Key Considerations for the Use of Seaweed to Reduce Enteric Methane Emissions From Cattle.

Enteric methane emissions are the single largest source of direct greenhouse gas emissions (GHG) in beef and dairy value chains and a substantial contributor to anthropogenic methane emissions globally. In late 2019, the World Wildlife Fund (WWF), the Advanced Research Projects Agency-Energy (ARPA-E) and the Foundation for Food and Agriculture Research (FFAR) convened approximately 50 stakeholders representing research and production of seaweeds, animal feeds, dairy cattle, and beef and dairy foods to discuss challenges and opportunities associated with the use of seaweed-based ingredients to reduce enteric methane emissions. This Perspective article describes the considerations identified by the workshop participants and suggests next steps for the further development and evaluation of seaweed-based feed ingredients as enteric methane mitigants. Although numerous compounds derived from sources other than seaweed have been identified as having enteric methane mitigation potential, these mitigants are outside the scope of this article.

Correction to: Molecular, biochemical and kinetic analysis of a novel, thermostable lipase (LipSm) from Stenotrophomonas maltophilia Psi­1, the first member of a new bacterial lipase family (XIX).

[This corrects the article DOI: 10.1186/s40709-018-0074-6.].

Molecular, biochemical and kinetic analysis of a novel, thermostable lipase (LipSm) from Stenotrophomonas maltophilia Psi-1, the first member of a new bacterial lipase family (XVIII).

BACKGROUND: Microbial lipases catalyze a broad spectrum of reactions and are enzymes of considerable biotechnological interest. The focus of this study was the isolation of new lipase genes, intending to discover novel lipases whose products bear interesting biochemical and structural features and may have a potential to act as valuable biocatalysts in industrial applications. RESULTS: A novel lipase gene (lipSm), from a new environmental Stenotrophomonas maltophilia strain, Psi-1, originating from a sludge sample from Psittaleia (Greece), was cloned and sequenced. lipSm was further overexpressed in E. coli BL21(DE3) and the overproduced enzyme LipSm was purified and analyzed in respect to its biochemical and kinetic properties. In silico analysis of LipSm revealed that it is taxonomically related to several uncharacterized lipases from different genera, which constitute a unique clade, markedly different from all other previously described bacterial lipase families. All members of this clade displayed identical, conserved consensus sequence motifs, i.e. the catalytic triad (S, D, H), and an unusual, amongst bacterial lipases, Y-type oxyanion hole. 3D-modeling revealed the presence of a lid domain structure, which allows LipSm to act on small ester substrates without interfacial activation. In addition, the high percentage of alanine residues along with the occurrence of the AXXXA motif nine times in LipSm suggest that it is a thermostable lipase, a feature verified experimentally, since LipSm was still active after heating at 70 °C for 30 min. CONCLUSIONS: The phylogenetic analysis of LipSm suggests the establishment of a new bacterial lipase family (XVIII) with LipSm being its first characterized member. Furthermore, LipSm is alkaliphilic, thermostable and lacks the requirement for interfacial activation, when small substrates are used. These properties make LipSm a potential advantageous biocatalyst in industry and biotechnology.

Novel FRET-substrates of Rhizomucor pusillus rennin: Activity and mechanistic studies.

The development of sensitive, easy and reliable methods for the determination of Rhizomucor pusillus rennin (MPR) activity, in free and in immobilized form, along with the elucidation of the mechanism of action, represent challenges for the widespread use of the enzyme in industrial cheese production. These could be accomplished by using highly specific and sensitive substrates, as well as direct assay methods. We designed and synthesized novel substrates based on Fluorescence Resonance Energy Transfer (FRET) for the MPR by employing computational simulation techniques and peptide synthesis in liquid phase. Three FRET-substrates (Abz-GFY-pNA, Abz-SFY-pNA and Abz-GFI-pNA) were found active, while the Abz-GFY-pNA showed the highest reliability, sensitivity and specificity among them. Subsequently, a novel mechanism of MPR action was elucidated, with the development of novel methods for assaying activity in free and immobilized form, which both may contribute in the wider use of rennin in cheese production and other biotechnological applications.

