Cyanobacterial Proteomics: Diversity and Dynamics.

Cyanobacteria (oxygenic photoautrophs) comprise a diverse group holding significance both environmentally and for biotechnological applications. The utilization of proteomic techniques has significantly influenced investigations concerning cyanobacteria. Application of proteomics allows for large-scale analysis of protein expression and function within cyanobacterial systems. The cyanobacterial proteome exhibits tremendous functional, spatial, and temporal diversity regulated by multiple factors that continuously modify protein abundance, post-translational modifications, interactions, localization, and activity to meet the dynamic needs of these tiny blue greens. Modern mass spectrometry-based proteomics techniques enable system-wide examination of proteome complexity through global identification and high-throughput quantification of proteins. These powerful approaches have revolutionized our understanding of proteome dynamics and promise to provide novel insights into integrated cellular behavior at an unprecedented scale. In this Review, we present modern methods and cutting-edge technologies employed for unraveling the spatiotemporal diversity and dynamics of cyanobacterial proteomics with a specific focus on the methods used to analyze post-translational modifications (PTMs) and examples of dynamic changes in the cyanobacterial proteome investigated by proteomic approaches.

Tools for genetic engineering and gene expression control in Novosphingobium aromaticivorans and Rhodobacter sphaeroides.

Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts that have the potential to contribute to the sustainable bioeconomy. Among the Alphaproteobacteria, Novosphingobium aromaticivorans, Rhodobacter sphaeroides, and Zymomonas mobilis show promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit for Z. mobilis, N. aromaticivorans and R. sphaeroides still need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools for N. aromaticivorans and R. sphaeroides to address these issues. We demonstrate that Tn7 transposition is an effective approach for introducing engineered DNA into the chromosome of N. aromaticivorans and R. sphaeroides. We screen a synthetic promoter library to identify isopropyl ß-D-1-thiogalactopyranoside-inducible promoters with regulated activity in both organisms (up to ~15-fold induction in N. aromaticivorans and ~5-fold induction in R. sphaeroides). Combining Tn7 integration with promoters from our library, we establish CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference systems for N. aromaticivorans and R. sphaeroides (up to ~10-fold knockdown in N. aromaticivorans and R. sphaeroides) that can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these species and other Alphaproteobacteria.IMPORTANCEIt is important to increase our understanding of the microbial world to improve health, agriculture, the environment, and biotechnology. For example, building a sustainable bioeconomy depends on the efficient conversion of plant material to valuable biofuels and bioproducts by microbes. One limitation in this conversion process is that microbes with otherwise promising properties for conversion are challenging to genetically engineer. Here we report genetic tools for Novosphingobium aromaticivorans and Rhodobacter sphaeroides that add to the burgeoning set of tools available for genome engineering and gene expression in Alphaproteobacteria. Our approaches allow straightforward insertion of engineered pathways into the N. aromaticivorans or R. sphaeroides genome and control of gene expression by inducing genes with synthetic promoters or repressing genes using CRISPR interference. These tools can be used in future work to gain additional insight into these and other Alphaproteobacteria and to aid in optimizing yield of biofuels and bioproducts.

Lignocellulosic materials valorization in second generation biorefineries: an opportunity to produce fungal biopigments.

Second generation biorefineries play an important role in the production of renewable energy and fuels, utilizing forest and agro-industrial residues and by-products as raw materials. The integration of novel bioproducts, such as: xylitol, ß-carotene, xylooligosaccharides, and biopigments into the biorefinery's portfolio can offer economic benefits in the valorization of lignocellulosic materials, particularly cellulosic and hemicellulosic fractions. Fungal biopigments, known for their additional antioxidant and antimicrobial properties, are appealing to consumers and can have applications in various industrial sectors, including food and pharmaceuticals. The use of lignocellulosic materials as carbon and nutrient sources for the growth medium helps to reduce production costs, increasing the competitiveness of fungal biopigments in the market. In addition, the implementation of biopigment production in biorefineries allows the utilization of underutilized fractions, such as hemicellulose, for value-added bioproducts. This study deals with the potential of fungal biopigments production in second generation biorefineries in order to diversify the produced biomolecules together with energy generation. A comprehensive and critical review of the recent literature on this topic has been conducted, covering the major possible raw materials, general aspects of second generation biorefineries, the fungal biopigments and their potential for incorporation into biorefineries.

Extremophiles in a changing world.

