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At least 181 records · Page 10

Combining biotechnology with sustainability: feasibility of bioenergy sorghum in generating high-value bioproducts

Biofuels generated through fermentation of plant-derived lignocellulosic biomass are currently not competitive with fossil fuels. Here, a framework is described for the engineering of valuable coproducts to improve the economic feasibility of biofuel production. To accomplish this goal, the desirable traits of the bioenergy crop Sorghum bicolor are harnessed via modern engineering approaches. A robust analysis is provided to highlight the scientific basis, costs, regulatory challenges, and lucrative nature of coproduct development. Sustainable biofuel production could also have far-reaching societal impacts, especially in the context of our rapidly changing global climate. This perspective suggests that the path to profitable biofuel production is attainable and encourages consideration of similar approaches by the biotechnology industry.

09 BIOMASS FUELS↗

Impact of europium and samarium on growth and metabolism of two anaerobic wastewater microorganisms: Sporacetigenium mesophilum and Clostridium sporogenes

Increases in environmental fluxes of rare earth elements (REEs) stemming from increased demand for these technology critical elements may affect microbial ecosystem functions important for pollutant degradation and nutrient cycling. Here, this study investigates the responses of Sporacetigenium mesophilum and Clostridium sporogenes, two anaerobic fermenters, to varying concentrations of two REE, namely europium and samarium. Eu and Sm are adjacent lanthanides but differ significantly in their redox behavior. We tested Eu and Sm concentrations ranging from < 1 to 600 µM. Despite the close phylogenetic relatedness of the two bacteria, we observed species-specific as well as REE-specific and incubation time-dependent sensitivities. S. mesophilum exhibited pronounced inhibition of hydrogen production and growth with exposure to ≥ 60 µM of Eu, but with 6 µM Eu, a hormetic effect was observed—hydrogen production was enhanced relative to the control. With Sm, the only impact observed on S. mesophilum was inhibition at the highest concentration (600 µM) tested. For C. sporogenes, growth inhibition was observed only at 600 µM Eu or Sm, and no hormesis was observed with either REE. This study offers both significant benefits and novelty by addressing the emerging environmental concern of REE pollution, particularly the effects of Eu and Sm on anaerobic microorganisms relevant to wastewater treatment. It provides valuable insights into REE impacts under anaerobic conditions, which are essential for understanding potential disruptions to organic matter degradation and nutrient cycling processes.

60 - APPLIED LIFE SCIENCES↗

Long-term effects of cycle time and volume exchange ratio on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production from food waste digestate by Haloferax mediterranei cultivated in sequencing batch reactors for 450 days

Food waste digestate was fed into a sequencing batch reactor (SBR) for Haloferax mediterranei (HM) to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). This SBR was operated uninterruptedly for 450 days to test its stability, during which the cycle time and volume exchange ratio were varied to understand their impacts on the PHBV fermentation performance under ranged organic loading rates (OLR). Results showed that 1) PHBV productivity was proportional to OLR of food waste digestate; 2) substrate and product inhibitions were two limiting factors constraining substrate utilization and PHBV yields; 3) PHBV titer was dependent on the hydraulic retention time of the SBR while a volume exchange ratio lower than 0.5 is unfavorable due to the product inhibitor accumulation. Furthermore, this study for the first time demonstrated that the long-term stability of food waste-fed PHBV production by HM and revealed that inhibition effects could be barriers in SBR limiting the full-scale application of the technology.

09 BIOMASS FUELS↗

A “Sweet” Biorefinery: Sugar-derived ionic liquids for the pretreatment of lignocellulosic biomass

Sugar-based ionic liquids (ILs) derived from biomass-based cations (choline, betaine) and sugar-derived anions were developed as sustainable pretreatment solvents to advance the concept of self-reliant biorefinery. Cholinium gluconate ([Ch][GlcA]) enabled streamlined one-pot sorghum pretreatment without the need for pH adjustment or washing, while betainium gluconate ([Bet][GlcA]) achieved higher sugar yields, improving overall process efficiency and economics. Both ILs produced hydrolysates fully compatible with microbial fermentation, demonstrating their potential for efficient, simplified biomass conversion. This work positions sugar-based ILs as powerful platforms that unite sustainable chemistry with integrated bioprocessing, marking a pivotal step toward realizing the self-reliant biorefinery.