Scale-up for esters production from straw whiskers for biofuel applications.

Delignified wheat straw was fermented by a mixed bacterial anaerobic culture obtained from a UASB reactor to produce organic acids (OAs). Kissiris was used as immobilization carrier in a 2-compartment 82L bioreactor filled with 17L of fermentation broth for the first 7 fermentation batches and up to 40L for the subsequent batches. The amount of straw used was 30g/L and the temperature was set at 37°C for all experiments. The total OAs reached concentrations up to 17.53g/L and the produced ethanol ranged from 0.3 to 1mL/L. The main OAs produced was acetic acid (6-8g/L) and butyric acid (3-8g/L). The OAs were recovered from the fermentation broth by a downstream process using 1-butanol, which was the solvent with the best recovery yields and also served as the esterification alcohol. The enzymatic esterification of OAs resulted to 90% yield.

Sustainable production of a new generation biofuel by lipase-catalyzed esterification of fatty acids from liquid industrial waste biomass.

In this work we suggest a methodology comprising the design and use of cost-effective, sustainable, and environmentally friendly process for biofuel production compatible with the market demands. A new generation biofuel is produced using fatty acids, which were generated from acidogenesis of industrial wastes of bioethanol distilleries, and esterified with selected alcohols by immobilized Candida antarctica Lipase-B. Suitable reactors with significant parameters and conditions were studied through experimental design, and novel esterification processes were suggested; among others, the continuous removal of the produced water was provided. Finally, economically sustainable biofuel production was achieved providing high ester yield (<97%) along with augmented concentration (3.35M) in the reaction mixtures at relatively short esterification times, whereas the immobilized lipase maintained over 90% of its initial esterifying ability after reused for ten cycles.

Kinetic and computational analysis of the reversible inhibition of porcine pancreatic elastase: a structural and mechanistic approach.

Structural and mechanistic insights were revealed for the reversible inhibition of Porcine Pancreatic Elastase (PPE); the kinetics of uninhibited and inhibited hydrolysis of substrate Suc-AAA-pNA was analyzed thoroughly. Additionally, the interactions between PPE and its inhibitor were studied by computational techniques. The uninhibited hydrolysis of Suc-AAA-pNA by PPE proceeds through a virtual transition state, involving an inferior physical and another dominating chemical step, where two stabilized reactant states precede the predominant acyl-enzyme. Different kinds of bonding with the PPE-backbone residues, including those of the catalytic triad, were found during the MD simulation of 5 ns, as key interactions favoring a higher stabilization of the best ranked complex PPE-CF3C(O)-KA-NHPh-p-CF3. The proton inventories of the inhibited hydrolysis of Suc-AAA-pNA by PPE, were ruled out the existence of any virtual transition state and thus they argue for a different mode of catalysis involving a structurally disturbed PPE molecule. Thereafter, a novel inhibition mechanism was suggested.

Economic evaluation of technology for a new generation biofuel production using wastes.

An economic evaluation of an integrated technology for industrial scale new generation biofuel production using whey, vinasse, and lignocellulosic biomass as raw materials is reported. Anaerobic packed-bed bioreactors were used for organic acids production using initially synthetic media and then wastes. Butyric, lactic and acetic acid were predominately produced from vinasse, whey, and cellulose, respectively. Mass balance was calculated for a 16,000L daily production capacity. Liquid-liquid extraction was applied for recovery of the organic acids using butanol-1 as an effective extraction solvent which serves also as the alcohol for the subsequent enzyme-catalyzed esterification. The investment needed for the installation of the factory was estimated to about 1.7million€ with depreciation excepted at about 3months. For cellulosics, the installation investment was estimated to be about 7-fold higher with depreciation at about 1.5years. The proposed technology is an alternative trend in biofuel production.

