High-value Bioproducts Research Articles

AMSGE: advanced multiplex site-specific genome engineering with orthogonal modular recombinases in actinomycetes

Chromosomal integration of genes and pathways is of particular importance for large-scale and long-term fermentation in industrial biotechnology. However, stable, multi-copy integration of long DNA segments (e.g., large gene clusters) remains challenging. Here, we describe a plug-and-play toolkit that allows for high-efficiency, single-step, multi-locus integration of natural product (NP) biosynthetic gene clusters (BGCs) in actinomycetes, based on the innovative concept of “multiple integrases-multiple attB sites”. This toolkit consists of 27 synthetic modular plasmids, which contain single- or multi-integration modules (from two to four) derived from five orthogonal site-specific recombination (SSR) systems. The multi-integration modules can be readily ligated into plasmids containing large BGCs by Gibson assembly, which can be simultaneously inserted into multiple native attB sites in a single step. We demonstrated the applicability of this toolkit by performing stabilized amplification of acetyl-CoA carboxylase genes to facilitate actinorhodin biosynthesis in Streptomyces coelicolor. Furthermore, using this toolkit, we achieved a 185.6% increase in 5-oxomilbemycin titers (from 2.23 to 6.37 g/L) in Streptomyces hygroscopicus via the multi-locus integration of the entire 5-oxomilbemycin BGC (72 kb) (up to four copies). Compared with previously reported methods, the advanced multiplex site-specific genome engineering (aMSGE) method does not require the introduction of any modifications into host genomes before the amplification of target genes or BGCs, which will drastically simplify and accelerate efforts to improve NP production. Considering that SSR systems are widely distributed in a variety of industrial microbes, this novel technique also promises to be a valuable tool for the enhanced biosynthesis of other high-value bioproducts.

Pilot-scale isolation and characterization of extracellular polymeric substances (EPS) from cell-free medium of Spirulina sp. LEB-18 cultures under outdoor conditions

This objective of this work was to monitor the EPS production during the growth of Spirulina sp. LEB-18, evaluate the productivity and to characterize the exopolymers obtained on pilot-scale under outdoor conditions. The production of crude EPS occurred in all phases of biomass growth and was approximately ten folds higher than that biomass concentration of Spirulina sp. LEB-18 at the end cultivation, demonstrating the importance of the use of supernatant after harvesting of Spirulina to obtain high value bioproducts. The EPS extracted by Spirulina sp. LEB-18 are typically heteropolymers with one high molecular weight fraction (polysaccharides) with potential to be utilized as an alternative bioflocculant and another fraction of lower molecular mass (proteins). The presence of uronic acids, pyruvates and acyl groups of carbohydrates or carboxylic groups of amino acids in protein moiety is the main responsible for overall negative charge of EPS, which is also of biotechnological importance. Moreover, due to the pseudoplastic behavior of the solutions and high thermal stability, the obtained EPS can be widely applied in several industrial sectors, thus determining its technological and market potentiality.

Methacrylation of kraft lignin for UV-curable coatings: Process optimization using response surface methodology

Lignin, as the world's second most abundant renewable polymer, is an under-utilized sustainable resource. Most technical lignin, in particular kraft lignin (KL) – a byproduct from pulping process, is used as a fuel for recovery boilers. Thus, developing approaches to transform this under-utilized sustainable resource into a high-value bio-product, such as methacrylated lignin (ML) for UV-curable coatings, is of utmost importance to sustainability. In this work, methacrylated lignin (ML) was produced by methacrylation of KL, and the process was optimized using response surface methodology employing a central composite design (CCD). Three variables were examined and optimized via the CCD: reaction time, reaction temperature, and catalyst/lignin molar ratio, and their corresponding effects on the product yield were investigated. The mathematical model that was derived from the employed CCD was accurate in predicting the optimal reaction conditions. At the optimal reaction conditions (54 min, 54 °C and catalyst/lignin molar ratio of 0.225), the ML product yield of 146.5% was achieved. The ML materials were characterized using 1H NMR and FTIR spectroscopies, as well as thermogravimetric analysis. A UV-curable coating system containing 30 wt% ML with a hexafunctional siloxane-based crosslinker and photoinitiator was shown to be promising.