Biomass-based ions↗

Engineering Clostridium Tyrobutyricum for High Butanol Production through Induction Expression of Exogenous NADPH-Dependent HBD

Clostridium tyrobutyricum Δ cat 1:: adh E2 is a promising cell factory for butanol production because of its robustness, high butanol tolerance, and minimal butyrate production. However, excessive acetate and ethanol production remains a major bottleneck limiting its butanol yield. Coexpressing an exogenous hbd ( Ck ) encoding the NADPH-dependent 3-hydroxybutyryl-CoA dehydrogenase (HBD) from Clostridium kluyveri with adh E2 could increase the C4 carbon flux, resulting in increased butanol and decreased acetate and ethanol production. However, constitutively overexpressing hbd ( Ck ) in Δ cat 1:: adh E2 shows little improvement in butanol yield, productivity, and selectivity, which might be caused by redox imbalance and growth inhibition. To alleviate this problem, C. tyrobutyricum MΔ cat 1:: adh E2- Pbgal-hbd ( Ck ) with a dynamic expression of hbd ( Ck ) controlled by an inducible promoter was developed. In serum bottle fermentation at 37 °C, when the hbd ( Ck ) expression was induced at 12 h or in the early exponential phase, butanol production increased ∼20% in yield (from 0.22 to 0.27 g/g glucose), 87.5% in productivity (from 0.16 to 0.30 g/L·h), and 52% in selectivity (from 0.46 to 0.70 g/g total products) compared to the control strain without expressing any hbd(Ck), whereas hbd ( Ck ) expression induced at 0 or 24 h in MΔ cat 1:: adh E2-P bgal - hbd ( Ck ) or constitutively in MΔ cat 1:: adh E2-P cat 1- hbd ( Ck ) showed significantly lower butanol yield and productivity. At 25 °C, MΔ cat 1:: adh E2-P bgal - hbd ( Ck ) with 12 h induction produced the highest butanol titer of 23 g/L with 0.32 g/g yield, 0.16 g/L·h productivity, and 0.83 g/g product selectivity due to much reduced acetate formation. Subsequent scale-up to a stirred-tank bioreactor at 37 °C increased productivity to 0.39 g/L·h while also achieving high butanol titer (21.8 g/L), yield (0.30 g/g), and selectivity (0.67 g/g). The optimized induction timing resulted in a balanced NAD(P)H pool, effectively channeling substrates toward butanol biosynthesis. It was concluded that the timing for hbd ( Ck ) expression was critical as it affected glucose catabolism, cell growth, redox balance, and carbon flux distribution. These findings underscore the potential of dynamic metabolic regulation to overcome bottlenecks in biobutanol production, providing a scalable and economically viable bioprocess for industrial application.

Clostridium tyrobutyricum↗

Phenotypic and genomic characterization of Methanothermobacter wolfeii strain BSEL, a CO 2 -capturing archaeon with minimal nutrient requirements

A new variant of Methanothermobacter wolfeii was isolated from an anaerobic digester using enrichment cultivation in anaerobic conditions. Here, the new isolate was taxonomically identified via 16S rRNA gene sequencing and tagged as M. wolfeii BSEL. The whole genome of the new variant was sequenced and de novo assembled. Genomic variations between the BSEL strain and the type strain were discovered, suggesting evolutionary adaptations of the BSEL strain that conferred advantages while growing under a low concentration of nutrients. M. wolfeii BSEL displayed the highest specific growth rate ever reported for the wolfeii species (0.27 ± 0.03 h –1 ) using carbon dioxide (CO 2 ) as unique carbon source and hydrogen (H 2 ) as electron donor. M. wolfeii BSEL grew at this rate in an environment with ammonium (NH 4 + ) as sole nitrogen source. The minerals content required to cultivate the BSEL strain was relatively low and resembled the ionic background of tap water without mineral supplements. Optimum growth rate for the new isolate was observed at 64°C and pH 8.3. In this work, it was shown that wastewater from a wastewater treatment facility can be used as a low-cost alternative medium to cultivate M. wolfeii BSEL. Continuous gas fermentation fed with a synthetic biogas mimic along with H 2 in a bubble column bioreactor using M. wolfeii BSEL as biocatalyst resulted in a CO 2 conversion efficiency of 97% and a final methane (CH 4 ) titer of 98.5%v, demonstrating the ability of the new strain for upgrading biogas to renewable natural gas.

