Polyhydroxyalkanoates production in biorefineries: A review on current status, challenges and opportunities.

The need for a sustainable and circular bioeconomy model is imperative due to petroleum non-renewability, scarcity and environmental impacts. Biorefineries systems explore biomass to its maximum, being an important pillar for the development of circular bioeconomy. Polyhydroxyalkanoates (PHAs) can take advantage of biorefineries, as they can be produced using renewable feedstocks, and are potential substitutes for petrochemical plastics. The present work aims to evaluate the current status of the industrial development of PHAs production in biorefineries and PHAs contributions to the bioeconomy, along with future development points. Advancements are noticed when PHA production is coupled in wastewater treatment systems, when residues are used as substrate, and also when analytical methodologies are applied to evaluate the production process, such as the Life Cycle and Techno-Economic Analysis. For the commercial success of PHAs, it is established the need for dedicated investment and policies, in addition to proper collaboration of different society actors.

Emerging contaminants bioremediation by enzyme and nanozyme-based processes - A review.

Due to their widespread occurrence and the inadequate removal efficiencies by conventional wastewater treatment plants, emerging contaminants (ECs) have recently become an issue of great concern. Current ongoing studies have focused on different physical, chemical, and biological methods as strategies to avoid exposing ecosystems to significant long-term risks. Among the different proposed technologies, the enzyme-based processes rise as green biocatalysts with higher efficiency yields and lower generation of toxic by-products. Oxidoreductases and hydrolases are among the most prominent enzymes applied for bioremediation processes. The present work overviews the state of the art of recent advances in enzymatic processes during wastewater treatment of EC, focusing on recent innovations in terms of applied immobilization techniques, genetic engineering tools, and the advent of nanozymes. Future trends in the enzymes immobilization techniques for EC removal were highlighted. Research gaps and recommendations on methods and utility of enzymatic treatment incorporation in conventional wastewater treatment plants were also discussed.

Polyhydroxybutyrate production by Cupriavidus necator in a corn biorefinery concept.

The high costs of bioplastics' production may hinder their commercialization. Development of new processes with high yields and in biorefineries can enhance diffusion of these materials. This work evaluated the production of polyhydroxybutyrate (PHB) from the combination of milled corn starchy fraction hydrolysate and crude glycerol as substrates by the strain Cupriavidus necator LPB 1421. After optimization steps, maximum accumulation of 62 % of PHB was obtained, which represents 11.64 g.L-1 and productivity of 0.162 g.Lh-1. In a stirred tank bioreactor system with 8 L of operational volume, 70 % of PHB accumulation was reported, representing 14.17 g.L-1 of the biopolymer with 0.197 g.Lh-1 productivity. PHB recovery was conducted using a chemical digestion method, reaching >99 % purity. Therefore, the potential application of milled corn as substrate for PHB production was confirmed. The developed bioplastic process could be coupled to a bioethanol producing unit creating the opportunity of a sustainable and economic biorefinery.

A non-waste strategy for enzymatic hydrolysis of cellulose recovered from domestic wastewater.

Cellulose is a potential resource to be recovered from wastewater treatment plants (WWTP). Enzyme formulations can be employed to hydrolyze cellulose into fermentable sugars, to be further used as biochemical building blocks or reducing its recalcitrance to further treatment processes. This study proposed the production, recovery and formulation of cellulase using domestic wastewater as culture medium and its application for the hydrolysis of cellulosic residues recovered from WWTPs. Cellulose was recovered from raw sanitary wastewater using a fine-mesh sieve (0.35 mm) and quantified through enzymatic hydrolysis and thermogravimetric analysis. The production, concentration and formulation of cellulase enzyme resulted in an enzymatic blend of endoglucanases (7.3 UFP/mL), cellobiohydrolases (7.4 UCMC/mL) and beta-glucosidases (4.4 UBGL/mL). The content of the recovered cellulosic material was 21.3% according to enzymatic hydrolysis and 27.7 for thermogravimetric results. The enzymatic hydrolysis of the WWTP residue using the produced cellulase (107.6 ± 10.2 mgreduc/gresidue) showed better results than using the commercial cellulase complex (66.4 ± 2.5 mgreduc/gresidue). This fact showed the potential of application of the produced enzyme for the hydrolysis of cellulosic residues recovered from WWTP processes. In a non-waste biorefinery approach, the generated hydrolysate can be further used for producing added-value biomolecules including biofuels and biochemicals.

