear relation between the intensity of a pixel in biofilm images grabbed on the x-y plane and the corresponding number of cells in the z direction, which allows the calculation of the biofilm thickness. The method is intended to overcome the need for expensive microscopes to study biofilms.

Preventing biofilm formation: promoting cell separation with terpenes.

Both carveol and carvone were effective in dispersing Rhodococcus erythropolis cells that were being stimulated to aggregate by the presence of organic solvents. The two terpenes influenced the fatty acid composition of the cell membrane, decreasing the percentage of fatty acids with more than 16 carbon atoms, and thus cell hydrophobicity, and also the degree of saturation of the fatty acids. In the presence of 250 micromol of terpene, the volume of biofilm was reduced by one third in comparison with biofilms in the absence of terpenes. The percentage of aggregated cells was also found to depend on carvone concentration during the bioconversion of carveol to carvone, in a membrane reactor. The extent of cell aggregation decreased from 90% to 10% when carvone concentration reached ca. 48 mM in the organic phase.

Biotransformation of terpenes.

The main application of terpenes as fragrances and flavors depends on the absolute configuration of the compounds because enantiomers present different organoleptic properties. Biotransformations allow the production of regio- and stereoselective compounds under mild conditions. These products may be labeled as "natural". Commercially useful chemical building-blocks and pharmaceutical stereo isomers can also be produced by bioconversion of terpenes. Enzymes and extracts from bacteria, cyanobacteria, yeasts, microalgae, fungi, plants, and animal cells have been used for the production and/or bioconversion of terpenes. In addition, whole cell catalysis has also been used. A variety of media and reactors have been assessed for these biotransformations and have produced encouraging results, as discussed in this review.

Degradation of hydrocarbons and alcohols at different temperatures and salinities by Rhodococcus erythropolis DCL14.

Rhodococcus erythropolis DCL14 cells were able to metabolise C5-C16 hydrocarbons and C1-C12 alcohols as sole carbon and energy sources, both at 15 and 28 degrees C. Metabolic activity was also observed at 1.00%, 1.95% and 2.50% sodium chloride. Almost complete degradation of n-, iso- and cyclo-alkanes and aromatic compounds present in fuel oil was achieved after 9 months, 60% being consumed in the first three months. The results from the conditions tested here suggest that this type of bacterium could be involved in bioremediation processes in marine environments such as the Atlantic, Pacific and Indian Ocean.

A simple imaging method for biomass determination.

An inexpensive and fast method based on images taken during growth of bacterial cells on multi-well plates was developed for biomass quantification. A correlation of 85% between the results obtained by image analysis and optical density measurements was obtained. This simple method allows the assessment of growth with highly aggregated cell cultures and the rapid screening of a large number of carbon sources.

Mycobacterium sp., Rhodococcus erythropolis, and Pseudomonas putida behavior in the presence of organic solvents.

This work aimed at studying the behavior and tolerance of Mycobacterium sp. NRRL B-3805, Rhodococcus erythropolis DCL14 and Pseudomonas putida S12 cells in the presence of various concentrations of water miscible (ethanol, butanol, and dimethylformamide, up to 50% v/v) and water immiscible solvents (dodecane, bis(2-ethylhexyl) phthalate and toluene, up to 5% v/v). When incubated in the presence of these solvents, the cells were found to have lower tolerance to butanol and toluene than to the remaining solvents. Nevertheless, the concentrations of solvents endured by the tested strains show that they are quite solvent-tolerant, confirming their potential as biocatalysts in nonconventional systems. Microscopic observation of samples showed that the hydrophobic Mycobacterium sp. and R. erythropolis cells were able to aggregate to protect the population under stress conditions. Comparison of the results obtained at the single cell level by fluorescence microscopy and colony development on agar plates indicated that the primary effects of most solvents tested were on the cell membrane and replicating capability of the cells.

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.

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).

Adaptation of Rhodococcus erythropolis cells to high concentrations of toluene.

Cells of Rhodococcus erythropolis DCL14 were adapted to increasing toluene concentrations in a mechanically stirred reactor. When the initial non-adapted cells were placed in contact with toluene, only 10.5% of cells remained viable after 1 h in the presence of 20% (v/v) toluene, while 8.6% of cells were viable after 28 h in the presence of an organic phase containing 80% (v/v) toluene in n-dodecane. Cell adaptation was studied by following the toluene consumption rate, the viability of the cell population, and the composition of the bacteria cellular membrane in the presence of increasing concentrations of toluene in the reactor. A maximum toluene concentration of 4.9 M, which corresponds to 52.4% (v/v) toluene in the organic phase, was achieved, toluene being consumed at 10.7 mg/(h mg protein). The adapted cells showed a substantially increased resistance to 50% ethanol and to concentrations of Betadine and Micropur tablets currently used in water purification, when compared to non-adapted cells.

The remarkable Rhodococcus erythropolis.

Rhodococcus erythropolis cells contain a large set of enzymes that allow them to carry out an enormous number of bioconversions and degradations. Oxidations, dehydrogenations, epoxidations, hydrolysis, hydroxylations, dehalogenations and desulfurisations have been reported to be performed by R. erythropolis cells or enzymes. This large array of enzymes fully justifies the prospective application of this bacterium in biotechnology.

Cell adaptation to solvent, substrate and product: a successful strategy to overcome product inhibition in a bioconversion system.

Carvone has previously been found to highly inhibit its own production at concentrations above 50 mM during conversion of a diastereomeric mixture of (-)-carveol by whole cells of Rhodococcus erythropolis. Adaptation of the cells to the presence of increasing concentrations of carveol and carvone in n-dodecane prior to biotransformation proved successful in overcoming carvone inhibition. By adapting R. erythropolis cells for 197 h, an 8.3-fold increase in carvone production rate compared to non-adapted cells was achieved in an air-driven column reactor. After an incubation period of 268 h, a final carvone concentration of 1.03 M could be attained, together with high productivity [0.19 mg carvone h(-1) (ml organic phase)(-1)] and high yield (0.96 g carvone g carveol(-1)).