Harvesting effectiveness of Chlorella sp. biomass using different flocculation treatments of Moringa oleifera extract and pH conditions

Chlorella sp. is microalgae species that is commonly found in seawater. Chlorella biomass have been produced worldwide and considered as sources for commercial high value bioproducts and biofuels for the last few decades. However, its microscopic size and negatively charged cell surface causes the difficulties of harvesting process or separation of its biomass from the culture medium. The common techniques for harvesting microalgae biomass are centrifugation, filtration, and flocculation. The determination of efficient harvesting methods was the background of this study to lower the cost of harvesting. In this study, the flocculation harvesting method was used by applying several flocculant doses of moringa seeds extract (Moringa oleifera) and adjusted pH. Moringa seed was used as a flocculant to reduce the use of synthetic flocculant materials, especially metals, in order to achieve an environmentally friendly, more efficient, and safe biomass harvesting method for uses as aquaculture feed. The results showed that the moringa seeds are considered efficient for use as flocculants of Chlorella biomass. This flocculant achieved the optimum performance in the treatment of pH 11 with a dose of 0.15 grams moringa seeds flocculant in 1 Liter of Chlorella sp. culture.

Production of microbial mutan polysaccharide by expression of a mutansucrase gene (gtfI) in sugarcane

Due to its high productivity and sucrose content, sugarcane (Saccharum officinarum) is becoming the source of high-value bioproducts. Expression of bacterial extracellular polysaccharide genes in non-biopolymer accumulating plants is an excellent resource for production of added-value products. To this end, an expression cassette containing a full-length glucosyltransferase (gtfI) gene from Streptococcus downei driven by a CaMV promoter was expressed in a commercial sugarcane cultivar (CP48-103) using a biolistic approach. Copy number was assessed for a number of selected transgenic sugarcane lines by DNA blot analysis, where it was corroborated that each transgenic line contained at least two gtfI copies. The southern blot analysis of gtfI-expressing lines showed that the number of integrated copies ranged from two to four. The expression of gtfI in transgenic sugarcane plants was confirmed by mRNA blot analysis and qRT-PCR analysis. The expression of gtfI in transgenic sugarcane plants resulted in an approximate 30% reduction in sucrose accumulation, suggesting that mutansucrase actively converted sucrose to mutan polymer. In internodal stalk tissues, mutan polymer accumulated up to 55.9 mg/g FW, which apparent through glucan staining. The levels of glucose and fructose increased nearly by twofold, suggesting that mutansucrase may also have hydrolyzing activity.

Terpenes and isoprenoids: a wealth of compounds for global use.

Role of terpenes and isoprenoids has been pivotal in the survival and evolution of higher plants in various ecoregions. These products find application in the pharmaceutical, flavor fragrance, and biofuel industries. Fitness of plants in a wide range of environmental conditions entailed (i) evolution of secondary metabolic pathways enabling utilization of photosynthate for the synthesis of a variety of biomolecules, thereby facilitating diverse eco-interactive functions, and (ii) evolution of structural features for the sequestration of such compounds away from the mainstream primary metabolism to prevent autotoxicity. This review summarizes features and applications of terpene and isoprenoid compounds, comprising the largest class of secondary metabolites. Many of these terpene and isoprenoid biomolecules happen to be high-value bioproducts. They are essential components of all living organisms that are chemically highly variant. They are constituents of primary (quinones, chlorophylls, carotenoids, steroids) as well as secondary metabolism compounds with roles in signal transduction, reproduction, communication, climatic acclimation, defense mechanisms and more. They comprise single to several hundreds of repetitive five-carbon units of isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In plants, there are two pathways that lead to the synthesis of terpene and isoprenoid precursors, the cytosolic mevalonic acid (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway. The diversity of terpenoids can be attributed to differential enzyme and substrate specificities and to secondary modifications acquired by terpene synthases. The biological role of secondary metabolites has been recognized as pivotal in the survival and evolution of higher plants. Terpenes and isoprenoids find application in pharmaceutical, nutraceutical, synthetic chemistry, flavor fragrance, and possibly biofuel industries.

Functions of aldehyde reductases from Saccharomyces cerevisiae in detoxification of aldehyde inhibitors and their biotechnological applications.