Extremophiles and their products have been a major focus of research interest for over 40 years. Through this period, studies of these organisms have contributed hugely to many aspects of the fundamental and applied sciences, and to wider and more philosophical issues such as the origins of life and astrobiology. Our understanding of the cellular adaptations to extreme conditions (such as acid, temperature, pressure and more), of the mechanisms underpinning the stability of macromolecules, and of the subtleties, complexities and limits of fundamental biochemical processes has been informed by research on extremophiles. Extremophiles have also contributed numerous products and processes to the many fields of biotechnology, from diagnostics to bioremediation. Yet, after 40 years of dedicated research, there remains much to be discovered in this field. Fortunately, extremophiles remain an active and vibrant area of research. In the third decade of the twenty-first century, with decreasing global resources and a steadily increasing human population, the world's attention has turned with increasing urgency to issues of sustainability. These global concerns were encapsulated and formalized by the United Nations with the adoption of the 2030 Agenda for Sustainable Development and the presentation of the seventeen Sustainable Development Goals (SDGs) in 2015. In the run-up to 2030, we consider the contributions that extremophiles have made, and will in the future make, to the SDGs.

A comprehensive review on versatile microalga Tetraselmis: Potentials applications in wastewater remediation and bulk chemical production.

Microalgae are considered sustainable resources for the production of biofuel, feed, and bioactive compounds. Among various microalgal genera, the Tetraselmis genus, containing predominantly marine microalgal species with wide tolerance to salinity and temperature, has a high potential for large-scale commercialization. Until now, Tetraselmis sp. are exploited at smaller levels for aquaculture hatcheries and bivalve production. However, its prolific growth rate leads to promising areal productivity and energy-dense biomass, so it is considered a viable source of third-generation biofuel. Also, microbial pathogens and contaminants are not generally associated with Tetraselmis sp. in outdoor conditions due to faster growth as well as dominance in the culture. Numerous studies revealed that the metabolite compositions of Tetraselmis could be altered favorably by changing the growth conditions, taking advantage of its acclimatization or adaptation ability in different conditions. Furthermore, the biorefinery approach produces multiple fractions that can be successfully upgraded into various value-added products along with biofuel. Overall, Tetraselmis sp. could be considered a potential strain for further algal biorefinery development under the circular bioeconomy framework. In this aspect, this review discusses the recent advancements in the cultivation and harvesting of Tetraselmis sp. for wider application in different sectors. Furthermore, this review highlights the key challenges associated with large-scale cultivation, biomass harvesting, and commercial applications for Tetraselmis sp.

The Role of Lignin Molecular Weight on Activated Carbon Pore Structure.

Over the past decade, the production of biofuels from lignocellulosic biomass has steadily increased to offset the use of fuels from petroleum. To make biofuels cost-competitive, however, it is necessary to add value to the "ligno-" components (up to 30% by mass) of the biomass. The properties of lignin, in terms of molecular weight (MW), chemical functionality, and mineral impurities often vary from biomass source and biorefinery process, resulting in a challenging precursor for product development. Activated carbon (AC) is a feasible target for the lignin-rich byproduct streams because it can be made from nearly any biomass, and it has a market capacity large enough to use much of the lignin generated from the biorefineries. However, it is not known how the variability in the lignin affects the key properties of AC, because, until now, they could not be well controlled. In this work, various fractions of ultraclean (<0.6% ash) lignin are created with refined MW distributions using Aqueous Lignin Purification using Hot Agents (ALPHA) and used as precursors for AC. AC is synthesized via zinc chloride activation and characterized for pore structure and adsorption capacity. We show that AC surface area and the adsorption capacity increase when using lignin with increasing MW, and, furthermore, that reducing the mineral content of lignin can significantly enhance the AC properties. The surface area of the AC from the highest MW lignin can reach ~1830 m2/g (absorption capacity). Furthermore, single step activation carbonization using zinc chloride allows for minimal carbon burn off (<30%), capturing most of the lignin carbon compared to traditional burn off methods in biorefineries for heat generation.

Integration of physiologically relevant photosynthetic energy flows into whole genome models of light-driven metabolism.