09 BIOMASS FUELS↗

Clostridium autoethanogenum alters cofactor synthesis, redox metabolism, and lysine-acetylation in response to elevated H 2 :CO feedstock ratios for enhancing carbon capture efficiency

Clostridium autoethanogenum is an acetogenic bacterium that autotrophically converts carbon monoxide (CO) and carbon dioxide (CO 2 ) gases into bioproducts and fuels via the Wood–Ljungdahl pathway (WLP). To facilitate overall carbon capture efficiency, the reaction stoichiometry requires supplementation of hydrogen at an increased ratio of H 2 :CO to maximize CO 2 utilization; however, the molecular details and thus the ability to understand the mechanism of this supplementation are largely unknown. In order to elucidate the microbial physiology and fermentation where at least 75% of the carbon in ethanol comes from CO 2 , we established controlled chemostats that facilitated a novel and high (11:1) H 2 :CO uptake ratio. We compared and contrasted proteomic and metabolomics profiles to replicate continuous stirred tank reactors (CSTRs) at the same growth rate from a lower (5:1) H 2 :CO condition where ~ 50% of the carbon in ethanol is derived from CO 2 . Our hypothesis was that major changes would be observed in the hydrogenases and/or redox-related proteins and the WLP to compensate for the elevated hydrogen feed gas. Our analyses did reveal protein abundance differences between the two conditions largely related to reduction–oxidation (redox) pathways and cofactor biosynthesis, but the changes were more minor than we would have expected. While the Wood–Ljungdahl pathway proteins remained consistent across the conditions, other post-translational regulatory processes, such as lysine-acetylation, were observed and appeared to be more important for fine-tuning this carbon metabolism pathway. Metabolomic analyses showed that the increase in H 2 :CO ratio drives the organism to higher carbon dioxide utilization resulting in lower carbon storages and accumulated fatty acid metabolite levels. This research delves into the intricate dynamics of carbon fixation in C. autoethanogenum, examining the influence of highly elevated H 2 :CO ratios on metabolic processes and product outcomes. The study underscores the significance of optimizing gas feed composition for enhanced industrial efficiency, shedding light on potential mechanisms, such as post-translational modifications (PTMs), to fine-tune enzymatic activities and improve desired product yields.

09 BIOMASS FUELS↗

Cultivation of novel Atribacterota from oil well provides new insight into their diversity, ecology, and evolution in anoxic, carbon-rich environments

Background: The Atribacterota are widely distributed in the subsurface biosphere. Recently, the first Atribacterota isolate was described and the number of Atribacterota genome sequences retrieved from environmental samples has increased significantly; however, their diversity, physiology, ecology, and evolution remain poorly understood. Results: We report the isolation of the second member of Atribacterota, Thermatribacter velox gen. nov., sp. nov., within a new family Thermatribacteraceae fam. nov., and the short-term laboratory cultivation of a member of the JS1 lineage, Phoenicimicrobium oleiphilum HX-OS.bin.34 TS , both from a terrestrial oil reservoir. Physiological and metatranscriptomics analyses showed that Thermatribacter velox B11 T and Phoenicimicrobium oleiphilum HX-OS.bin.34 TS ferment sugars and n-alkanes, respectively, producing H 2 , CO 2 , and acetate as common products. Comparative genomics showed that all members of the Atribacterota lack a complete Wood-Ljungdahl Pathway (WLP), but that the Reductive Glycine Pathway (RGP) is widespread, indicating that the RGP, rather than WLP, is a central hub in Atribacterota metabolism. Ancestral character state reconstructions and phylogenetic analyses showed that key genes encoding the RGP (fdhA, fhs, folD, glyA, gcvT, gcvPAB, pdhD) and other central functions were gained independently in the two classes, Atribacteria (OP9) and Phoenicimicrobiia (JS1), after which they were inherited vertically; these genes included fumarate-adding enzymes (faeA; Phoenicimicrobiia only), the CODH/ACS complex (acsABCDE), and diverse hydrogenases (NiFe group 3b, 4b and FeFe group A3, C). Finally, we present genome-resolved community metabolic models showing the central roles of Atribacteria (OP9) and Phoenicimicrobiia (JS1) in acetate- and hydrocarbon-rich environments. Conclusion: Our findings expand the knowledge of the diversity, physiology, ecology, and evolution of the phylum Atribacterota. This study is a starting point for promoting more incisive studies of their syntrophic biology and may guide the rational design of strategies to cultivate them in the laboratory.