Bioconversion of lignocellulosic biomass to high-value bioproducts by fermentative microorganisms has drawn extensive attentions worldwide. Lignocellulosic biomass cannot be efficiently utilized by microorganisms, such as Saccharomyces cerevisiae, but has to be pretreated prior to fermentation. Aldehyde compounds, as the by-products generated in the pretreatment process of lignocellulosic biomass, are considered as the most important toxic inhibitors to S. cerevisiae cells for their growth and fermentation. Aldehyde group in the aldehyde inhibitors, including furan aldehydes, aliphatic aldehydes, and phenolic aldehydes, is identified as the toxic factor. It has been demonstrated that S. cerevisiae has the ability to in situ detoxify aldehydes to their corresponding less or non-toxic alcohols. This reductive reaction is catalyzed by the NAD(P)H-dependent aldehyde reductases. In recent years, detoxification of aldehyde inhibitors by S. cerevisiae has been extensively studied and a huge progress has been made. This mini-review summarizes the classifications and structural features of the characterized aldehyde reductases from S. cerevisiae, their catalytic abilities to exogenous and endogenous aldehydes and effects of metal ions, chemical protective additives, and salts on enzyme activities, subcellular localization of the aldehyde reductases and their possible roles in protection of the subcellular organelles, and transcriptional regulation of the aldehyde reductase genes by the key stress-response transcription factors. Cofactor preference of the aldehyde reductases and their molecular mechanisms and efficient supply pathways of cofactors, as well as biotechnological applications of the aldehyde reductases in the detoxification of aldehyde inhibitors derived from pretreatment of lignocellulosic biomass, are also included or supplemented in this mini-review.

A biolistic-based genetic transformation system applicable to a broad-range of sugarcane and energycane varieties

ABSTRACTSugarcane and energycane (Saccharum spp. hybrids) are prominent sources of sugar, ethanol, as well as high-value bioproducts globally. Genetic analysis for trait improvement of sugarcane is greatly hindered by its complex genome, limited germplasm resources, long breeding cycle, as well as recalcitrance to genetic transformation. Here, we present a biolistic-based transformation and bioreactor-based micro-propagation system that has been utilized successfully to transform twelve elite cane genotypes, yielding transformation efficiencies of up to 39%. The system relies on the generation of embryogenic callus from sugarcane and energycane apical shoot tissue, followed by DNA bombardment of embryogenic leaf roll discs (approximately one week) or calli (approximately 4 weeks). We present optimal criteria and practices for selection and regeneration of independent transgenic lines, molecular characterization, as well as a bioreactor-based micro-propagation technique, which can aid in rapid multiplication and analysis of transgenic lines. The cane transformation and micro-propagation system described here, although built on our previous protocols, has significantly accelerated the process of producing and multiplying transgenic material, and it is applicable to other varieties. The system is highly reproducible and has been successfully used to engineer multiple commercial sugarcane and energycane varieties. It will benefit worldwide researchers interested in genomics and genetics of sugarcane photosynthesis, cell wall, and bioenergy related traits.

Hydrodynamics, mass transfer and cell growth characteristics in a novel microbubble stirred bioreactor employing sintered porous metal plate impeller as gas sparger

Stirred tank bioreactors are one of the most widely used bio-fermentation systems at both the laboratory and factory scale for the production of high value bioproducts. However, conventional systems suffer from irreconcilable contradictions to ensure adequate volumetric oxygen mass transfer rates (kLa) the necessary operating conditions to increase the energy consumption and the shear conditions. Adaptation of systems that incorporate microbubbles (MBs) generated by microporous sparger are advantageous for high viscosity and low shear stress bio-fermentation systems due to their superior kLa’s, high gas-liquid interfacial area to volume ratios, high gas holdup, and slow coalescence rates. Herein, we describe the development of a novel microbubble-based stirred tank bioreactor (MSTBR) using a sintered porous metal plate impeller as gas sparger that produces MBs in both Newtonian and non-Newtonian fluids. The MSTBR showed increased kLa and improved the gas holdup by forming an abundance of MBs, while avoiding the high shear stress and reducing the mixing energy consumption that are typically caused by utilization of Rushton turbine impellers. To validate the robustness of the MSTBR, arachidonic acid (ARA) -rich oil was produced from the shear-sensitive, high oxygen consuming filamentous fungus Mortierella alpine. The total ARA content produced using the MSTBR system was found to be 2.82 times that of the control bioreactor. Taken together, our data indicates that the new MSTBR bio-fermentation system can be broadly utilized in applications requiring elevated oxygen levels, shear-sensitive microorganisms, and high viscosity medium during the bio-fermentation process.