Characterizing photosynthetic productivity is necessary to understand the ecological contributions and biotechnology potential of plants, algae, and cyanobacteria. Light capture efficiency and photophysiology have long been characterized by measurements of chlorophyll fluorescence dynamics. However, these investigations typically do not consider the metabolic network downstream of light harvesting. By contrast, genome-scale metabolic models capture species-specific metabolic capabilities but have yet to incorporate the rapid regulation of the light harvesting apparatus. Here, we combine chlorophyll fluorescence parameters defining photosynthetic and non-photosynthetic yield of absorbed light energy with a metabolic model of the pennate diatom Phaeodactylum tricornutum. This integration increases the model predictive accuracy regarding growth rate, intracellular oxygen production and consumption, and metabolic pathway usage. Through the quantification of excess electron transport, we uncover the sequential activation of non-radiative energy dissipation processes, cross-compartment electron shuttling, and non-photochemical quenching as the rapid photoacclimation strategy in P. tricornutum. Interestingly, the photon absorption thresholds that trigger the transition between these mechanisms were consistent at low and high incident photon fluxes. We use this understanding to explore engineering strategies for rerouting cellular resources and excess light energy towards bioproducts in silico. Overall, we present a methodology for incorporating a common, informative data type into computational models of light-driven metabolism and show its utilization within the design-build-test-learn cycle for engineering of photosynthetic organisms.

Prevention and Detoxification of Mycotoxins in Human Food and Animal Feed using Bio-resources from South Mediterranean Countries: a Critical Review.

Mycotoxins, which are natural toxic compounds produced by filamentous fungi, are considered major contaminants in the food and feed chain due to their stability during processing. Their impacts in food and feedstuff pollution were accentuated due the climate change in the region. They are characterized by their toxicological effects on human and animal health but also by their harmful economic impact. Mediterranean countries: Algeria, Egypt, Libya, Morocco and Tunisia are characterized by high temperatures and high relative humidity, particularly in littoral regions that provide favorable conditions for fungal growth and toxinogenesis. Many scientific papers have been published recently in these countries showing mycotoxin occurrence in different commodities and an attempt at bio-detoxification using many bio-products. In order to minimize the bioavailability and/or to detoxify mycotoxins into less toxic metabolites (bio-transforming agents), safe and biological methods have been developed including the use of lactic acid bacteria, yeasts, plant extracts and clays minerals from Mediterranean regions. The aim of this review is to present the pollution of mycotoxins in food and feedstuff of humans and animals and to discuss the development of effective biological control for mycotoxin removal/detoxification and prevention using bio-products. This review will also elucidate the new used natural products to be considered as a new candidates for mycotoxins detoxification/prevention on animal feedstuffs.

Production of Trichoderma harzianum K179 bioagent for maize diseases control: complete laboratory stage bioprocess development.

AIMS: In order to be competitive on the market, the production of biopreparations needs to be optimized, modelled, and assessed in the early stages of its development. The aim of this paper was to optimize medium for the production of Trichoderma harzianum K179 biocontrol agent, to analyze its kinetics at enlarged laboratory scale and finally economic analysis of the production of this high-value product through simulation modelling. METHODS AND RESULTS: The results showed that the bioprocess of T. harzianum K179 bioagent production in a laboratory bioreactor on the medium with optimal composition (dextrose 10 g l-1, soy flour 6.87 g l-1, K2HPO4 1.51 g l-1, KCl 0.5 g l-1, and MgSO4 × 7H2O 0.5 g l-1), at stirring speed of 1.75 × g and aeration intensity of 1.5 vvm, can be shortened from 96 to 36 h. The results of bioprocess economic analysis showed that with a 25-year project lifetime and an investment payback time of 7.58 years, this project represents an economically viable system. CONCLUSIONS: Complete analysis of the bioprocess of T. harzianum K179 biocontrol agent production showed that the biologically produced preparation can be competitive on the market with synthetic preparations.

Insect biorefinery: A circular economy concept for biowaste conversion to value-added products.

With rapid growing world population and increasing demand for natural resources, the production of sufficient food, feed for protein and fat sources and sustainable energy presents a food insecurity challenge globally. Insect biorefinery is a concept of using insect as a tool to convert biomass waste into energy and other beneficial products with concomitant remediation of the organic components. The exploitation of insects and its bioproducts have becoming more popular in recent years. This review article presents a summary of the current trend of insect-based industry and the potential organic wastes for insect bioconversion and biorefinery. Numerous biotechnological products obtained from insect biorefinery such as biofertilizer, animal feeds, edible foods, biopolymer, bioenzymes and biodiesel are discussed in the subsequent sections. Insect biorefinery serves as a promising sustainable approach for waste management while producing valuable bioproducts feasible to achieve circular bioeconomy.

