An Alternative Exploitation of Synechocystis sp. PCC6803: A Cascade Approach for the Recovery of High Added-Value Products.

Microalgal biomass represents a very interesting biological feedstock to be converted into several high-value products in a biorefinery approach. In this study, the cyanobacterium Synechocystis sp. PCC6803 was used to obtain different classes of molecules: proteins, carotenoids and lipids by using a cascade approach. In particular, the protein extract showed a selective cytotoxicity towards cancer cells, whereas carotenoids were found to be active as antioxidants both in vitro and on a cell-based model. Finally, for the first time, lipids were recovered from Synechocystis biomass as the last class of molecules and were successfully used as an alternative substrate for the production of polyhydroxyalkanoate (PHA) by the native PHA producer Pseudomonas resinovorans. Taken together, our results lead to a significant increase in the valorization of Synechocystis sp. PCC6803 biomass, thus allowing a possible offsetting of the process costs.

Immobilization of carbonic anhydrase for CO2 capture and utilization.

Carbonic anhydrase (CA) is an excellent candidate for novel biocatalytic processes based on the capture and utilization of CO2. The setup of efficient methods for enzyme immobilization makes CA utilization in continuous bioreactors increasingly attractive and opens up new opportunities for the industrial use of CA. The development of efficient processes for CO2 capture and utilization (CCU) is one of the most challenging targets of modern chemical reaction engineering. In the general frame of CCU processes, the interest in the utilization of immobilized CA as a biocatalyst for augmentation of CO2 reactive absorption has grown consistently over the last decade. The present mini-review surveys and discusses key methodologies for CA immobilization aimed at the development of heterogeneous biocatalysts for CCU. Advantages and drawbacks of covalent attachment on fine granular solids, immobilization as cross-linked enzyme aggregates, and "in vivo" immobilization methods are presented. In particular, criteria for optimal selection of CA-biocatalyst and design of CO2 absorption units are presented and discussed to highlight the most effective solutions. Perspectives on biocatalytic CCU processes that can include the use of CA in an enzymatic reactive CO2 absorption step are eventually presented with a special focus on two examples of CO2 fixation pathways: hybrid enzyme-microalgae process and enzyme cascade for the production of carboxylic acids. KEY POINTS: • Covalent immobilization techniques applied to CA are effective for CO2 ERA. • Biocatalyst type and morphology must be selected considering CO2 ERA conditions. • Immobilized CA can offer novel routes to CO2 capture and direct utilization.

Efficient succinic acid production from high-sugar-content beverages by Actinobacillus succinogenes.

This study presents the production of succinic acid (SA) by Actinobacillus succinogenes using high-sugar-content beverages (HSCBs) as feedstock. The aim of this study was the valorization of a by-product stream from the beverage industry for the production of an important building block chemical, such as SA. Three types of commercial beverages were investigated: fruit juices (pineapple and ace), syrups (almond), and soft drinks (cola and lemon). They contained mainly glucose, fructose, and sucrose at high concentration-between 50 and 1,000 g/L. The batch fermentation tests highlighted that A. succinogenes was able to grow on HSCBs supplemented with yeast extract, but also on the unsupplemented fruit juices. Indeed, the bacteria did not grow on the unsupplemented syrup and soft drinks because of the lack of indispensable nutrients. About 30-40 g/L of SA were obtained, depending on the type of HSCB, with yield ranging between 0.75 and 1.00 gSA /gS . The prehydrolysis step improved the fermentation performance: SA production was improved by 6-24%, depending on the HSCB, and sugar conversion was improved of about 30-50%.

Clostridial conversion of corn syrup to Acetone-Butanol-Ethanol (ABE) via batch and fed-batch fermentation.

Corn syrup - a commercial product derived from saccharification of corn starch - was used to produce acetone-butanol-ethanol (ABE) by Clostridium spp. Screening of commercial Clostridium spp., substrate inhibition tests and fed-batch experiments were carried out to improve ABE production using corn syrup as only carbon source. The screening tests carried out in batch mode using a production media containing 50 g/L corn syrup revealed that C. saccharobutylicum was the best performer in terms of total solvent concentration (12.46 g/L), yield (0.30 g/g) and productivity (0.19 g/L/h) and it was selected for successive experiments. Concentration of corn syrup higher than 50 g/L resulted in no solvents production. Fed-batch fermentation improved ABE production with respect to batch fermentation: the butanol and solvent concentration increased up to 8.70 and 16.68 g/L, respectively. The study demonstrated the feasibility of producing solvents via ABE fermentation using corn syrup as a model substrate of concentrated sugar mixtures.