59 BASIC BIOLOGICAL SCIENCES↗

Zero-Carbon Biofuels: An Optimized Two-Stage System for High Productivity Conversion of CO2 to Liquid Fuels

In MIT’s Zero-Carbon Biofuels work, under ARPAe’s EcoSynBio funding, we capitalized on our previously developed technology of the oleaginous yeast, Yarrowia lipolytica, engineered to produce high titer biodiesel-like lipids and alkanes, and set out to reduce or eliminate CO 2 generation during lipid production. We set out to do this by both engineering Y. lipolytica with the necessary enzymes to allow the generation of reducing equivalents from H 2 , formic acid, or methanol, and, in parallel, installing the non-oxidative glycolysis (NOG) pathway. The goal was that combination of these two would enable stoichiometric conversion of glucose and acetate to biodiesel products. Furthermore, our ARPAe Electrofuels funded research, yielded a very innovative two-stage system where gases comprising CO 2 and H 2 are used for the production of liquid fuels. Acetate is produced from CO 2 and H 2 , via acetogenic fermentation, in the first stage as the intermediary product, then fed to Y. lipolytica in the second stage, for conversion to lipids and alkanes. To increase the commercial competitiveness of the two-stage system, by improvement to the overall productivity, we recently showed that synergistic substrate co-feeding drastically enhances CO 2 fixation rates and set out to explore additional co-substrate pairs, including methanol and formic acid, in order to maximize acetate productivity. Finally, given the attractiveness as a biofuel, we also set out to attempt an important increase in the production of alkanes over lipids in Yarrowia.

10 SYNTHETIC FUELS↗

Feedstock/Pretreatment Screening for Bioconversion of Sugars and Lignin Residues

This project will conduct biomass deconstruction (pretreatment and enzymatic saccharification) on two representative biomass feedstocks and four different pretreatment processes, including three high temperature steam/chemical pretreatments and one low temperature chemical/mechanical pretreatment. A third biomass feedstock will undergo biomass deconstruction with three different pretreatment processes, including two high temperature chemical pretreatments and one low temperature chemical/mechanical pretreatment. Several pretreatment conditions will be performed in a screening study using NLR pilot-scale pretreatment equipment to generate a range of pretreated biomass substrates. A selected number of these substrates will be chosen for enzymatic saccharification evaluation, based on standard compositional analysis of the pretreated substrates as a primary indicator of pretreatment efficacy. Resulting enzymatic hydrolysis slurries will be analyzed to determine overall biomass sugar yields. Additional compositional analysis will be performed to determine oligomeric sugar composition and structure, to analyze structural characteristics of solids fractions on native, pretreated, and enzymatically saccharified biomass residues for one of the biomass feedstocks, corn stover. The enzymatically saccharified materials will undergo 2,3-butanediol fermentation in a shaker flask as bench scale. Using relevant process performance data collected in these various conversion steps, technoeconomic analysis activities will be performed to compare the economic potential of the various biomass feedstock and pretreatment processes and to identify key economic drivers and sustainability metrics.

09 BIOMASS FUELS↗

Biohydrogen: prospects for industrial utilization and energy resiliency in rural communities

Biohydrogen (bioH 2 ) production in rural regions of the United States leveraged from existing biomass waste streams serves two extant needs: rural energy resiliency and decarbonization of heavy industry, including the production of ammonia and other H 2 -dependent nitrogenous products. We consider bioH 2 production using two different strategies: (1) dark fermentation (DF) and (2) anaerobic digestion followed by steam methane reforming of the biogas (AD-SMR). Production of bioH 2 from biomass waste streams is a potentially ‘greener’ pathway in comparison to natural gas-steam methane reforming (NG-SMR), especially as fugitive emissions from these wastes are avoided. It also provides a decarbonizing potential not found in water-splitting technologies. Based on literature on DF and AD of crop residues, woody biomass residues from forestry wastes, and wastewaters containing fats, oils, and grease (FOG), we outline scenarios for bioH 2 production and displacement of fossil fuel derived methane. Finally, we compare the costs and carbon intensity (CI) of bioH 2 production with those of other H 2 production pathways.