Harnessing the Value of Tripolium pannonicum and Crithmum maritimum Halophyte Biomass through Integrated Green Biorefinery.

Bioactive extracts are often the target fractions in bioprospecting, and halophyte plants could provide a potential source of feedstock for high-value applications as a part of integrated biorefineries. Tripolium pannonicum (Jacq.) Dobrocz. (sea aster) and Crithmum maritimum L. (sea fennel) are edible plants suggested for biosaline halophyte-based agriculture. After food production and harvesting of fresh leaves for food, the inedible plant fractions could be utilized to produce extracts rich in bioactive phytochemicals to maximize feedstock application and increase the economic feasibility of biomass processing to bioenergy. This study analyzed fresh juice and extracts from screw-pressed sea aster and sea fennel for their different phenolic compounds and pigment concentrations. Antioxidant and enzyme inhibition activities were also tested in vitro. Extracts from sea aster and sea fennel had phenolic contents up to 45.2 mgGAE/gDM and 64.7 mgGAE/gDM, respectively, and exhibited >70% antioxidant activity in several assays. Ethanol extracts also showed >70% inhibition activity against acetylcholinesterase and >50% inhibition of tyrosinase and α-glucosidase. Therefore, these species can be seen as potential feedstocks for further investigations.

Sequential Extraction of Valuable Bio-Products from Snow Crab (Chionoecetes opilio) Processing Discards Using Eco-Friendly Methods.

Green extraction methods using a combination of mechanical, enzymatic, and green chemical treatments were evaluated for the sequential extraction of carotenoid pigments, protein, and chitin from crab processing discards. Key objectives included avoiding the use of hazardous chemical solvents, conducting as close to a 100% green extraction as possible, and developing simple processes to facilitate implementation into processing plants without the need for complicated and expensive equipment. Three crab bio-products were obtained: pigmented vegetable oil, pigmented protein powder, and chitin. Carotenoid extractions were performed using vegetable oils (corn, canola, and sunflower oils), giving between 24.85% and 37.93% astaxanthin recovery. Citric acid was used to demineralize the remaining material and afforded a pigmented protein powder. Three different proteases were used to deproteinate and isolate chitin in yields between 17.06% and 19.15%. The chitin was still highly colored and therefore decolorization was attempted using hydrogen peroxide. Characterization studies were conducted on each of the crab bio-products isolated including powder X-ray diffraction analysis on the chitin (80.18% crystallinity index, CI, achieved using green methods). Overall, three valuable bio-products could be obtained but further research is needed to obtain pigment-free chitin in an environmentally friendly manner.

Physiological responses to pH in the freshwater microalga Limnomonas gaiensis.

The ecological niche of the recently described limnic microalga Limnomonas gaiensis (Chlamydomonadales) in Northern Europe remains unknown. To decipher the species tolerance capacity to pH, the effects of hydrogen ions on the physiological response of L. gaiensis were investigated. Results showed that L. gaiensis could tolerate exposure from pH 3 up to pH 11, with an optimal survival at pH 5-8. Its physiological response to pH was strain specific. Globally the southernmost strain was more alkaliphilic, had a slightly rounder shape, a slowest growth rate, and a lowest carrying capacity. Despite strain discrepancies among lakes, Swedish strains exhibited similar growth rates, faster at more acidic conditions. The extreme pH conditions affected its morphological features such as the eye spot and papilla shape, especially at acidic pH, and the cell wall integrity, at more alkaline pH. The wide range tolerance of L. gaiensis to pH would not be a hindrance to its dispersal in Swedish lakes (pH 4-8). Notably, the storage of high-energetic reserves over a wide range of pH conditions, as numerous starch grains and oil droplets, makes L. gaiensis a good candidate for bioethanol/fuel industrial production and a key resource to sustain aquatic food chain and microbial loop.

Supercritical Carbon Dioxide Technology for Recovering Valuable Phytochemicals from Cannabis sativa L. and Valorization of Its Biomass for Food Applications.

Supercritical carbon dioxide (CO2) extraction techniques meet all-new consumer market demands for health-promoting phytochemical compound-rich extracts produced from green and sustainable technology. In this regard, this review is dedicated to discussing is the promise of integrating high-pressure CO2 technologies into the Cannabis sativa L. processing chain to valorize its valuable pharmaceutical properties and food biomass. To do this, the cannabis plant, cannabinoids, and endocannabinoid system were reviewed to understand their therapeutic and side effects. The supercritical fluid extraction (SFE) technique was presented as a smart alternative to producing cannabis bioproducts. The impact of SFE operating conditions on cannabis compound extraction was examined for aerial parts (inflorescences, stems, and leaves), seeds, and byproducts. Furthermore, the opportunities of using non-thermal supercritical CO2 processing on cannabis biomass were addressed for industrial hemp valorization, focusing on its biorefinery to simultaneously produce cannabidiol and new ingredients for food applications as plant-based products.

