Effect Of Substrate Concentration Research Articles

Multi-phase CFD modelling for hydroformylation of 1-hexene with microbubbles

Based on the micro-bubbling process as an intensification method, an experimental study on hydroformylation of 1-hexene was carried out. Effects of different total pressures, temperatures, partial pressures and substrate concentrations on the conversion rate of olefin and normal-to-iso ratio were investigated and the reaction kinetic coefficients were determined by fitting the experimental data with the model. Using the Euler two-fluid and standard k-ε turbulence model, a computational fluid dynamics model was established to describe the multi-phase flow mass transfer and reaction of olefin hydroformylation. The simulation results were in good agreement with the experimental results. Furthermore, the effects of microbubble sizes and internal components on the overall gas holdup, mass transfer rate, turbulent mixing and reaction efficiency of the multi-phase 1-hexene hydroformylation reaction system were studied, which could be applied to the process intensification and scale-up of the various multi-phase gas-liquid hydroformylation reactions.

Potato waste as feedstock to produce biohydrogen and organic acids: A comparison of acid and alkaline pretreatments using response surface methodology

The effects of physicochemical pre-treatment were evaluated on hydrogen (H2) production and organic acids from hydrolyzed potato peel. Central composite design (CCD) and response surface methodology (RSM) were used to evaluate the effects of different substrate concentrations on a wet basis (38.8–81.2 g.L−1) and hydrolyser ratios (6M NaOH and 30% HCl: 1.6–4.4% v.v−1; and H2SO4: 2.2–7.8% v.v−1). The experiments were conducted in batch reactors at 37 °C, using a heat-treated microbial consortium. The maximum H2 production potential (P), lag phase (λ), and maximum H2 production rate (Rm) were evaluated for untreated and pre-treated potato peel waste. H2 production was positively influenced under hydrolyzed substrate concentrations ≥75 g.L−1 in the three CCDs performed. Only the increase in the H2SO4 proportions (≥5% v.v−1) had a negative influence on H2 production. Increasing the 30% HCl and 6M NaOH proportions did not significantly influence the cumulative H2 production. The highest hydrogen production was obtained after alkaline pre-treatment by dark fermentation (P: 762.09 mL H2.L−1; λ: 14.56 h; Rm: 38.39 mL H2.L−1.h−1). Based on the CCD and RSM, the highest H2 production (1060.10 mL H2.L−1) was observed with 81.2 g.L−1 hydrolyzed potato peel with 3.0% v.v−1 of 6M NaOH. The highest yield liquid metabolites were acetic (513.70 mg. g−1 COD) and butyric acids (491.90 mg. g−1 COD).

Novel efficient enzymatic synthesis of the key-reaction intermediate of PET depolymerization, mono(2-hydroxyethyl terephthalate) – MHET

Poly(ethylene terephthalate) (PET) is one of the main synthetic plastics produced worldwide. The extensive use of this polymer causes several problems due to its low degradability. In this scenario, biocatalysts dawn as an alternative to enhance PET recycling. The enzymatic hydrolysis of PET results in a mixture of terephthalic acid (TPA), ethylene glycol (EG), mono-(2-hydroxyethyl) terephthalate (MHET) and bis-(2-hydroxyethyl) terephthalate (BHET) as main products. This work developed a new methodology to quantify the hydrolytic activity of biocatalysts, using BHET as a model substrate. The protocol can be used in screening enzymes for PET depolymerization reactions, amongst other applications. The very good fitting (R2 = 0.993) between experimental data and the mathematical model confirmed the feasibility of the Michaelis-Menten equation to analyze the effect of BHET concentration (8–200 mmol L−1) on initial hydrolysis rate catalyzed by Humicola insolens cutinase (HiC). In addition to evaluating the effects of enzyme and substrate concentration on the enzymatic hydrolysis of BHET, a novel and straightforward method for MHET synthesis was developed using an enzyme load of 0.025 gprotein gBHET−1 and BHET concentration of 60 mmol L−1 at 40 °C. MHET was synthesized with high selectivity (97 %) and yield (82 %). The synthesized MHET properties were studied using differential scanning calorimetry (DSC), thermogravimetry (TGA), and proton nuclear magnetic resonance (1H NMR), observing the high purity of the final product (86.7 %). As MHET is not available commercially, this synthesis using substrate and enzyme from open suppliers adds new perspectives to monitoring PET hydrolysis reactions.