08 HYDROGEN↗

Gramicidin S production by Bacillus brevis in simulated microgravity

In a continuing study of microbial secondary metabolism in simulated microgravity, we have examined gramicidin S (GS) production by Bacillus brevis strain Nagano in NASA High Aspect Rotating Vessels (HARVs), which are designed to simulate some aspects of microgravity. Growth and GS production were found to occur under simulated microgravity. When performance under simulated microgravity was compared with that under normal gravity conditions in the bioreactors, GS production was found to be unaffected by simulated microgravity. The repressive effect of glycerol in flask fermentations was not observed in the HARV. Thus the negative effect of glycerol on specific GS formation is dependent on shear and/or vessel geometry, not gravity.

NASA Discipline Number 04-10↗

Bionutrients: Microbial Production of on-Demand Nutrients on the International Space Station

Providing adequate nutrition to crew members is essential as deep-space missions cannot rely on consistent Earth-based resupply. The current NASA pre-packaged food system is designed for low-earth-orbit missions with a stated shelf-life of two years and notable vitamin degradation over time. One strategy to mitigate nutrient loss is to implement bioregenerative food sources to supplement the pre-packaged food system. The BioNutrients project is designed to provide targeted production of short shelf-life nutrients in a single-use production pack. BioNutrients-1 includes two strains of edible yeast that have been genetically engineered to produce carotenoids, β-carotene, and zeaxanthin. This five-year mission on the International Space Station (ISS), launched in 2019, tests the long-term storage and nutrient production of the microorganisms of interest. Additional microorganisms are also stored in stasis packs to determine the effects of long-duration storage on the ISS. These organisms may be useful for production of fermented foods, pharmaceuticals, or biomanufacturing processes. Improving on the BioNutrients-1 project, BioNutrients-2 has optimized the production pack hardware by reducing the overall mass and volume of the system. Furthermore, BioNutrients-2 has expanded the projects scope by investigating novel products and microbial food sources. BioNutrients-2 is a six-month mission, launched in 2022, which tests production of fresh foods such as yogurt and kefir. The BioNutrients missions seek to provide advances for in-space biomanufacturing by addressing safe and reliable production of high-value nutrients and on-demand foods for future exploration efforts.

BioNutrients↗

Bionutrients: Microbial Production of on-Demand Nutrients on the International Space Station

Providing adequate nutrition to crew members is essential as deep-space missions cannot rely on consistent Earth-based resupply. The current NASA pre-packaged food system is designed for low-earth-orbit missions with a stated shelf-life of two years and notable vitamin degradation over time. One strategy to mitigate nutrient loss is to implement bioregenerative food sources to supplement the pre-packaged food system. The BioNutrients project is designed to provide targeted production of short shelf-life nutrients in a single-use production pack. BioNutrients-1 includes two strains of edible yeast that have been genetically engineered to produce carotenoids, β-carotene, and zeaxanthin. This five-year mission on the International Space Station (ISS), launched in 2019, tests the long-term storage and nutrient production of the microorganisms of interest. Additional microorganisms are also stored in stasis packs to determine the effects of long-duration storage on the ISS. These organisms may be useful for production of fermented foods, pharmaceuticals, or biomanufacturing processes. Improving on the BioNutrients-1 project, BioNutrients-2 has optimized the production pack hardware by reducing the overall mass and volume of the system. Furthermore, BioNutrients-2 has expanded the projects scope by investigating novel products and microbial food sources. BioNutrients-2 is a six-month mission, launched in 2022, which tests production of fresh foods such as yogurt and kefir. The BioNutrients missions seek to provide advances for in-space biomanufacturing by addressing safe and reliable production of high-value nutrients and on-demand foods for future exploration efforts.