Bioreactors for succinic acid production processes.

Succinic acid (SA) has been recognized as one of the most important bio-based building block chemicals due to its numerous potential applications. Fermentation SA production from renewable carbohydrate feedstocks can have the economic and sustainability potential to replace petroleum-based production in the future, not only for existing markets, but also for new larger volume markets. Design and operation of bio-reactors play a key role. During the last 20 years, many different fermentation strategies for SA production have been described in literature, including utilization of immobilized biocatalysts, integrated fermentation and separation systems and batch, fed-batch, and continuous operation modes. This review is an overview of different fermentation process design developed over the past decade and provides a perspective on remaining challenges for an economically feasible succinate production processes. The analysis stresses the idea of improving the efficiency of the fermentation stage by improving bioreactor design and by increasing bioreactor performance.

Butanol production from laccase-pretreated brewer's spent grain.

BACKGROUND: Beer is the most popular alcoholic beverage worldwide. In the manufacture of beer, various by-products and residues are generated, and the most abundant (85% of total by-products) are spent grains. Thanks to its high (hemi)cellulose content (about 50% w/w dry weight), this secondary raw material is attractive for the production of second-generation biofuels as butanol through fermentation processes. RESULTS: This study reports the ability of two laccase preparations from Pleurotus ostreatus to delignify and detoxify milled brewer's spent grains (BSG). Up to 94% of phenols reduction was achieved. Moreover, thanks to the mild conditions of enzymatic pretreatment, the formation of other inhibitory compounds was avoided allowing to apply the sequential enzymatic pretreatment and hydrolysis process (no filtration and washing steps between the two phases). As expected, the high detoxification and delignification yields achieved by laccase pretreatment resulted in great saccharification. As a fact, no loss of carbohydrates was observed thanks to the novel sequential strategy, and thus the totality of polysaccharides was hydrolysed into fermentable sugars. The enzymatic hydrolysate was fermented to acetone-butanol-ethanol (ABE) by Clostridium acetobutilycum obtaining about 12.6 g/L ABE and 7.83 g/L butanol within 190 h. CONCLUSIONS: The applied sequential pretreatment and hydrolysis process resulted to be very effective for the milled BSG, allowing reduction of inhibitory compounds and lignin content with a consequent efficient saccharification. C. acetobutilycum was able to ferment the BSG hydrolysate with ABE yields similar to those obtained by using synthetic media. The proposed strategy reduces the amount of wastewater and the cost of the overall process. Based on the reported results, the potential production of butanol from the fermentation of BSG hydrolysate can be envisaged.

Combined antioxidant-biofuel production from coffee silverskin.

Biorefinery concept asks for an integrated processing approach to exploit all biomass components. The self-sustainability target may be approached if molecules characterized by high added value and fermentable sugars are produced simultaneously. In the present study, sequential (i) mild hydrothermal pretreatment to produce antioxidants and (ii) NaOH pretreatment to produce a fermentable sugar solution were carried out on coffee silverskin. Twenty-minute treatment and biomass to liquid ratio 1:30 (g mL-1) were identified as optimal operating conditions to extract bioactive compounds characterized by antioxidant activity (22.2 mgGAE/gCSS; 13.9 mgTE/gCSS). Twenty-minutes and biomass to liquid ratio of 1:20 (g mL-1) were identified as optimal operating conditions to maximize sugar recovery and ABE production (solvent yield YABE/Sugars and ABE productivity of 0.21 g g-1 and 0.12 g L-1 h-1 were obtained, respectively). The study marks the highly economic potential of the process aimed to exploit the CSS as feesdstock for antioxidant and biofuel production.

Continuous Succinic Acid Fermentation by Actinobacillus Succinogenes: Assessment of Growth and Succinic Acid Production Kinetics.

Succinic acid is one of the most interesting platform chemicals that can be produced in a biorefinery approach. The paper reports the characterization of the growth kinetics of Actinobacillus succinogenes DSM 22257 using glucose as carbon source. Tests were carried out in a continuous bioreactor operated under controlled pH. Under steady-state conditions, the conversion process was characterized in terms of concentration of glucose, cells, acids, and pH. The effects of acid-succinic, acetic, and formic-concentration in the medium on fermentation performance were investigated. The fermentation was interpreted according to several models characterized by substrate and product inhibition. The selected kinetic model of biomass growth and of metabolite production described the microorganism growth rate under a broad interval of operating conditions. Under the investigated operating conditions, results pointed out that: no substrate inhibition was observed; acetic acid did not inhibit the cell growth and succinic acid production.

