Thermo resistant antioxidants from photoautotrophic microorganisms: screening and characterization.

The demand for natural antioxidants to be used in food industry is increasing, as synthetic antioxidants are toxic and have high production costs. Specifically, food processing and preservation require antioxidants resistant to thermal sterilization processes. In this study, twenty-five strains among microalgae and cyanobacteria were screened as antioxidants producers. The species Enallax sp., Synechococcus bigranulatus and Galdieria sulphuraria showed the highest content of chlorophyll a and total carotenoids. In vitro stability and antioxidant activity of the ethanolic extracts were performed. The results revealed that pigments present in the extracts, obtained from the previously mentioned species, were stable at room temperature and exhibited in vitro free radical scavenging potential with IC50 values of 0.099 ± 0.001, 0.048 ± 0.001 and 0.13 ± 0.02 mg mL-1, respectively. Biocompatibility assay showed that the extracts were not toxic on immortalized cell lines. The antioxidant activity was also tested on a cell-based model by measuring intracellular ROS levels after sodium arsenite treatment. Noteworthy, extracts were able to exert the same protective effect, before and after the pasteurization process. Results clearly indicate the feasibility of obtaining biologically active and thermostable antioxidants from microalgae. Green solvents can be used to obtain thermo-resistant antioxidants from cyanobacteria and microalgae which can be used in the food industry. Thus, the substitution of synthetic pigments with natural ones is now practicable.

Design of Value Chains for Microalgal Biorefinery at Industrial Scale: Process Integration and Techno-Economic Analysis.

The objective of this work was to identify industrial scenarios for the most promising microalgal biorefinery value chains on the basis of product selection, yields, and techno-economic performance, using biological characteristics of algae species. The development, value creation, and validation of several new processing routes with applications in food, aquafeeds and non-food products were particularly considered in this work. The techno-economic performance of various single product value chains (SP) and multiproduct value chains (MP) was evaluated for four industrial microalgal strains. Cost-revenue optimization was done for a 10 kton microalgal dry weight y-1 simulated biorefinery plant, using flow sheeting software for equipment sizing, mass and energy flow modeling, and subsequent techno-economic evaluation. Data on yield, material and energy consumption were based on pre- and pilot size production plants (TRL 5-6). Revenue optimization was accomplished by first analyzing the performance of single product value chains of the microalgal strains. Subsequently, a strategy was developed to exploit almost all biomass based on the most promising microalgal strains. The cultivation costs are most of the time the major costs of the value chains. For the single product value chains common process bottlenecks are low product yields, especially for soluble proteins where only a small fraction of the biomass is leading to economic value. The biorefinery costs (excluding cultivation) vary significantly for various species, due to the species-specific operating conditions as well as differences in product yields. For the evaluated single product value chain scenarios the costs for utilities and other inputs were in general the highest contributing expenses. A biorefinery approach significantly increases the biomass utilization potential to marketable products from 7-28% to more than 97%. Although the cascading approach increases the total production costs of the multiproduct value chains significantly, this is more than compensated by the increased overall biomass revenue. For all selected multiproduct chains there is a significant potential to become profitable at a relevant industrial scale of 10 kton per year. Additional insights in the product functionality, quality, and their market size are needed to narrow down the wide range of foreseen product revenues and resulting profits.

Towards green extraction methods from microalgae learning from the classics.

Microalgae started receiving attention as producers of third generation of biofuel, but they are rich in many bioactive compounds. Indeed, they produce many molecules endowed with benefic effects on human health which are highly requested in the market. Thus, it would be important to fractionate algal biomass into its several high-value compounds: this represents the basis of the microalgal biorefinery approach. Usually, conventional extraction methods have been used to extract a single class of molecules, with many side effects on the environment and on human health. The development of a green downstream platform could help in obtaining different class of molecules with high purity along with low environmental impact. This review is focused on technical advances that have been performed, from classic methods to the newest and green ones. Indeed, it is fundamental to set up new procedures that do not affect the biological activity of the extracted molecules. A comparative analysis has been performed among the conventional methods and the new extraction techniques, i.e., switchable solvents and microwave-assisted and compressed fluid extractions.

Comparison of Galdieria growth and photosynthetic activity in different culture systems.