Citric acid bioproduction and downstream processing: Status, opportunities, and challenges.

Citric acid (CA) has been widely used in different industrial sectors, being produced through fermentation of low-cost feedstock. The development of downstream processes, easier to operate, environmentally friendly, and more economic than precipitation, is certainly a challenge in CA bioproduction. Large volumes of by-products generated in precipitation require treatment before disposal. Adsorption, extraction, and membrane separation have been shown to have a lower environmental impact than precipitation, but the technological maturity of these methods is still limited. However, reactive extraction and adsorption have great potential for industrial applications. This review shows that there is still much to be explored, both about the factors that are intrinsic to the techniques, but also in their combination for new processes' development. This review reports the most recent advances on CA bioproduction, with significant information about recovery and purification methods involving this highly industrially demanded organic acid.

Current developments and challenges of green technologies for the valorization of liquid, solid, and gaseous wastes from sugarcane ethanol production.

The sugarcane industry is one of the largest in the world and processes huge volumes of biomass, especially for ethanol and sugar production. These processes also generate several environmentally harmful solid, liquid, and gaseous wastes. Part of these wastes is reused, but with low-added value technologies, while a large unused fraction continues to impact the environment. In this review, the classic waste reuse routes are outlined, and promising green and circular technologies that can positively impact this sector are discussed. To remain competitive and reduce its environmental impact, the sugarcane industry must embrace technologies for bagasse fractionation and pyrolysis, microalgae cultivation for both CO2 recovery and vinasse treatment, CO2 chemical fixation, energy generation through the anaerobic digestion of vinasse, and genetically improved fermentation yeast strains. Considering the technological maturity, the anaerobic digestion of vinasse emerges as an important solution in the short term. However, the greatest environmental opportunity is to use the pure CO2 from fermentation. The other opportunities still require continued research to reach technological maturity. Intensifying the processes, the exploration of driving-change technologies, and the integration of wastes through biorefinery processes can lead to a more sustainable sugarcane processing industry.

Selenium-Enriched Probiotic Saccharomyces boulardii CCT 4308 Biomass Production Using Low-Cost Sugarcane Molasses Medium

Abstract Food supplements have been increasingly investigated. Probiotics have several benefits for human and animal health and selenium (Se) is widely recommended against oxidative stress. In this context, the aim of this study was to develop a low-cost bioprocess to produce a functional food product comprising both probiotic and Se accumulation. Yeast cells of Saccharomyces boulardii CCT 4308 were cultivated using sugarcane molasses as substrate. Optimization studies were performed to evaluate the best medium composition for biomass production and Se-accumulation in batch and fed-batch systems. Optimized conditions were defined with a medium composed of 150 g L-1 sugarcane molasses and 12 g L-1 yeast extract, with feeding of 100 g L-1 sugarcane molasses and 100 μg mL-1 of Se incorporation after 4 h and 10 h of fermentation, respectively, during 48 h in STR (stirred tank reactor). Best biomass production reached 14.52 g L-1 with 3.20 mg Se g-1 biomass at 12 h. Process optimization led to 4.82-fold increase in biomass production compared to initial condition. A final Se-enriched S. boulardii CCT 4308 biomass was obtained, which is comparable to commercial products. An alternative probiotic yeast biomass was efficiently produced as a new food-form of Se supplement in a sustainable process using an inexpensive agro-industrial residue.