Comparative Exergy and Environmental Assessment of the Residual Biomass Gasification Routes for Hydrogen and Ammonia Production.

The need to reduce the dependency of chemicals on fossil fuels has recently motivated the adoption of renewable energies in those sectors. In addition, due to a growing population, the treatment and disposition of residual biomass from agricultural processes, such as sugar cane and orange bagasse, or even from human waste, such as sewage sludge, will be a challenge for the next generation. These residual biomasses can be an attractive alternative for the production of environmentally friendly fuels and make the economy more circular and efficient. However, these raw materials have been hitherto widely used as fuel for boilers or disposed of in sanitary landfills, losing their capacity to generate other by-products in addition to contributing to the emissions of gases that promote global warming. For this reason, this work analyzes and optimizes the biomass-based routes of biochemical production (namely, hydrogen and ammonia) using the gasification of residual biomasses. Moreover, the capture of biogenic CO2 aims to reduce the environmental burden, leading to negative emissions in the overall energy system. In this context, the chemical plants were designed, modeled, and simulated using Aspen plus™ software. The energy integration and optimization were performed using the OSMOSE Lua Platform. The exergy destruction, exergy efficiency, and general balance of the CO2 emissions were evaluated. As a result, the irreversibility generated by the gasification unit has a relevant influence on the exergy efficiency of the entire plant. On the other hand, an overall negative emission balance of -5.95 kgCO2/kgH2 in the hydrogen production route and -1.615 kgCO2/kgNH3 in the ammonia production route can be achieved, thus removing from the atmosphere 0.901 tCO2/tbiomass and 1.096 tCO2/tbiomass, respectively.

Advances in sustainable approaches utilizing orange peel waste to produce highly value-added bioproducts.

Orange peel waste (OPW), a discarded part of orange fruit, is a rich source of essential constituents that can be transformed into highly value-added bioproducts. OPW is being generated in million tonnes globally and returns to the environment without complete benefit. Thus, a high volume of annually produced OPW in the industry requires effective valorization. In this regard, limited data is available that summarizes the broader spectrum for the sustainable fate of OPW to produce value-added bioproducts. The main objective of this treatise is to explore the sustainable production of bioproducts from OPW. Therefore, this review covers all the aspects of OPW, from its production to complete valorization. The review encompasses the extraction technologies employed for extracting different valuable bioactive compounds, such as: essential oil (EO), pectin, and carotenoids, from OPW. Furthermore, the suitability of bioconversion technologies (digestion/fermentation) in transforming OPW to other useful bioproducts, such as: biochemicals (lactic acid and succinic acid), biopolysaccharides (xanthan and curdlan gum), and bioenergy (biomethane and bioethanol) is discussed. Also, it includes the concept of OPW-based biorefineries and their development that shall play a definite role in future to cover demands for: food, chemicals, materials, fuels, power, and heat. Lastly, this review focuses on OPW-supplemented functional food products such as: beverages, yogurts, and extruded products. In conclusion, insights provided in this review maximize the potential of OPW for commercial purposes, leading to a safe, and waste-free environment.

Isolation and agricultural potential of penicillic acid against citrus canker.

AIMS: The control of Xanthomonas citri subsp. citri (X. citri), causal agent of citrus canker, relies heavily on integrated agricultural practices involving the use of copper-based chemicals. Considering the need for alternatives to control this disease and the potential of fungi from extreme environments as producers of bioactive metabolites, we isolated and identified a bioactive compound from Penicillium sp. CRM 1540 isolated from Antarctica marine sediment. METHODS AND RESULTS: The potential of compound as an antibacterial agent against X. citri was assessed through in vitro and greenhouse experiments. Molecular taxonomy indicates that this fungus is a possible new species of Penicillium. Results revealed 90% bacterial inhibition in vitro at 25 µg ml- 1 and a decrease in 75.37% of citrus canker symptoms emergency in vivo in treated leaves of Citrus sinensis (L.) Osbeck considering the number of lesions per cm2 (p < 0.05) in comparison with the control. The structure of the active agent was identified as penicillic acid based on a detailed spectroscopic analysis. CONCLUSION: Penicillic acid can be an alternative against citrus canker. SIGNIFICANCE AND IMPACT OF STUDY: Research into extremophile micro-organisms can identify molecules with biotechnological potential and alternatives to current agricultural practices.