In the last years, the acidothermophilic red microalga Galdieria sulphuraria has been increasingly studied for industrial applications such as wastewater treatment, recovery of rare earth elements, production of phycobilins. However, even now it is not possible an industrial cultivation of this organism because biotechnological research on G. sulphuraria and allied species is relatively recent and fragmented. Having in mind a possible scale-up for commercial applications, we have compared the growth and photosynthetic performance of G. sulphuraria in four suspended systems (Inclined bubble column, Decanter Laboratory Flask, Tubular Bioreactor, Ultra-flat plate bioreactor) and one immobilized system (Twin Layer Sytem). The results showed that G. sulphuraria had the highest growth, productivity and photosynthetic performance, when grown on the immobilized system, which also offers some economics advantages.

Biomass and phycobiliprotein production of Galdieria sulphuraria, immobilized on a twin-layer porous substrate photobioreactor.

The extremophile red alga Galdieria sulphuraria was successfully grown immobilized in a twin-layer porous substrate bioreactor (TL-PSBR). A maximal biomass growth rate of 10 g dry weight m-2 day-1 was measured at a photon fluence rate of 200 µmol photons m-2 s-1 with addition of 1% CO2 and a temperature of 34 °C. Under these conditions, a maximal biomass value of 232 g m-2 was attained after 33 days of growth. Phycobilin productivity, however, was highest at a lower photon fluence rate of 100 µmol photons m-2 s-1 and reached a phycobilin value of 14 g m-2, a phycobilin content in the biomass of 63 mg g-1 and a phycobilin growth rate of 0.28 g m-2 day-1 for phycocyanin and 0.23 g m-2 day-1 for allophycocyanin. Addition of CO2 was essential to enhance growth and phycobilin production in G. sulphuraria and further optimization of the cultivation process in the TL-PSBR appears possible using a multi-phase approach, higher growth temperatures and optimization of nutrient supply. It is concluded that autotrophic cultivation of G. sulphuraria in a TL-PSBR is an attractive alternative to suspension cultivation for phycobilin production and applications in bioremediation.

A thermophilic C-phycocyanin with unprecedented biophysical and biochemical properties.

C-phycoyanins are abundant light-harvesting pigments which have an important role in the energy transfer cascade of photosystems in prokaryotic cyanobacteria and eukaryotic red algae. These proteins have important biotechnological applications, since they can be used in food, cosmetics, nutraceutical, pharmaceutical industries and in biomedical research. Here, C-phycocyanin from the extremophilic red alga Galdieria phlegrea (GpPC) has been purified and characterized from a biophysical point of view by SDS-PAGE, mass spectrometry, UV-Vis absorption spectroscopy, circular dichroism and intrinsic fluorescence. Stability against pH variations, addition of the oxidizing agent hydrogen peroxide and the effects of temperature have been also investigated, together with its in cell antioxidant potential and antitumor activity. GpPC is stable under different pHs and unfolds at a temperature higher than 80 °C within the pH range 5.0-7.0. Its fluorescence spectra present a maximum at 650 nm, when excited at 589 nm. The protein exerts interesting in cell antioxidant properties even after high temperature treatments, like the pasteurization process, and is cytotoxic for A431 and SVT2 cancer cells, whereas it is not toxic for non-malignant cells. Our results assist in the development of C-phycocyanin as a multitasking protein, to be used in the food industry, as antioxidant and anticancer agent.

Green Compressed Fluid Technologies To Extract Antioxidants and Lipids from Galdieria phlegrea in a Biorefinery Approach.

A green cascade approach was used to recover phycocyanins, carotenoids and lipids from Galdiera phlegrea. Phycocyanin extraction was performed by high pressure homogenization and purified by ultrafiltration, whereas carotenoids were obtained by a pressurized liquid extraction and lipids by supercritical fluid extraction. The second step of this innovative, green, and cost-effective procedure is able to improve the recovery of zeaxanthin and ß-carotene up to 40%, without affecting the quality of compounds and avoiding the use of organic solvents and the drying processes. The isolated carotenoids were active as antioxidants, as clearly shown by their protective activity on a cell-based model. The lipid yield was increased by 12% with respect to conventional methods.

A cascade extraction of active phycocyanin and fatty acids from Galdieria phlegrea.

