Unlocking bacterial potential to reduce farmland N2O emissions.

Farmed soils contribute substantially to global warming by emitting N2O (ref. 1), and mitigation has proved difficult2. Several microbial nitrogen transformations produce N2O, but the only biological sink for N2O is the enzyme NosZ, catalysing the reduction of N2O to N2 (ref. 3). Although strengthening the NosZ activity in soils would reduce N2O emissions, such bioengineering of the soil microbiota is considered challenging4,5. However, we have developed a technology to achieve this, using organic waste as a substrate and vector for N2O-respiring bacteria selected for their capacity to thrive in soil6-8. Here we have analysed the biokinetics of N2O reduction by our most promising N2O-respiring bacterium, Cloacibacterium sp. CB-01, its survival in soil and its effect on N2O emissions in field experiments. Fertilization with waste from biogas production, in which CB-01 had grown aerobically to about 6 × 109 cells per millilitre, reduced N2O emissions by 50-95%, depending on soil type. The strong and long-lasting effect of CB-01 is ascribed to its tenacity in soil, rather than its biokinetic parameters, which were inferior to those of other strains of N2O-respiring bacteria. Scaling our data up to the European level, we find that national anthropogenic N2O emissions could be reduced by 5-20%, and more if including other organic wastes. This opens an avenue for cost-effective reduction of N2O emissions for which other mitigation options are lacking at present.

Optogenetic regulation of engineered cellular metabolism for microbial chemical production.

The optimization of engineered metabolic pathways requires careful control over the levels and timing of metabolic enzyme expression. Optogenetic tools are ideal for achieving such precise control, as light can be applied and removed instantly without complex media changes. Here we show that light-controlled transcription can be used to enhance the biosynthesis of valuable products in engineered Saccharomyces cerevisiae. We introduce new optogenetic circuits to shift cells from a light-induced growth phase to a darkness-induced production phase, which allows us to control fermentation with only light. Furthermore, optogenetic control of engineered pathways enables a new mode of bioreactor operation using periodic light pulses to tune enzyme expression during the production phase of fermentation to increase yields. Using these advances, we control the mitochondrial isobutanol pathway to produce up to 8.49 ± 0.31 g l-1 of isobutanol and 2.38 ± 0.06 g l-1 of 2-methyl-1-butanol micro-aerobically from glucose. These results make a compelling case for the application of optogenetics to metabolic engineering for the production of valuable products.

Assessing the efficiency of changes in land use for mitigating climate change.

Land-use changes are critical for climate policy because native vegetation and soils store abundant carbon and their losses from agricultural expansion, together with emissions from agricultural production, contribute about 20 to 25 per cent of greenhouse gas emissions1,2. Most climate strategies require maintaining or increasing land-based carbon3 while meeting food demands, which are expected to grow by more than 50 per cent by 20501,2,4. A finite global land area implies that fulfilling these strategies requires increasing global land-use efficiency of both storing carbon and producing food. Yet measuring the efficiency of land-use changes from the perspective of greenhouse gas emissions is challenging, particularly when land outputs change, for example, from one food to another or from food to carbon storage in forests. Intuitively, if a hectare of land produces maize well and forest poorly, maize should be the more efficient use of land, and vice versa. However, quantifying this difference and the yields at which the balance changes requires a common metric that factors in different outputs, emissions from different agricultural inputs (such as fertilizer) and the different productive potentials of land due to physical factors such as rainfall or soils. Here we propose a carbon benefits index that measures how changes in the output types, output quantities and production processes of a hectare of land contribute to the global capacity to store carbon and to reduce total greenhouse gas emissions. This index does not evaluate biodiversity or other ecosystem values, which must be analysed separately. We apply the index to a range of land-use and consumption choices relevant to climate policy, such as reforesting pastures, biofuel production and diet changes. We find that these choices can have much greater implications for the climate than previously understood because standard methods for evaluating the effects of land use4-11 on greenhouse gas emissions systematically underestimate the opportunity of land to store carbon if it is not used for agriculture.

Molecular mechanisms and highly functional development for stress tolerance of the yeast Saccharomyces cerevisiae.