The Antihypertensive, Antimicrobial and Anticancer Peptides from Arthrospira with Therapeutic Potential: A Mini Review.

The interest in biological peptides from Arthrospira sp. (syn Spirulina) is increasing due to its Generally Recognised as Safe "GRAS" status, the high concentration of proteins and the history of its use as a supplement and nutraceutical agent. Arthrospira peptides can be generated by the controlled hydrolysis of proteins, using proteases, followed by fractionation. The peptides obtained have a range of therapeutic effects. Amongst these bioactive peptides, three classes are of major importance: the antihypertensive (AHP), antimicrobial (AMP) and anticancer (ACP) peptides. AHPs have the ability to work as inhibitors of angiotensin-converting enzyme (ACE), and help to control several diseases such as hypertension, obesity, and cardiovascular issues, AMPs play a crucial role in the immune response, inhibiting the development of pathogens such as bacteria, fungi, viruses and others, while ACPs can aid in tumour control by the induction of apoptosis or necrosis, or the inhibition of angiogenesis. Thus, bioactive peptides are of great significance to the pharmaceutical industry. However, they can show secondary effects. This paper reviews the inhibition mechanism of antimicrobial, hypertensive and anticancer peptides from Arthrospira sp., and the possible structures of the peptides according to the type of activity and its intensity. In addition, this paper describes the purification methods of absorption mechanisms, and reviews databases for designing peptides.

Sequential chemical and enzymatic pretreatment of palm empty fruit bunches for Candida pelliculosa bioethanol production.

Second-generation bioethanol production process was developed using pretreated empty fruit bunches (EFB). Consecutive acid/alkali EFB pretreatment was performed, first with HCl and then with NaOH with final washing steps for phenolic compounds elimination. Scanning electron microscopy images showed that EFB chemical treatments indeed attacked the cellulose fibers and removed the silica from surface pores. The optimization of enzymatic hydrolysis of EFB's cellulosic fraction was performed with 0.5%-4% v/v of Cellic® CTec2/Novozymes, different EFB concentrations (5%-15%, w/v), and hydrolysis time (6-72 H). Optimization essays were carried out in Erlenmeyer flasks and also in a 1 L stirred tank reactor. After enzymatic hydrolysis, a hydrolysate with 66 g/L of glucose was achieved with 2.2% (v/v) Cellic® CTec2, 15% (m/v) acid/alkaline pretreated EFB after 39 H of hydrolysis. A gain of 11.2% was then obtained in the 1 L stirred tank promoted by the agitation (72.2 g/L glucose). The hydrolysate was employed in bioethanol production by a new isolate Candida pelliculosa CCT 7734 in a separate hydrolysis and fermentation process reaching 16.6 and 23.0 g/L of bioethanol through batch and fed-batch operation, respectively. An integrated biorefinery process was developed for EFB processing chain.

Sustainability of sugarcane lignocellulosic biomass pretreatment for the production of bioethanol.

The sustainability of a biofuel is severely affected by the technological route of its production. Chemical pretreatment can be considered the traditional method of decomposition of the lignocellulose into its mono and oligomeric units, which can be further bioconverted to ethanol. The evaluation of the recent advances in chemical pretreatments of sugarcane bagasse, especially diluted acids, alkaline, organosolv and ionic liquids, identified the critical points for sustainability. In this context, chemicals recovery and reutilization or their substitution by green solvents, heat and electricity generation through bioenergy, reutilization of water from evaporators, vinasse concentration and the upgrading of lignin were discussed as strategic routes for developing sustainable chemical-based lignocellulose pretreatment. The advances in the technologies that allow greater fractionation of lignocellulosic biomass should be focused on the minimization of the use of natural resources, effluent generation and energy expenditure.

Definition of Liquid and Powder Cellulase Formulations Using Domestic Wastewater in Bubble Column Reactor.