Replacement of Trypsin by Proteases for Medical Applications.

Cell culture has a crucial role in many applications in biotechnology. The production of vaccines, recombinant proteins, tissue engineering, and stem cell therapy all need cell culture. Most of these activities needed adherent cells to move, which should be trypsinized several times until received on a large scale. Although trypsin is manufactured from the bovine or porcine pancreas, the problem of contamination by unwanted animal proteins, unwanted immune reactions, or contamination to pathogen reagents is the main problem. This study investigated microbial proteases as a safe alternative for trypsin replacement in cell culture experiments for the detachment of adherent cells. The bacteria were isolated from the leather industry effluent based on their protease enzymes. After sequencing their 16S ribosomal deoxyribonucleic acid, their protease enzymes were purified, and their enzyme activities were assayed. The alteration of enzymatic activities using different substrates and the effect of substrate concentrations on enzyme activities were determined. The purified proteases were evaluated for cell detachment in the L929 fibroblast cells compared to trypsin. The separated cells were cultured again, and cell proliferation was determined by the MTT assay. The results showed that the isolated bacteria were Bacillus pumilus, Stenotrophomonas sp., Klebsiella aerogenes, Stenotrophomonas maltophilia, and Bacillus licheniformis. Among the isolated bacteria, the highest and the lowest protease activity belonged to Stenotrophomonas sp. and K. aerogenes, with 60.34 and 11.09 U/mL protease activity, respectively. All the isolated microbial proteases successfully affected L929 fibroblast cells' surface proteins and detached the cells. A significant induction in cell proliferation was observed in the cells treated with K. aerogenes protease and B. pumilus protease, respectively (P < 0.05). The obtained results suggested that microbial proteases can be used as safe and efficient alternatives to trypsin in cell culture in biopharmaceutical applications.

Nitrification upon Nitrogen Starvation and Recovery: Effect of Stress Period, Substrate Concentration and pH on Ammonia Oxidizers’ Performance

Nitrification has been widely applied in wastewater treatment, however gaining more insight into the nitrifiers’ physiology and stress response is necessary for the optimization of nutrient removal and design of advanced processes. Since nitrification initiates with ammonia oxidation performed by ammonia-oxidizing bacteria (AOB), the purpose of this study was to investigate the effects of short-term ammonia starvation on nitrogen uptake and transformation efficiency, as well as the performance of starved nitrifiers under various initial substrate concentrations and pH values. Ammonium deprivation for 3 days resulted in fast ammonium/ammonia accumulation upon nitrogen availability, with a maximum uptake rate of 3.87 mmol gprotein−1 min−1. Furthermore, a delay in the production of nitrate was observed with increasing starvation periods, resulting in slower recovery and lower nitrification rate compared to non-starved cells. The maximum accumulation capacity observed was 8.51% (w/w) independently of the external nitrogen concentration, at a range of 250–750 mg N L−1, while pH significantly affected ammonia oxidizers’ response, with alkaline values enhancing nitrogen uptake. In total, ammonia accumulation after short-term starvation might serve as an important strategy that helps AOB restore their activity, while concurrently it could be applied in wastewater treatment for effective nitrogen removal and subsequent biomass utilization.

Modelling of anaerobic digester for the conversion of organic waste into hydrogen & methane

Multistage Anaerobic digestion (AD) nowadays plays a beneficial role compared to single-stage AD to achieve a high rate of waste conversion with higher energy recovery. Hence, obtaining a stable operation with optimal operating conditions is of paramount importance in the modeling study. In the current study, modeling of two stages continuous stirred anaerobic digesters was considered using mass balances over the substrates (organic waste) and biomass in the respective reactor. All the simulation work was performed using MATLAB, from which steady-state concentration values were taken for the calculation of Hydrogen flow rate (QH2) and Methane flow rate (QCH4). The operating volumes of both the reactors were computed by looking at the effect on the production rates (QH2 &QCH4) as a function of dilution rate of first (D1) and second (D2) reactors for constant initial substrate concentration (S0). At last, the effect of Initial substrate concentration on both QH2 &QCH4 was studied by considering different S0 values as 4 g/L, 8 g/L, and 12 g/L. From our modeling result, it was found out that for Maximum Hydrogen production, operating D1 (in day-1) was 0.8, 0.9 & 1 and the Methane production rate was Maximum at operating D2 (inday-1) of 0.25, for S0 values of 4 g/L, 8 g/L, and 12 g/L respectively.