In response to environmental stress, microorganisms adapt to drastic changes while exerting cellular functions by controlling gene expression, metabolic pathways, enzyme activities, and protein-protein interactions. Microbial cells that undergo a fermentation process are subjected to stresses, such as high temperature, freezing, drying, changes in pH and osmotic pressure, and organic solvents. Combinations of these stresses that continue over long terms often inhibit cells' growth and lead to their death, markedly limiting the useful functions of microorganisms (eg their fermentation ability). Thus, high stress tolerance of cells is required to improve productivity and add value to fermented/brewed foods and biofuels. This review focuses on stress tolerance mechanisms, including l-proline/l-arginine metabolism, ubiquitin system, and transcription factors, and the functional development of the yeast Saccharomyces cerevisiae, which has been used not only in basic science as a model of higher eukaryotes but also in fermentation processes for making alcoholic beverages, food products, and bioethanol.

The genome of the recently domesticated crop plant sugar beet (Beta vulgaris).

Sugar beet (Beta vulgaris ssp. vulgaris) is an important crop of temperate climates which provides nearly 30% of the world's annual sugar production and is a source for bioethanol and animal feed. The species belongs to the order of Caryophylalles, is diploid with 2n = 18 chromosomes, has an estimated genome size of 714-758 megabases and shares an ancient genome triplication with other eudicot plants. Leafy beets have been cultivated since Roman times, but sugar beet is one of the most recently domesticated crops. It arose in the late eighteenth century when lines accumulating sugar in the storage root were selected from crosses made with chard and fodder beet. Here we present a reference genome sequence for sugar beet as the first non-rosid, non-asterid eudicot genome, advancing comparative genomics and phylogenetic reconstructions. The genome sequence comprises 567 megabases, of which 85% could be assigned to chromosomes. The assembly covers a large proportion of the repetitive sequence content that was estimated to be 63%. We predicted 27,421 protein-coding genes supported by transcript data and annotated them on the basis of sequence homology. Phylogenetic analyses provided evidence for the separation of Caryophyllales before the split of asterids and rosids, and revealed lineage-specific gene family expansions and losses. We sequenced spinach (Spinacia oleracea), another Caryophyllales species, and validated features that separate this clade from rosids and asterids. Intraspecific genomic variation was analysed based on the genome sequences of sea beet (Beta vulgaris ssp. maritima; progenitor of all beet crops) and four additional sugar beet accessions. We identified seven million variant positions in the reference genome, and also large regions of low variability, indicating artificial selection. The sugar beet genome sequence enables the identification of genes affecting agronomically relevant traits, supports molecular breeding and maximizes the plant's potential in energy biotechnology.

Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform.

The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of brown macroalgae as feedstocks for commercial-scale fuel ethanol production, however, requires extensive re-engineering of the alginate and mannitol catabolic pathways in the standard industrial microbe Saccharomyces cerevisiae. Here we present the discovery of an alginate monomer (4-deoxy-L-erythro-5-hexoseulose uronate, or DEHU) transporter from the alginolytic eukaryote Asteromyces cruciatus. The genomic integration and overexpression of the gene encoding this transporter, together with the necessary bacterial alginate and deregulated native mannitol catabolism genes, conferred the ability of an S. cerevisiae strain to efficiently metabolize DEHU and mannitol. When this platform was further adapted to grow on mannitol and DEHU under anaerobic conditions, it was capable of ethanol fermentation from mannitol and DEHU, achieving titres of 4.6% (v/v) (36.2 g l(-1)) and yields up to 83% of the maximum theoretical yield from consumed sugars. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-added renewable chemicals in a manner that is comparable to traditional arable-land-based feedstocks.

Production of Methane, Hydrogen and Ethanol from Secale cereale L. Straw Pretreated with Sulfuric Acid.