Raw domestic wastewater was used as a culture medium for cellulase production in a bubble column reactor (6.2 UFP/mL, 64.6 U/L h) using the strain Trichoderma harzianum TRIC03-LPBII. Cellulases presented optimum pH and temperature between 4 and 5 and 50 and 70 °C, respectively. Enzymatic extract was concentrated through ultrafiltration and then a cellulolytic formulation was prepared with the addition of sorbitol (50% w/v) and benzoic acid (0.05% w/v). High cellulase stability of around 100% was reached after 30 days at 4 °C. The concentrated extract was also dried in a spray-dryer with the addition of maltodextrin at 20% (w/v), resulting in powder enzymatic formulation with 85% stability after 60 days. With these characteristics, the liquid and powder cellulase products have potential to be used in different industrial applications.

Microalgal biorefineries: Integrated use of liquid and gaseous effluents from bioethanol industry for efficient biomass production.

A new method for CO2 recovery was proposed for cultivation of different microalgae. First, a chemical fixation, where CO2 was injected in alkalinized vinasse to form (bi)carbonate salts, was performed. In addition, biological fixation with CO2-enriched air injection was also accomplished for evaluation of the most promising results. Two bioreactor systems, a stirred-tank reactor and a bubble column reactor, were employed. A higher carbon transfer rate (43.35 g.L-1.h-1) was achieved in the bubble column reactor using NaOH-alkalinized vinasse, along with reductions of the chemical oxygen demand (COD), biological oxygen demand (BOD) and turbidity (TD). This allowed the cultivation of microalgae and cyanobacteria at vinasse concentrations between 70 and 100%, reaching a biomass production of 2.25 g.L-1 in 15 days of culture. The viability of chemical CO2 fixation together with the use of 100% treated vinasse from a bioethanol production unit for microalgae cultivation has been demonstrated in a successfully integrated biorefinery approach.

Microscale direct transesterification of microbial biomass with ethanol for screening of microorganisms by its fatty acid content

Abstract We present an improved method of direct transesterification suitable for the quantitative analysis of multiple dry samples for its fatty acid content, using a minimal amount of biomass and reactants. The method features an acid-catalyzed direct alcoholysis of microgram samples of dry biomass; the rationale behind the solvent and reagent proportions chosen is discussed. The method was validated using seven microbial strains with diverse lipid content (Saccharomyces cerevisiae, Saccharomyces boulardii, Candida tropicalis, Haematococcus pluvialis, Chlorella vulgaris, Spirulina platensis and Schizochytrium limacinum), and compared with a macroscale direct transesterification method, and with gravimetric analysis of lipids extracted with solvents. The microscale method showed a conversion of 98.06 ± 0.87% of the lipids, using approximately 3 mg of dry biomass, 1mL of 0.2M H2SO4 dissolved in anhydrous ethanol (the acid is the catalyzer and ethanol the reactant)). The mixture was maintained at 70 °C for 20 h with periodic mixing, and then extracted with 2mL n-heptane and analyzed by GC-FID. The lipid content was then calculated considering dilution and sample mass. This method is effective, reliable, and technically attractive for analytical and comparative purposes.

Crude Fermented Extract Containing Gibberellic Acid Produced by Fusarium moniliforme is an Alternative to Cost Reduction in Biofactories

ABSTRACT Nidularium procerum and Nidularium innocentii (Bromeliaceae) were cultivated in vitro on media supplemented with different sources and levels of GA3 (gibberellic acid). These sources were the commercial powder (analytical degree) and fermented extract obtained by Fusarium moniliforme via solid state fermentation. The in vitro elongation and rooting of these plants were evaluated after 50 days of cultivation. The GA3 present in the fermented extract possess the same effect of purified GA3 (analytical degree) for the increase of the height of aerial part of shoots of N. innocentii, but not for the N. procerum being the GA3 fermented extract in a lesser degree. The GA3 fermented extract influences negatively the rooting in N. innocentii, while GA3 analytical degree practically does not interfere in the rooting. On the other hand, in N. procerum, both the GA3 sources reduce the root number and do not interfere in rooting percentage. GA3 crude fermented extract is an alternative to reduce costs, however, its results can vary depending on the species and parameter evaluated. The fermented extract was stored at temperature during 260 days and its shelf life presented a suitable stability, maintaining 92% of its initial GA3 amount.