Current Bottlenecks and Challenges of the Microalgal Biorefinery.

Microalgae are increasingly considered as sources of renewable feedstocks for industrial production, and microalgae production now focuses on the multiproduct microalgal biorefinery. However, such a biorefinery presents several bottlenecks that are mainly associated with downstream processes. This reduced downstream efficiency results from unsolved problems related to the culture strategy for the accumulation of different products - the protein versus lipid dilemma - and the dilute nature of the microalgal culture. We identify new trends and propose promising solutions for realizing microalgal biorefineries at industrial scale. New perspectives and challenges are identified in protein properties and in the integration and cooptimization of culture and downstream processes.

Continuous succinic acid fermentation by Actinobacillus succinogenes in a packed-bed biofilm reactor.

BACKGROUND: Succinic acid is one of the most interesting platform chemicals that can be produced in a biorefinery approach. In this study, continuous succinic acid production by Actinobacillus succinogenes fermentation in a packed-bed biofilm reactor (PBBR) was investigated. RESULTS: The effects of the operating conditions tested, dilution rate (D), and medium composition (mixture of glucose, xylose, and arabinose-that simulate the composition of a lignocellulosic hydrolysate)-on the PBBR performances were investigated. The maximum succinic acid productivity of 35.0 g L-1 h-1 and the maximum SA concentration were achieved at a D = 1.9 h-1. The effect of HMF and furfural on succinic acid production was also investigated. HMF resulted to reduce succinic acid production by 22.6%, while furfural caused a reduction of 16% in SA production at the same dilution rate. CONCLUSION: Succinic acid production by A. succinogenes fermentation in a packed-bed reactor (PBBR) was successfully carried out for more than 5 months. The optimal results were obtained at the dilution rate 0.5 h-1: 43.0 g L-1 of succinic acid were produced, glucose conversion was 88%; and the volumetric productivity was 22 g L-1 h-1.

Deep Eutectic Solvents pretreatment of agro-industrial food waste.

BACKGROUND: Waste biomass from agro-food industries are a reliable and readily exploitable resource. From the circular economy point of view, direct residues from these industries exploited for production of fuel/chemicals is a winning issue, because it reduces the environmental/cost impact and improves the eco-sustainability of productions. RESULTS: The present paper reports recent results of deep eutectic solvent (DES) pretreatment on a selected group of the agro-industrial food wastes (AFWs) produced in Europe. In particular, apple residues, potato peels, coffee silverskin, and brewer's spent grains were pretreated with two DESs, (choline chloride-glycerol and choline chloride-ethylene glycol) for fermentable sugar production. Pretreated biomass was enzymatic digested by commercial enzymes to produce fermentable sugars. Operating conditions of the DES pretreatment were changed in wide intervals. The solid to solvent ratio ranged between 1:8 and 1:32, and the temperature between 60 and 150 °C. The DES reaction time was set at 3 h. Optimal operating conditions were: 3 h pretreatment with choline chloride-glycerol at 1:16 biomass to solvent ratio and 115 °C. Moreover, to assess the expected European amount of fermentable sugars from the investigated AFWs, a market analysis was carried out. The overall sugar production was about 217 kt yr-1, whose main fraction was from the hydrolysis of BSGs pretreated with choline chloride-glycerol DES at the optimal conditions. CONCLUSIONS: The reported results boost deep investigation on lignocellulosic biomass using DES. This investigated new class of solvents is easy to prepare, biodegradable and cheaper than ionic liquid. Moreover, they reported good results in terms of sugars' release at mild operating conditions (time, temperature and pressure).

Identification of an industrial microalgal strain for starch production in biorefinery context: The effect of nitrogen and carbon concentration on starch accumulation.

The recent trends in microalgal cultures are focused on the biorefinery of the biomass components. Some of them are not completely valorised, for example starch. Since there is a wide market for starch products in food and non-food industries, the exploitation of microalgal starch fractions could improve the economic sustainability of microalgae production. In this perspective, the optimization of nitrogen and carbon source uptake for starch accumulation is a critical point for reducing the nitrogen requirement footprint and to increase CO2 capture. In this study, four robust microalgal strains, already known as starch-accumulating strain, were investigated: Chlorella sorokiniana, Scenedesmus vacuolatus, Dunaliella tertiolecta, and Tetraselmis chuii. C. sorokiniana was selected as the best starch producer in the biorefinery context, and the role nitrogen and CO2 concentration had on the starch production was investigated. For light irradiance of 300µmolm-2s-1 the optimal nitrogen concentration for growth and starch accumulation resulted 32mgL-1. The CO2 concentration clearly does not influence the starch accumulation, but concentrations distant from 2% negatively influence microalgal growth, affecting the final starch productivity. The biomass composition during the batch growth of C. sorokiniana was also analysed in order to explicitly characterise the dynamic of starch accumulation during the different growth phases. Protein content decreased during N-depletion, carbohydrates were mainly produced during the early N-depletion, followed by the accumulation of lipids in the late depletion.