The setup of an economic and sustainable method to increase the production and commercialization of products from microalgae, beyond niche markets, is a challenge. Here, a cascade approach has been designed to optimize the recovery of high valuable bioproducts starting from the wet biomass of Galdieria phlegrea. This unicellular thermo-acidophilic red alga can accumulate high-value compounds and can live under conditions considered hostile to most other species. Extractions were performed in two sequential steps: a conventional high-pressure procedure to recover phycocyanins and a solvent extraction to obtain fatty acids. Phycocyanins were purified to the highest purification grade reported so far and were active as antioxidants on a cell-based model. Fatty acids isolated from the residual biomass contained high amount of PUFAs, more than those recovered from the raw biomass. Thus, a simple, economic, and high effective procedure was set up to isolate phycocyanin at high purity levels and PUFAs.

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%.

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.

Cost-effective production of recombinant peptides in Escherichia coli.

Among bioactive peptides, cationic antimicrobial peptides (AMPs), also referred to as host defence peptides (HDPs), are valuable tools to treat infections, being able to kill a wide variety of microbes directly and/or modulate host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites. However, high manufacturing costs have greatly limited their development as drugs, thus highlighting the need to develop novel and competitive production strategies. Here, a cost-effective procedure was established to produce the high amounts of peptides required for basic and clinical research. Firstly, a novel culture medium was designed, which was found to support significantly higher cell densities and recombinant expression levels of peptides under test compared to conventional media. The procedure has been also efficiently scaled up by using a 5 L fermenter, while the costs have been lowered significantly by developing a successful auto-induction strategy, which has been found to support higher yields of target constructs and cell biomass compared to conventional strategies based on expression induction by IPTG. Interestingly, it was estimated that by increasing production scale from 100 to 1000 mg/batch, unit costs decreased strongly from 253 to 42 €/mg. These costs appear highly competitive when compared to chemical synthesis strategies. Altogether, the data indicate that the strategy represents an important starting point for the future development of large-scale manufacture of HDPs.

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.

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: 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.

From Current Algae Products to Future Biorefinery Practices: A Review.

Microalgae are considered to be one of the most promising next generation bio-based/food feedstocks with a unique lipid composition, high protein content, and an almost unlimited amount of other bio-active molecules. High-value components such as the soluble proteins, (poly) unsaturated fatty acids, pigments, and carbohydrates can be used as an important ingredient for several markets, such as the food/feed/chemical/cosmetics and health industries. Although cultivation costs have decreased significantly in the last few decades, large microalgae production processes become economically viable if all complex compounds are optimally valorized in their functional state. To isolate these functional compounds from the biomass, cost-effective, mild, and energy-efficient biorefinery techniques need to be developed and applied. In this review we describe current microalgae biorefinery strategies and the derived products, followed by new technological developments and an outlook toward future products and the biorefinery philosophy.

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.

Growth and biomass productivity of Scenedesmus vacuolatus on a twin layer system and a comparison with other types of cultivations.

Scenedesmus is a genus of microalgae employed for several industrial uses. Industrial cultivations are performed in open ponds or in closed photobioreactors (PBRs). In the last years, a novel type of PBR based on immobilized microalgae has been developed termed porous substrate photobioreactors (PSBR) to achieve significant higher biomass density during cultivation in comparison to classical PBRs. This work presents a study of the growth of Scenedesmus vacuolatus in a Twin Layer System PSBR at different light intensities (600 µmol photons m-2 s-1 or 1000 µmol photons m-2 s-1), different types and concentrations of the nitrogen sources (nitrate or urea), and at two CO2 levels in the gas phase (2% or 0.04% v/v). The microalgal growth was followed by monitoring the attached biomass density as dry weight, the specific growth rate and pigment accumulation. The highest productivity (29 g m-2 d-1) was observed at a light intensity of 600 µmol photons m-2 s-1 and 2% CO2. The types and concentrations of nitrogen sources did not influence the biomass productivity. Instead, the higher light intensity of 1000 µmol photons m-2 s-1 and an ambient CO2 concentration (0.04%) resulted in a significant decrease of productivity to 18 and 10-12 g m-2 d-1, respectively. When compared to the performance of similar cultivation systems (15-30 g m-2 d-1), these results indicate that the Twin Layer cultivation System is a competitive technique for intensified microalgal cultivation in terms of productivity and, at the same time, biomass density.