4-Hydroxybenzoic acid-a versatile platform intermediate for value-added compounds.

4-Hydroxybenzoic acid (4-HBA) has recently emerged as a promising intermediate for several value-added bioproducts with potential biotechnological applications in food, cosmetics, pharmacy, fungicides, etc. Over the past years, a variety of biosynthetic techniques have been developed for producing the 4-HBA and 4-HBA-based products. At this juncture, synthetic biology and metabolic engineering approaches enabled the biosynthesis of 4-HBA to address the increasing demand for high-value bioproducts. This review summarizes the biosynthesis of a variety of industrially pertinent compounds such as resveratrol, muconic acid, gastrodin, xiamenmycin, and vanillyl alcohol using 4-HBA as the starting feedstock. Moreover, potential research activities with a close-up look at the future perspectives to produce new compounds using 4-HBA have also been discussed.

Characterization of coffee (Coffea arabica) husk lignin and degradation products obtained after oxygen and alkali addition

The full use of biomass in future biorefineries has stimulated studies on utilization of lignin from agricultural crops, such as coffee husk, a major residue from coffee processing. This study focuses on characterizing the lignin obtained from coffee husk and its further wet oxidation products as a function of alkali loading, temperature and residence time. The lignin fraction after diluted acid and alkali pretreatments is composed primarily of p-hydroxylphenyl units (≥49%), with fewer guaiacyl and syringyl units. Linkages appear to be mainly β-O-4 ether linkages. Thermal degradation of pretreated lignin during wet oxidation occurred in two stages. Carboxylic acids were the main degradation product. Due to the condensed structure of this lignin, relatively low yields of aromatic aldehydes were achieved, except with temperatures over 210 °C, 5 min residence time and 11.7 wt% NaOH. Optimization of the pretreatment and oxidation parameters are important to maximizing yield of high-value bioproducts from lignin.

Biowaste Valorisation in a Future Circular Bioeconomy

The biowaste refinery concept has received significant attention in recent years as a sustainable alternative the petroleum refinery, exploiting the biowaste for producing high value bioproducts. However, waste-based biorefineries mainly apply homogeneous waste streams from agriculture and food processing as feedstock. This paper presents the state of the art of mixed-biowaste biorefinery concepts. We identified 20 studies that use the organic fraction of municipal solid waste (OFMSW) as feedstock, producing enzymes, bioplastics, biopesticides and other high value products. Valorisation efficiency (output product per kg OFMSW) and potential revenue by valorising the OFMSW into different products (Euro/ton OFMSW) was analysed for the identified studies. It was found that enzymes have the highest potential revenue followed by biopesticides and bioplastics. Developing biorefineries applying OFMSW as feedstock presents a promising opportunity for moving up the waste hierarch through coupling the waste and production sector in a future circular bioeconomy.

Rational engineering of photosynthetic electron flux enhances light-powered cytochrome P450 activity.

In this study, we exploited a modified photosynthetic electron transfer chain (PET) in the model cyanobacterium Synechococcus PCC 7002, where electrons derived from water-splitting are used to power reactions catalyzed by a heterologous cytochrome P450 (CYP1A1). A simple in vivo fluorescent assay for CYP1A1 activity was employed to determine the impact of rationally engineering of photosynthetic electron flow. This showed that knocking out a subunit of the type I NADH dehydrogenase complex (NDH-1), suggested to be involved in cyclic photosynthetic electron flow (ΔndhD2), can double the activity of CYP1A1, with a concomitant increase in the flux of electrons from photosynthesis. This also resulted in an increase in cellular adenosine triphosphate (ATP) and the ATP/nicotinamide adenine dinucleotide phosphate (NADPH) ratio, suggesting that expression of a heterologous electron sink in photosynthetic organisms can be used to modify the bioenergetic landscape of the cell. We therefore demonstrate that CYP1A1 is limited by electron supply and that photosynthesis can be re-engineered to increase heterologous P450 activity for the production of high-value bioproducts. The increase in cellular ATP achieved could be harnessed to support metabolically demanding heterologous processes. Furthermore, this experimental system could provide valuable insights into the mechanisms of photosynthesis.