BioNutrients↗

BioNutrients Bioreactors: Design and Materials Considerations for Synthetic Biology

The BioNutrients project aims to develop an on-demand biological production system capable of producing dietary nutrients and other products of interest. A key element of this system is the bioreactor itself, which needs to provide a suitable environment for growth of the selected microorganism(s). To support respiration of the organisms, the bioreactor should have gas-permeable elements to allow metabolically required gases (e.g., oxygen) to enter and byproduct gases (e.g., carbon dioxide) to escape. For systems utilizing yeast for precision fermentation, the system also needs to prevent or minimize the release of ethanol, which has impacts on the Environmental Control and Life Support System (ECLSS). Additionally, the bioreactor must include interfaces for hydrating the microorganism(s), sampling, and crew interface. For experiments on the International Space Station (ISS), the bioreactor must also comply with ISS-levied offgassing, cold stowage, and other safety requirements. This presentation aims to explore these design considerations, using the examples of the bioreactors used for the BioNutrients ISS payload series.

vitamins↗

Simultaneous optimization of simulated moving bed adsorption and distillation for 2,3‐butanediol recovery

Abstract A combined simulated moving bed (SMB) and distillation separation scheme is developed to recover 2,3‐butanediol (BDO) from a dilute fermentation broth. The scheme was integrated into a lignocellulosic biorefinery that produces hydrocarbon fuels from corn stover with BDO as an intermediate. BDO recovery is one of the most challenging processes in this biorefinery; and given the high associated energy duties, direct distillation is considered cost‐prohibitive. An alternative separation is SMB adsorption in nanoporous materials, which can reject 90% of the water and reduce subsequent distillation costs. Rigorous models were used to optimize the SMB and distillation simultaneously. The separation can be added to the biorefinery while keeping the projected minimum fuel selling price (MFSP) below $0.66 USD (US dollars) per liter gasoline‐equivalent ($2.50/GGE, gallon gasoline equivalent). Finally, sensitivity analyses were conducted to assess the effects of cost and lifetime of the adsorbent, titer concentration, and BDO purity.

09 BIOMASS FUELS↗

An argument for using anaerobes as microbial cell factories to advance synthetic biology and biomanufacturing

Anaerobes thrive in the absence of oxygen and are an untapped reservoir of biotechnological potential. Therefore, bioprospecting efforts focused on anaerobic microbial diversity could rapidly uncover new enzymes, pathways, and chassis organisms to drive biotechnology innovation. Despite their potential utility, anaerobic fermenters are viewed as inefficient from a biochemical perspective because their metabolisms produce fewer ATP (~2) per molecule of glucose processed than heterotrophic respirers (~32–38 ATP). While aerobes excel at ATP generation, they are often less efficient than anaerobes at processes that compete with ATP generation for cellular resources. This perspective highlights how anaerobic adaptations are advantageous for synthetic biology and biomanufacturing applications through the engineering of microbial cell factories. We further highlight emerging applications of anaerobic bioprocessing, including the use of anaerobic metabolisms for lignocellulosic bioprocessing, human and environmental health, and value-added bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗

Carbon‐negative hydrogen from ethanol via catalytic oxidative reforming

Abstract This study evaluated a commercial technology for producing low‐ or negative‐carbon hydrogen through ethanol catalytic oxidative reforming, focusing on the life cycle greenhouse gas emissions, or carbon intensity (CI). Various scenarios were analyzed: (a) comparing corn ethanol (first‐generation or Gen1 ethanol) and cellulosic ethanol (second‐generation or Gen2 ethanol) as feedstocks; (b) assessing carbon capture and sequestration (CCS) for CO 2 from upstream fermentation; and (c) evaluating oxygen sourcing via air separation units vs. on‐site or off‐site water electrolysis using a proton exchange membrane. Findings indicate that the CI for hydrogen production using Gen2 ethanol from corn stover is lower than that of Gen1 corn ethanol. Additionally, using proton exchange membrane‐generated oxygen results in a lower CI than air separation unit‐generated oxygen, regardless of the sourcing method. Implementing CCS for the hydrogen production plant's evolved CO 2 is essential for achieving a net‐negative CI for hydrogen from Gen1 ethanol. All examined scenarios, including both ethanol generations, oxygen sources, and CCS applications, demonstrated a net‐negative carbon intensity, surpassing the life cycle greenhouse gas emissions threshold of 0.45 kg CO 2 e/kg to enable policy credits as outlined in the Inflation Reduction Act §45V. In comparison, the CI for hydrogen from steam methane reforming stands at 3.4 kg CO 2 e/kg with CCS and 9.4 kg CO 2 e/kg without CCS.

08 HYDROGEN↗