The study describes sulfuric acid pretreatment of straw from Secale cereale L. (rye straw) to evaluate the effect of acid concentration and treatment time on the efficiency of biofuel production. The highest ethanol yield occurred after the enzyme treatment at a dose of 15 filter paper unit (FPU) per gram of rye straw (subjected to chemical hydrolysis with 2% sulfuric acid (SA) at 121 °C for 1 h) during 120 h. Anaerobic digestion of rye straw treated with 10% SA at 121 °C during 1 h allowed to obtain 347.42 L methane/kg volatile solids (VS). Most hydrogen was released during dark fermentation of rye straw after pretreatment of 2% SA, 121 °C, 1 h and 1% SA, 121 °C, 2 h-131.99 and 134.71 L hydrogen/kg VS, respectively. If the rye straw produced in the European Union were processed into methane, hydrogen, ethanol, the annual electricity production in 2018 could reach 9.87 TWh (terawatt-hours), 1.16 TWh, and 0.60 TWh, respectively.

Development of Robust Yeast Strains for Lignocellulosic Biorefineries Based on Genome-Wide Studies.

Lignocellulosic biomass has been widely studied as the renewable feedstock for the production of biofuels and biochemicals. Budding yeast Saccharomyces cerevisiae is commonly used as a cell factory for bioconversion of lignocellulosic biomass. However, economic bioproduction using fermentable sugars released from lignocellulosic feedstocks is still challenging. Due to impaired cell viability and fermentation performance by various inhibitors that are present in the cellulosic hydrolysates, robust yeast strains resistant to various stress environments are highly desired. Here, we summarize recent progress on yeast strain development for the production of biofuels and biochemical using lignocellulosic biomass. Genome-wide studies which have contributed to the elucidation of mechanisms of yeast stress tolerance are reviewed. Key gene targets recently identified based on multiomics analysis such as transcriptomic, proteomic, and metabolomics studies are summarized. Physiological genomic studies based on zinc sulfate supplementation are highlighted, and novel zinc-responsive genes involved in yeast stress tolerance are focused. The dependence of host genetic background of yeast stress tolerance and roles of histones and their modifications are emphasized. The development of robust yeast strains based on multiomics analysis benefits economic bioconversion of lignocellulosic biomass.

Sustainable bioenergy production from marginal lands in the US Midwest.

Legislation on biofuels production in the USA and Europe is directing food crops towards the production of grain-based ethanol, which can have detrimental consequences for soil carbon sequestration, nitrous oxide emissions, nitrate pollution, biodiversity and human health. An alternative is to grow lignocellulosic (cellulosic) crops on 'marginal' lands. Cellulosic feedstocks can have positive environmental outcomes and could make up a substantial proportion of future energy portfolios. However, the availability of marginal lands for cellulosic feedstock production, and the resulting greenhouse gas (GHG) emissions, remains uncertain. Here we evaluate the potential for marginal lands in ten Midwestern US states to produce sizeable amounts of biomass and concurrently mitigate GHG emissions. In a comparative assessment of six alternative cropping systems over 20 years, we found that successional herbaceous vegetation, once well established, has a direct GHG emissions mitigation capacity that rivals that of purpose-grown crops (-851 ± 46 grams of CO(2) equivalent emissions per square metre per year (gCO(2)e m(-2) yr(-1))). If fertilized, these communities have the capacity to produce about 63 ± 5 gigajoules of ethanol energy per hectare per year. By contrast, an adjacent, no-till corn-soybean-wheat rotation produces on average 41 ± 1 gigajoules of biofuel energy per hectare per year and has a net direct mitigation capacity of -397 ± 32 gCO(2)e m(-2) yr(-1); a continuous corn rotation would probably produce about 62 ± 7 gigajoules of biofuel energy per hectare per year, with 13% less mitigation. We also perform quantitative modelling of successional vegetation on marginal lands in the region at a resolution of 0.4 hectares, constrained by the requirement that each modelled location be within 80 kilometres of a potential biorefinery. Our results suggest that such vegetation could produce about 21 gigalitres of ethanol per year from around 11 million hectares, or approximately 25 per cent of the 2022 target for cellulosic biofuel mandated by the US Energy Independence and Security Act of 2007, with no initial carbon debt nor the indirect land-use costs associated with food-based biofuels. Other regional-scale aspects of biofuel sustainability, such as water quality and biodiversity, await future study.

Microbial engineering for the production of advanced biofuels.