Carbon fibre: preparing for the middle ground

Lignocellulosic biomass is the most abundant renewable feedstock on Earth, rich in polysaccharides and the phenolic polymer lignin. Valorization of renewable feedstocks delivers opportunities to improve the economics of advanced biofuels and high-value bioproducts. This chapter presents a general overview of lignocellulosic composition, presenting cellulose, hemicelluloses and their decorations, pectins, and lignin, and how the cell-wall polymers can be depolymerized into monomeric sugars. In addition, the fundamental knowledge and constraints on the biosynthesis of succinic acid (SA) and 3-hydroxypropionic acid by microbial platforms are integrated into industrial processing. Metabolic engineering of industrial strains can improve the productivity of SA and 3-hydroxypropionic acid and grow the biotransformation process toward biorefineries increasing the value derived from feedstocks.

Valorisation of chicken feathers: a review on recycling and recovery route—current status and future prospects

Worldwide, the poultry meat processing industry generates large quantities of feather by-products that amount to 40 × 109 kg annually. The feathers are considered wastes although small amounts are often processed into valuable products such as feather meal and fertilisers. The remaining waste is disposed of by incineration or by burial in controlled landfills. Improper disposal of these biological wastes contributes to environmental damage and transmission of diseases. Economic pressures, environmental pressures, increasing interest in using renewable and sustainable raw materials, and the need to decrease reliance on non-renewable petroleum resources behove the industry to find better ways of dealing with waste feathers. A closer look at the structure and composition of feathers shows that the whole part of a chicken feather (rachis and barb) can be used as a source of a pure structural protein called keratin which can be exploited for conversion into a number of high-value bioproducts. Additionally, several technologies can be used to convert other biological components of feathers into high value-added products. Thus, conversion of the waste into valuable products can make feathers an attractive raw material for the production of bioproducts. In this review, possible applications of chicken feathers in a variety of technologies and products are discussed. Thus, using waste feathers as a valuable resource can help the poultry industry to dispose of the waste feathers in an environmentally sustainable manner that also generates extra income for the industry. Their valorisation can result in their sustainable conversion into high-value materials and products on the proviso of existence or development of cost-effective technologies for converting this waste into the useful products.

Simultaneous production of intracellular triacylglycerols and extracellular polyol esters of fatty acids by Rhodotorula babjevae and Rhodotorula aff. paludigena.

Microbial oils have been analyzed as alternatives to petroleum. However, just a handful of microbes have been successfully adapted to produce chemicals that can compete with their petroleum counterparts. One of the reasons behind the low success rate is the overall economic inefficiency of valorizing a single product. This study presents a lab-scale analysis of two yeast species that simultaneously produce multiple high-value bioproducts: intracellular triacylglycerols (TG) and extracellular polyol esters of fatty acids (PEFA), two lipid classes with immediate applications in the biofuels and surfactant industries. At harvest, the yeast strain Rhodotorula aff. paludigena UCDFST 81-84 secreted 20.9±0.2g L-1 PEFA and produced 8.8±1.0g L-1 TG, while the yeast strain Rhodotorula babjevae UCDFST 04-877 secreted 11.2±1.6g L-1 PEFA and 18.5±1.7g L-1 TG. The overall glucose conversion was 0.24 and 0.22g(total lipid) g (glucose)-1 , respectively. The results present a stable and scalable microbial growth platform yielding multiple co-products.

Enhanced growth and total fatty acid production of microalgae under various lighting conditions induced by flashing light.