Response of simultaneous sulfide and nitrate removal process on acute toxicity of substrate concentration and salinity: Single toxicity and combined toxicity.

Simultaneous sulfide and nitrate removal process has performed excellent to treat nitrogen and sulfur pollutants in wastewater treatment. A high salinity stress poses a great challenge to the treatment of highly saline wastewater containing nitrate and sulfide. In addition, sulfide and nitrates are also toxic for the process, and their high concentration would inhibit the process. Therefore, the current work explores the single acute toxic effect and combined toxic effect of salinity and substrate concentration on the performance of the process from the perspective of toxicology. Considering sulfide and nitrate removal performance as an indicator, the IC50 values of sulfide were 293.20 mg S/L and 572.30 mg S/L, respectively; while those of salinity were 6.14% wt (91.78 mS/cm) and 6.63% wt (98.73 mS/cm), respectively. High substrate concentration or salinity resulted in elemental sulfur generation. The molar ratio of generated elemental sulfur to consumed sulfide(R-Sulfate) was close to 1. The response of nitrate reduction product to the elevating substrate concentration was not obvious, while its response to increasing salinity was on the contrary. With the increasing salinity (1.2% wt to 9.6% wt), molar ratio of generated nitrogen gas to consumed nitrate (R-Nitrogen gas) increased from 0.58 to 1, while molar ratio of generated nitrite to consumed nitrate (R-Nitrite) decreased from 0.43 to 0. Factorial analysis test revealed that the combined acute toxicity of substrate and salinity on sulfide oxidization and nitrate reduction were both antagonistic effects.

Pectin Derived from Hydrolysis of Ripe Kepok Kuning Banana Peel Powder Employing Crude Pectinases Produced by Aspergillus niger

Banana fruits consumption generates about 35% weight of peel waste containing approximately 10.61 to 24 w/w% of pectin. Hence, improper banana peel waste management may induce various environmental and health issues. The objectives of this work were to study the effect of substrate concentration, pH, temperature, and duration on the yield of pectin extracted from enzymatic hydrolysis of banana peel powder. In this work, the crude enzymes were obtained via submerged fermentation of Kepok Kuning banana peel powder utilising Aspergillus niger and directly used without prior purification. Pectin extraction from banana peel powder was performed through hydrolysis using crude pectinases at various substrate concentrations (0.033 to 0.123 g/mL), pH (4.0 to 6.0), and temperature (40 to 70°C) for 180 min. The increase of extraction parameters enhanced the pectin yield to a maximum value and then declined. High substrate concentration, temperature, pH, and monomeric pectin compounds formation at long hydrolysis duration were found to reduce enzyme activity. A recommended extraction condition is using 0.103 g/mL substrate concentration, pH 5.0, and 55 °C for 120 min to achieve 10.80% weight yield. Commercial implementations of the results can be worthwhile in solving the environmental problem and enhance the economic value of pectin-rich fruit peels and other agricultural wastes.

Screening and characterizing flavone synthases and its application in biosynthesizing vitexin from naringenin by a one-pot enzymatic cascade

C-glycosylated flavonoids are important structural derivatives of flavonoids and have a variety of physiological activities. Flavone synthase is a key enzyme for producing C-glycosylated flavonoids. In this study, three flavone synthase genes were cloned, overexpressed and characterized in E. coli. By analyzing the enzymatic properties of the enzymes, Aethusa cynapium flavone synthase (AcFNS) was better than Apium graveolens flavone synthase (AgFNS) and Petroselinum crispum flavone synthase (PcFNS) in terms of catalytic ability, organic solvent tolerance and stability. Then, a one-pot enzymatic cascade was developed to synthesize vitexin from naringenin by using AcFNS, C-glycosyltransferase (TcCGT) from Trollius chinensis, and sucrose synthase (GmSUS) from Glycine max. The effects of enzyme ratios, substrate concentrations, cofactors, and reaction conditions on vitexin production were determined. The highest vitexin production reached 935.6 mg/L with a corresponding molar conversion of 78.7 % for (2 S)-naringenin. Thus, this is the first report of a one-pot enzymatic cascade for vitexin production from naringenin in vitro.