Optimization of the production of extracellular α-amylase by Kluyveromyces marxianus IF0 0288 by response surface methodology

The aim of this work was to study the production of extracellular α-amylase by Kluyveromyces marxianus IF0 0288 using optimized nutritional and cultural conditions in a complex yeast medium under aerobic batch fermentation. By applying the conventional "one-variable-at-a-time" approach and the response surface methodology, the effect of four fermentation parameters (type of carbon source, initial culture pH, temperature, and incubation time) on the growth and α-amylase production was evaluated. The production of α-amylase during 60 h of fermentation increased 13-fold under optimized conditions (1% starch, pH 6.0, 30ºC) in comparison to the conventional optimization method. The initial pH value of 6.13 and temperature of 30.3ºC were optimal conditions by the response surface methodology, leading to further improvement (up to 13-fold) in the production of extracellular α-amylase. These results constituted first evidence that K. marxianus could be potentially used as an effective source of extracellular α-amylase.

Advances in lipase-catalyzed esterification reactions.

Lipase-catalyzed esterification reactions are among the most significant chemical and biochemical processes of industrial relevance. Lipases catalyze hydrolysis as well as esterification reactions. Enzyme-catalyzed esterification has acquired increasing attention in many applications, due to the significance of the derived products. More specifically, the lipase-catalyzed esterification reactions attracted research interest during the past decade, due to an increased use of organic esters in biotechnology and the chemical industry. Lipases, as hydrolyzing agents are active in environments, which contain a minimum of two distinct phases, where all reactants are partitioned between these phases, although their distribution is not fixed and changes as the reaction proceeds. The kinetics of the lipase-catalyzed reactions is governed by a number of factors. This article presents a thorough and descriptive evaluation of the applied trends and perspectives concerning the enzymatic esterification, mainly for biofuel production; an emphasis is given on essential factors, which affect the lipase-catalyzed esterification reaction. Moreover, the art of using bacterial and/or fungal strains for whole cell biocatalysis purposes, as well as carrying out catalysis by various forms of purified lipases from bacterial and fungal sources is also reviewed.

Purification, kinetic characterization and properties of a novel thermo-tolerant extracellular protease from Kluyveromyces marxianus IFO 0288 with potential biotechnological interest.

A novel extracellular hydrolase of ∼45kDa molecular mass was purified from Kluyveromyces marxianus IFO 0288 cultures and characterized as serine protease. The K(m)-value of protease (designated protease-KM-IFO-0288-A), which was found active in media containing elevated [NaCl] but lacking EDTAK(2), decreased with increasing [Ca(2+)]. The protease maintained considerable activity at the range of 10-60°C and pH 6.00-10.25, with optimum k(cat)/K(m)-value at 35.5°C and pH 7.75. It was strongly affected by specific irreversible inhibitors of serine proteases while was unaffected by inhibitors of cysteine proteases. Significant rate constants, activation energies, and proton inventories were estimated from the profiles of Michaelis-Menten parameters, versus pH, temperature and deuterium atom fraction, in the hydrolysis of Suc-AAPF-pNA showing that protease-KM-IFO-0288-A performs catalysis via a charge-relay system. The properties of protease-KM-IFO-0288-A suggest that K. marxianus represents a valuable source of extracellular protease of biotechnological interest which, given its GRAS status, could find several important applications.

Experimental investigation and optimization of process variables affecting the production of extracellular lipase by Kluyveromyces marxianus IFO 0288.

In this study, the production and optimization of extracellular lipase from Kluyveromyces marxianus IFO 0288 was investigated by using optimized nutritional and cultural conditions in a yeast medium containing glucose as the carbon source in fully aerobic batch fermentation (150 rpm). The influence of four fermentation parameters (type of lipidic source, initial culture pH, temperature, and length of fermentation) on growth and lipase production was investigated and evaluated using the conventional "one variable at a time" approach and response surface methodology. An 18-fold increase in lipase production during 65 h of fermentation was obtained with optimized nutritional (0.5 % olive oil) and cultivation (pH 6.5, 35 °C) conditions by employing the conventional optimization method. By applying the response surface methodology technique the initial pH value of 6.4 and temperature of 32.5 °C were identified as optimal and led to further improvements (up to 18-fold) of extracellular lipase production. The results provide, for the first time, evidence that K. marxianus has the potential to be used as an efficient producer of extracellular lipase with prospective application in a variety of industrial and biotechnological areas.