Microalgae are gaining importance as a source of high-value bioproducts. However, data regarding optimization of algal productivity via variation of environmental factors are lacking. Here, we evaluated a novel lighting method for the enhancement of biomass and total fatty acid (TFA) productivities during algal cultivation. We cultivated six different algal strains (Chlorella vulgaris KCTC AG10002, Acutodesmus obliquus KGE18, Uronema sp. KGE03, Micractinium reisseri KGE19, Fragilaria sp., and Spirogyra sp.) under various lighting conditions-continuous light (CL), light-dark cycle (LD), and continuous dark (CD)-with or without additional flashing light. We monitored dry cell weight (DCW) and TFA concentrations during cultivation. For each algal strain, the growth rate showed markedly different responses to the various lighting modes. The growth rates of C. vulgaris KCTC AG10002 (1.34-fold DCW increase, LD with flash), A. obliquus KGE18 (5.16-fold DCW increase, LD with flash), Uronema sp. KGE03 (2.77-fold DCW increase, CL with flash), and M. reisseri KGE19 (1.52-fold DCW increase, CL with flash) markedly increased in response to flashing light. Additionally, in some algal strains cultivated under the LD mode, the flashing light treatment induced increased TFA concentrations (C. vulgaris, 1.19-fold increase; A. obliquus, 2.59-fold increase; and M. reisseri, 3.31-fold increase). Phytohormone analysis of M. reisseri revealed increases in growth rate and TFA concentrations, associated with phytohormone induction via flashing light (e.g. 2.93-fold increase in gibberellic acid); hence, flashing light can promote substantial alterations in algal metabolism.

Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes.

ABSTRACTThe breakdown of plant biomass to simple sugars is essential for the production of second-generation biofuels and high-value bioproducts. Currently, enzymes produced from filamentous fungi are used for deconstructing plant cell wall polysaccharides into fermentable sugars for biorefinery applications. A post-translational N-terminal pyroglutamate modification observed in some of these enzymes occurs when N-terminal glutamine or glutamate is cyclized to form a five-membered ring. This modification has been shown to confer resistance to thermal denaturation for CBH-1 and EG-1 cellulases. In mammalian cells, the formation of pyroglutamate is catalyzed by glutaminyl cyclases. Using the model filamentous fungus Neurospora crassa, we identified two genes (qc-1 and qc-2) that encode proteins homologous to mammalian glutaminyl cyclases. We show that qc-1 and qc-2 are essential for catalyzing the formation of an N-terminal pyroglutamate on CBH-1 and GH5-1. CBH-1 and GH5-1 produced in a Δqc-1 Δqc-2 mutant, and thus lacking the N-terminal pyroglutamate modification, showed greater sensitivity to thermal denaturation, and for GH5-1, susceptibility to proteolytic cleavage. QC-1 and QC-2 are endoplasmic reticulum (ER)-localized proteins. The pyroglutamate modification is predicted to occur in a number of additional fungal proteins that have diverse functions. The identification of glutaminyl cyclases in fungi may have implications for production of lignocellulolytic enzymes, heterologous expression, and biotechnological applications revolving around protein stability.

Sugarcane-Biorefinery.

Concepts such as biorefinery and green chemistry focus on the usage of biomass, as with the oil value chain. However, it can cause less negative impact on the environment. A biorefinery based on sugarcane (Saccharum spp.) as feedstock is an example, because it can integrate into the same physical space, of processes for obtaining biofuels (ethanol), chemicals (from sugars or ethanol), electricity, and heat.The use of sugarcane as feedstock for biorefineries is dictated by its potential to supply sugars, ethanol, natural polymers or macromolecules, organic matter, and other compounds and materials. By means of conversion processes (chemical, biochemical, and thermochemical), sugarcane biomass can be transformed into high-value bioproducts to replace petrochemicals, as a bioeconomy model.

Valorisation to biogas of macroalgal waste streams: a circular approach to bioproducts and bioenergy in Ireland

Seaweeds (macroalgae) have been recently attracting more and more interest as a third generation feedstock for bioenergy and biofuels. However, several barriers impede the deployment of competitive seaweed-based energy. The high cost associated to seaweed farming and harvesting, as well as their seasonal availability and biochemical composition currently make macroalgae exploitation too expensive for energy production only. Recent studies have indicated a possible solution to aforementioned challenges may lay in seaweed integrated biorefinery, in which a bioenergy and/or biofuel production step ends an extractions cascade of high-value bioproducts. This results in the double benefit of producing renewable energy while adopting a zero waste approach, as fostered by recent EU societal challenges within the context of the Circular Economy development. This study investigates the biogas potential of residues from six indigenous Irish seaweed species while discussing related issues experienced during fermentation. It was found that Laminaria and Fucus spp. are the most promising seaweed species for biogas production following biorefinery extractions producing 187–195 mL CH4 gVS−1 and about 100 mL CH4 gVS−1 , respectively, exhibiting overall actual yields close to raw un-extracted seaweed.