Low-energy biomass pretreatment with deep eutectic solvents for bio-butanol production.

Waste lettuce leaves - from the "fresh cut vegetable" industry - were pretreated with the deep eutectic solvent (DES) made of choline chloride - glycerol. Reaction time (3-16h) and the operation temperature (80-150°C) were investigated. Enzymatic glucose and xylose yields of 94.9% and 75.0%, respectively were obtained when the biomass was pretreated at 150°C for 16h. Sugars contained in the biomass hydrolysate were fermented in batch cultures of Clostridium acetobutylicum DSMZ 792. The energy consumption and the energy efficiency related to the DES pretreatment were calculated and compared to the most common lignocellulosic pretreatment processes reported in the literature. The DES pretreatment process was characterized by lower energy required (about 28% decrease and 72% decrease) than the NAOH pretreatment and steam explosion process respectively. The Net Energy Ratio (NER) value related to butanol production via DES biomass pretreatment was assessed.

Exploitation of Trametes versicolor for bioremediation of endocrine disrupting chemicals in bioreactors.

Endocrine disrupting chemicals (EDCs) are environmental contaminants causing increasing concerns due to their toxicity, persistence and ubiquity. In the present study, degradative capabilities of Trametes versicolor, Pleurotus ostreatus and Phanerochaete chrysosporium to act on five EDCs, which represent different classes of chemicals (phenols, parabens and phthalate) and were first applied as single compounds, were assessed. T. versicolor was selected due to its efficiency against target EDCs and its potentialities were exploited against a mixture of EDCs in a cost-effective bioremediation process. A fed-batch approach as well as a starvation strategy were applied in order to reduce the need for input of 'fresh' biomass, and avoid the requirement for external nutrients. The fungus was successfully operated in two different bioreactors over one week. Semi-batch cultures were carried out by daily adding a mixture of EDCs to the bioreactors in a total of five consecutive degradation cycles. T. versicolor was able to efficiently remove all compounds during each cycle converting up to 21 mg L-1 day-1 of the tested EDCs. The maintained ability of T. versicolor to remove EDCs without any additional nutrients represents the main outcome of this study, which enables to forecast its application in a water treatment process.

Biosuccinic Acid from Lignocellulosic-Based Hexoses and Pentoses by Actinobacillus succinogenes: Characterization of the Conversion Process.

Succinic acid (SA) is a well-established chemical building block. Actinobacillus succinogenes fermentation is by far the most investigated route due to very promising high SA yield and titer on several sugars. This study contributes to include the SA production within the concept of biorefinery of lignocellulose biomass. The study was focused on the SA production by A. succinogenes DSM 22257 using sugars representative from lignocellulose hydrolysis-glucose, mannose, arabinose, and xylose-as carbon source. Single sugar batch fermentation tests and mixture sugar fermentation tests were carried out. All the sugars investigated were converted in succinic acid by A. succinogenes. The best fermentation performances were measured in tests with glucose as carbon source. The bacterial growth kinetics was characterized by glucose inhibition. No inhibition phenomena were observed with the other sugar investigated. The sugar mixture fermentation tests highlighted the synergic effects of the co-presence of the four sugars. Under the operating conditions tested, the final concentration of succinic acid in the sugar mixture test was larger (27 g/L) than that expected (25.5 g/L) by combining the fermentation of the single sugar. Moreover, the concentration of acetic and formic acid was lower, consequently obtaining an increment in the succinic acid specificity.

Valorization of Apple Pomace by Extraction of Valuable Compounds.

Apple pomace is a promising source of carbohydrates, proteins, amino acids, fatty acids, phenolic compounds, vitamins, and other compounds with a vast range of food applications. This review focuses on the valorization of apple pomace towards the recovery of the main compounds, namely pectin and polyphenols. Applications, advantages, and drawbacks of conventional extraction (acidic medium under high temperatures) compared with novel extraction technologies are presented. The comparison is based on an extensive literature review of research on extraction of valuable compounds from plant matrixes, particularly apple pomace. Novel extraction techniques involving enzymes, electric field, ultrasound, microwave heating, pressurized liquid, and super/subcritical fluid are also discussed. These techniques offer several advantages, including shorter extraction time, increased yield, reduction-or suppression-of solvents, and minimization of the environmental impact. This paper may help researchers and food industry professionals on the scaling-up and optimization of eco-friendly extractions of pectin and phenolic compounds.