Gibberellic acid-induced fatty acid metabolism and ABC transporters promote astaxanthin production in Phaffia rhodozyma.

AIMS: Astaxanthin is an important natural antioxidant with various biological functions; however, the production of astaxanthin does not meet the requirements for industrialization. The aim of the present study was to identify an inducer that increases astaxanthin yield and to evaluate the regulatory mechanism of the induction of astaxanthin synthesis in Phaffia rhodozyma. METHODS AND RESULTS: The effects of indole-3-acetic acid (IAA), jasmonic acid (JA) and gibberellic acid (GA) on astaxanthin synthesis were studied by fermentation kinetics analysis. Then, combined transcriptomics and metabolomics approaches were used to analyse differential metabolites and expressed genes involved in astaxanthin synthesis induced by GA. The results indicated that GA significantly increased astaxanthin production; however, IAA and JA had no significant effect on astaxanthin synthesis. The induction by GA significantly enhanced fatty acid metabolism and ABC transporters, increased the expression of fatty acid desaturase and ABC transporter genes, and elevated the contents of unsaturated fatty acids. CONCLUSIONS: These results suggested that fatty acid saturation plays an important role in astaxanthin accumulation and that ABC transporters may be the efflux pumps for astaxanthin. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study reveals metabolic mechanism of GA-induced astaxanthin synthesis and proposes a new strategy of transporter engineering to improve astaxanthin production.

Improved high-throughput screening technique to rapidly isolate Chlamydomonas transformants expressing recombinant proteins.

The single-celled eukaryotic green alga Chlamydomonas reinhardtii has long been a model system for developing genetic tools for algae, and is also considered a potential platform for the production of high-value recombinant proteins. Identifying transformants with high levels of recombinant protein expression has been a challenge in this organism, as random integration of transgenes into the nuclear genome leads to low frequency of cell lines with high gene expression. Here, we describe the design of an optimized vector for the expression of recombinant proteins in Chlamydomonas, that when transformed and screened using a dual antibiotic selection, followed by screening using fluorescence activated cell sorting (FACS), permits rapid identification and isolation of microalgal transformants with high expression of a recombinant protein. This process greatly reduces the time required for the screening process, and can produce large populations of recombinant algae transformants with between 60 and 100% of cells producing the recombinant protein of interest, in as little as 3 weeks, that can then be used for whole population sequencing or individual clone analysis. Utilizing this new vector and high-throughput screening (HTS) process resulted in an order of magnitude improvement over existing methods, which normally produced under 1% of algae transformants expressing the protein of interest. This process can be applied to other algal strains and recombinant proteins to enhance screening efficiency, thereby speeding up the discovery and development of algal-derived recombinant protein products. KEY POINTS: • A protein expression vector using double-antibiotic resistance genes was designed • Double antibiotic selection causes fewer colonies with more positive for phenotype • Coupling the new vector with FACS improves microalgal screening efficiency > 60.

Rice recycling: a simple strategy to improve conidia production in solid-state cultures.

Here we studied at a laboratory scale a potential strategy to revalorize the residual rice remaining at the end of a conventional conidia production process in solid-state culture. The conidia production of Trichoderma asperellum Th-T4 (3) and Metarhizium robertsii Xoch-8.1 started with the use of fresh rice (unrecycled rice) as the substrate (cycle one), and continued with the use of recycled rice in successive cycles of conidia production. The rice remaining at the end of the first cycle was reused without any further sterilization or reinoculation. As a result, it was observed that the conidia production and productivity significantly increased in both fungi. Conidia production in T. asperellum Th-T4 (3) increased from 1 × 109 (first cycle) to 2·9 × 109 conidia per gram of initial dry substrate (con⋅gds-1 ) (second cycle using recycled rice), while in M. robertsii Xoch-8.1, this parameter increased form 5·7 × 108 to 1·4 × 109 con⋅gds-1 . Both fungi grew faster and conidiated earlier when recycled rice was used as the substrate, therefore, conidia productivity was also significantly improved. Furthermore, the use of recycled rice did not affect conidia viability. This is the first report about a recycling methodology completely free of extra-processing steps, and useful to increase conidia production and productivity.