Microbiological, biochemical, and functional aspects of sugary kefir fermentation - A review.

Sugary kefir beverage is produce by fermenting raw sugar solution with kefir grains, the latter consisting of polysaccharide and microorganisms. This beverage, with great consumption in countries such as USA, Japan, France, and Brazil, represents a promising market to functional cultured drinks. This paper reviews the microbial diversity and interaction, kinetics, safety, and bioactivities of sugary kefir fermentation. The literature reviewed here demonstrates that sugary kefir possesses a similar microbial association relative to traditional milk kefir fermentation, especially among lactic acid bacteria and yeast species, such as Lactobacillus, Leuconostoc, Kluyveromyces, Pichia, and Saccharomyces. However, a selective pressure at species level is generally observed, as, for example, the stimulation of Saccharomyces species metabolism, leading to a high content of alcohol in the final product. This also seems to stimulate the growth of acetic acid bacteria that benefit of increased ethanol production to acetic acid metabolism. Existing reports have suggested important bioactivities associated with sugary kefir beverage consumption, such as antimicrobial, antiedematogenic, anti-inflammatory, antioxidant, cicatrizing, and healing activities. Other alternative non-dairy substrates, such as fruits, vegetables, and molasses, have also been tested for adaptation of kefir grains and production of functional beverages with distinct sensory characteristics. This diversification is of crucial importance for the production of new probiotic products to provide people with special needs (lactose intolerance) and vegan consumers.

Pilot scale biodiesel production from microbial oil of Rhodosporidium toruloides DEBB 5533 using sugarcane juice: Performance in diesel engine and preliminary economic study.

A successful pilot-scale process for biodiesel production from microbial oil (Biooil) produced by Rhodosporidium toruloides DEBB 5533 is presented. Using fed-batch strategy (1000L working volume), a lipid productivity of 0.44g/L.h was obtained using a low-cost medium composed by sugarcane juice and urea. The microbial oil was used for biodiesel production and its performance was evaluated in diesel engine tests, showing very good performance, especially for the blend B20 SCO, when operating at 2500rpm with lower pollutant emissions (CO2 - 220% less; CO - 7-fold less; NOX 50% less and no detectable HC emissions (<0.11ppm)) when compared with the blends of standard biofuel from soybean oil. A preliminary analysis showed that microbial biodiesel is economically competitive (US$ 0.76/L) when compared to the vegetable biodiesel (US$ 0.81/L). Besides, the yield of biodiesel from microbial oil is higher (4172L/ha of cultivated sugarcane) that represents 6.3-fold the yield of standard biodiesel (661L/ha of cultivated soybean).

Production of Basidiomata and Ligninolytic Enzymes by the Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), in Licuri (Syagrus coronata) Wastes in Brazil.

Ganoderma lucidum is a medicinal mushroom with different forms of bioactivity that has been used in popular medicine for centuries. This study aimed to test the application of agricultural wastes (fruit shells, leaves, and bracts) from the endemic Brazilian palm tree Syagrus coronata (licuri) as substrates for the production of G. lucidum basidiomata and ligninolytic enzymes via solid-state fermentation. The best culture conditions were the same for all substrates (pH 6.5, carbon-to-nitrogen ratio = 40, and temperature 30°C) and were established from preliminary assays. The yield was not significantly different for bracts (33.53 g/kg) and leaves (37.48 g/kg), nor for the biological efficiency in these same substrates: bracts, 3.35%; leaves, 3.75%. The highest laccase (13.80 U/L) and manganese peroxidase (14.92 U/L) activities were achieved after 14 and 28 days of incubation, respectively, using bracts as the substrate. Licuri residues are then potential substrates to be used in the bioconversion process for mycelia, basidiomata, and ligninolytic enzyme production by G. lucidum.