Renewable feedstocks for biobutanol production by fermentation.

This paper reports a study of potential feedstock for butanol production via the biotechnological route. Several waste(water) streams rich in sugars and lignocellulosic biomass were studied: cheese-whey, leftovers of high sugar-content beverages, food lost or wasted, agriculture residues. The maximum butanol production rate from each type of feedstock was assessed according to the parameters indicated in the literature: feedstock availability rate, feedstock average composition and butanol yield. In Europe the potential biotechnological production of butanol from the feedstock studied was assessed to be about 39 Mt yr-1, which would be enough to meet the current European demand of biofuels. The potential butanol production at local level was also assessed taking into account the concentration of feedstock suppliers in the Campania region.

Alkaline direct transesterification of different species of Stichococcus for bio-oil production.

The cost of bio-oil refining from microalgal biomass can be significantly reduced by combining extraction and transesterification. The characterisation and optimisation of the combined steps have been carried out on strains of Stichococcus bacillaris, focusing on catalyst type and concentration, reaction time and temperature, methanol/biomass ratio, pre-mixing time and water content in the biomass. The bio-oil yield has been referenced as production of fatty acid methyl esters (FAMEs). The maximum yield (∼17%) was achieved using dried biomass with alkaline catalyst at 60°C and methanol/biomass weight ratio of 79:1. Alkaline catalyst conditions gave faster reaction rates and higher bio-oil yields than acid catalyst. Yield was also strongly affected by water content in the biomass. A mechanistic interpretation has been proposed to elucidate the effect of the different operating conditions. However, the structural characteristics of the Chlorophyta cell wall can be very different, leading to different bio-oil yields when the same protocol is applied. Therefore, the optimised protocol of direct transesterification for Stichococcus bacillaris strains was tested on other Stichococcus strains and several other Chlorophyta species characterised by a different cell wall structure. It was clearly demonstrated that different results for bio-oil yield were obtained within the same microalgal species and much more within different microalgal genera.

Stabilization of Candida antarctica Lipase B (CALB) Immobilized on Octyl Agarose by Treatment with Polyethyleneimine (PEI).

Lipase B from Candida antarctica (CALB) was immobilized on octyl agarose (OC) and physically modified with polyethyleneimine (PEI) in order to confer a strong ion exchange character to the enzyme and thus enable the immobilization of other enzymes on its surface. The enzyme activity was fully maintained during the coating and the thermal stability was marginally improved. The enzyme release from the support by incubation in the non-ionic detergent Triton X-100 was more difficult after the PEI-coating, suggesting that some intermolecular physical crosslinking had occurred, making this desorption more difficult. Thermal stability was marginally improved, but the stability of the OCCALB-PEI was significantly better than that of OCCALB during inactivation in mixtures of aqueous buffer and organic cosolvents. SDS-PAGE analysis of the inactivated biocatalyst showed the OCCALB released some enzyme to the medium during inactivation, and this was partially prevented by coating with PEI. This effect was obtained without preventing the possibility of reuse of the support by incubation in 2% ionic detergents. That way, this modified CALB not only has a strong anion exchange nature, while maintaining the activity, but it also shows improved stability under diverse reaction conditions without affecting the reversibility of the immobilization.

Photobioreactors for microalgal cultures: A Lagrangian model coupling hydrodynamics and kinetics.

Closed photobioreactors have to be optimized in terms of light utilization and overall photosynthesis rate. A simple model coupling the hydrodynamics and the photosynthesis kinetics has been proposed to analyze the photosynthesis dynamics due to the continuous shuttle of microalgae between dark and lighted zones of the photobioreactor. Microalgal motion has been described according to a stochastic Lagrangian approach adopting the turbulence model suitable for the photobioreactor configuration (single vs. two-phase flows). Effects of light path, biomass concentration, turbulence level and irradiance have been reported in terms of overall photosynthesis rate. Different irradiation strategies (internal, lateral and rounding) and several photobioreactor configurations (flat, tubular, bubble column, airlift) have been investigated. Photobioreactor configurations and the operating conditions to maximize the photosynthesis rate have been pointed out. Results confirmed and explained the common experimental observation that high concentrated cultures are not photoinhibited at high irradiance level.