Advanced biofuels produced by microorganisms have similar properties to petroleum-based fuels, and can 'drop in' to the existing transportation infrastructure. However, producing these biofuels in yields high enough to be useful requires the engineering of the microorganism's metabolism. Such engineering is not based on just one specific feedstock or host organism. Data-driven and synthetic-biology approaches can be used to optimize both the host and pathways to maximize fuel production. Despite some success, challenges still need to be met to move advanced biofuels towards commercialization, and to compete with more conventional fuels.

Exploiting diversity and synthetic biology for the production of algal biofuels.

Modern life is intimately linked to the availability of fossil fuels, which continue to meet the world's growing energy needs even though their use drives climate change, exhausts finite reserves and contributes to global political strife. Biofuels made from renewable resources could be a more sustainable alternative, particularly if sourced from organisms, such as algae, that can be farmed without using valuable arable land. Strain development and process engineering are needed to make algal biofuels practical and economically viable.

Lipase Immobilization on Silica Xerogel Treated with Protic Ionic Liquid and its Application in Biodiesel Production from Different Oils.

Treated silica xerogel with protic ionic liquid (PIL) and bifunctional agents (glutaraldehyde and epichlorohydrin) is a novel support strategy used in the effective immobilization of lipase from Burkholderia cepacia (LBC) by covalent binding. As biocatalysts with the highest activity recovery yields, LBC immobilized by covalent binding with epichlorohydrin without (203%) and with PIL (250%), was assessed by the following the hydrolysis reaction of olive oil and characterized biochemically (Michaelis⁻Menten constant, optimum pH and temperature, and operational stability). Further, the potential transesterification activity for three substrates: sunflower, soybean, and colza oils, was also determined, achieving a conversion of ethyl esters between 70 and 98%. The supports and the immobilized lipase systems were characterized using Fourier transform infrared spectra (FTIR), scanning electron microscopy (SEM), elemental analysis, and thermogravimetric (TG) analysis.

Physico-Chemical Conversion of Lignocellulose: Inhibitor Effects and Detoxification Strategies: A Mini Review.

A pretreatment of lignocellulosic biomass to produce biofuels, polymers, and other chemicals plays a vital role in the biochemical conversion process toward disrupting the closely associated structures of the cellulose-hemicellulose-lignin molecules. Various pretreatment steps alter the chemical/physical structure of lignocellulosic materials by solubilizing hemicellulose and/or lignin, decreasing the particle sizes of substrate and the crystalline portions of cellulose, and increasing the surface area of biomass. These modifications enhance the hydrolysis of cellulose by increasing accessibilities of acids or enzymes onto the surface of cellulose. However, lignocellulose-derived byproducts, which can inhibit and/or deactivate enzyme and microbial biocatalysts, are formed, including furan derivatives, lignin-derived phenolics, and carboxylic acids. These generation of compounds during pretreatment with inhibitory effects can lead to negative effects on subsequent steps in sugar flat-form processes. A number of physico-chemical pretreatment methods such as steam explosion, ammonia fiber explosion (AFEX), and liquid hot water (LHW) have been suggested and developed for minimizing formation of inhibitory compounds and alleviating their effects on ethanol production processes. This work reviews the physico-chemical pretreatment methods used for various biomass sources, formation of lignocellulose-derived inhibitors, and their contributions to enzymatic hydrolysis and microbial activities. Furthermore, we provide an overview of the current strategies to alleviate inhibitory compounds present in the hydrolysates or slurries.

Bio-Based Compounds from Grape Seeds: A Biorefinery Approach.

Food and agricultural waste represents a growing problem with negative effects on the economy, environment, and human health. Winemaking produces byproducts with high added value, which can be used for new productions in several application fields. From the perspective of biorefinery and circular economy, grape seeds could be exploited by extracting bioactive compounds with high added value before using biomass for energy purposes. The markets concerned are, in addition to the food, cosmetics, and pharmaceuticals sectors, which use bioactive compounds, the sector of biopolymeric materials and of energy for the production of biohydrogen and biomethane. Generally, bioactive components should be investigated through an integrated and multidisciplinary study approach based on emerging analytical techniques; in this context, attention is addressed towards green and sustainable procedures; an update of extraction techniques, innovative technologies, and chemometrics are described. Nowadays, processes so far tested on a pilot scale for grape waste are developed to enhance the extraction yields. Here, a picture of the Italian experience applied to the byproducts of the wine industry is given.