Optimization of Arundo donax Saccharification by (Hemi)cellulolytic Enzymes from Pleurotus ostreatus.

An enzymatic mixture of cellulases and xylanases was produced by Pleurotus ostreatus using microcrystalline cellulose as inducer, partially characterized and tested in the statistical analysis of Arundo donax bioconversion. The Plackett-Burman screening design was applied to identify the most significant parameters for the enzymatic hydrolysis of pretreated A. donax. As the most significant influence during the enzymatic hydrolysis of A. donax was exercised by the temperature (°C), pH, and time, the combined effect of these factors in the bioconversion by P. ostreatus cellulase and xylanase was analyzed by a 3(3) factorial experimental design. It is worth noting that the best result of 480.10 mg of sugars/gds, obtained at 45 °C, pH 3.5, and 96 hours of incubation, was significant also when compared with the results previously reached by process optimization with commercial enzymes.

Selection of the Strain Lactobacillus acidophilus ATCC 43121 and Its Application to Brewers' Spent Grain Conversion into Lactic Acid.

Six Lactobacillus strains were analyzed to select a bacterium for conversion of brewers' spent grain (BSG) into lactic acid. Among the investigated strains, L. acidophilus ATCC 43121 showed the highest yield of lactic acid production (16.1 g/L after 48 hours) when grown in a synthetic medium. It was then analyzed for its ability to grow on the hydrolysates obtained from BSG after acid-alkaline (AAT) or aqueous ammonia soaking (AAS) pretreatment. The lactic acid production by L. acidophilus ATCC 43121 through fermentation of the hydrolysate from AAS treated BSG was 96% higher than that from the AAT treated one, although similar yields of lactic acid per consumed glucose were achieved due to a higher (46%) glucose consumption by L. acidophilus ATCC 43121 in the AAS BSG hydrolysate. It is worth noting that adding yeast extract to the BSG hydrolysates increased both the yield of lactic acid per substrate consumed and the volumetric productivity. The best results were obtained by fermentation of AAS BSG hydrolysate supplemented by yeast extract, in which the strain produced 22.16 g/L of lactic acid (yield of 0.61 g/g), 27% higher than the value (17.49 g/L) obtained in the absence of a nitrogen source.

Evaluation of probiotic properties of Pediococcus acidilactici B14 in association with Lactobacillus acidophilus ATCC 4356 for application in a soy based aerated symbiotic dessert

The aim of this study was to evaluate the probiotic properties of Pediococcus acidilactici B14 and to study its resistance in the gastrointestinal system when combined with Lactobacillus acidophilus ATCC 4356 and used in a potentially symbiotic aerated soy based dessert. P. acidilactici B14 showed some important probiotic characteristics such as survival rate of 45.9% at pH 2.5; 72.4% in 0.3% bile salts and 95.8% after gastrointestinal transit at pH 4.0. Tolerance against the antibiotics cephalexin, neomycin, vancomycin, cefotaxime and penicillin G was also observed. The strain inhibited antagonism against the following cultures: Escherichia coli ATCC 25922, Bacillus cereus ATCC 33018, Staphylococcus aureus ATCC 6538P and Salmonella sp. The mixed culture of P. acidilactici B14 with L. acidophilus ATCC 4356 showed a survival rate of 92.4% after the passage through the gastrointestinal system at pH 4.0. Furthermore, in the presence of the food matrix, an average increase in cell viability, after being subjected to the gastrointestinal system of 9.9% at pH 2.0 and 6.1% at pH 4.0, was observed. This characterized the adequacy of the associated culture as probiotic in the development of a functional food such as soy based aerated symbiotic dessert.