Substrate Concentration and Thermal Effects During Polyhydroxyalkanoate Bioproduction

Polyhydroxyalkanoates (PHAs) have long been thought to have the potential to replace petrochemical-based polymers because their production cost has been a challenging factor hindering their production processes. Previously characterized quantities of Micrococcus flavus SS21B screened for polyhydroxyalkanoates (PHAs) were utilized. PHAs were then synthesized using corn cobs as a carbon source. Proximate analysis of corn cobs undertaken during this research reveals the presence of moisture and ash contents, crude fibre and protein, and carbohydrates. Optimization studies reveal an optimal PHA production at 8% substrate concentration and 37°C. This study shows that the carbon source has a more significant effect on the production of PHAs; i.e., the higher the carbon source in the production, the higher the PHAs synthesized, and the thermal effect required is average and not in excess.

Karakteristik HFS (High Fructose Syrup) dari umbi gembolo yang diproduksi secara hidrolisis enzimatis menggunakan amilase dan inulinase

HFS (High Fructose Syrup) from gembolo tubers is an innovation in to utilize gembolo tubers which so far have not been used optimally. The purpose of this study was to determine the effect of substrate concentration and duration of saccharification on the physicochemical characteristics of HFS (High Fructose Syrup). This study used a completely randomized design (CRD) with 2 factors consisting of 9 treatment levels. The factor I was the substrate concentration (15%, 20% and 25%) and factor II was the length of saccharification (24 hours, 36 hours and 48 hours). Data were analyzed using ANOVA level 5%. If there is a difference proceed with Duncan's Test (DMRT) 5%. Based on the results of the study, the best treatment was a substrate concentration of 25% with a saccharification period of 48 hours which produced HFS with the following characteristics: yield of 18.08%; reducing sugar 41, 28%; total dissolved solids 42.00oBrix; viscosity 58.30 cP; fructose content is 14.68% and organoleptic test got the highest score.

Selectivity Control in Photocatalytic Transfer Hydrogenation of Bio‐based Aldehydes

AbstractThe development of selectivity control strategies based on external operational conditions without modification of catalyst is an attractive but challenging issue in photocatalysis. Herein we reported the substrate concentration‐switched selectivity in photocatalytic transfer hydrogenation of biomass‐derived aromatic aldehydes. With the same catalyst, hydrodeoxygenation was realized at low aldehyde concentration and reductive etherification was realized at high aldehyde concentration. Mechanistic studies revealed that the substrate concentration affected the electron density on catalyst, thus controlled the initial reduction of formyl group and types of intermediates. Competitive adsorption of these intermediates on the catalyst affected hydrogenolysis of C−O bonds in the later stage. This method is easy to handle, and achieved selective hydrogenation of diverse bio‐based aldehydes to corresponding deoxygenated products and unsymmetric ether products as potential fuel additives under mild conditions. This work sheds light on the effect of substrate concentration on the selectivity in photocatalysis.

Effects of substrate concentration and glycine betaine on nitrogen removal and microbial communities in the sulfide autotrophic denitrification and Anammox coupling system

The effects of substance concentration and glycine betaine (GB) on nitrogen removal and microbial communities in the sulfide autotrophic denitrification (SAD) and Anammox coupling process were investigated. When the ratio of NH4+-N/NO3−-N/S2− was 1:1.26:0.79, the ammonia removal efficiency (ARE) was 96.8%, and total nitrogen removal efficiency (TNRE) was about 94.8%. When the substance concentration increased gradually, ARE and TNRE decreased to 53.7% and 71.2%, respectively. However, when 0.5 mM GB was added, ARE and TNRE improved to 62.1% and 77.6%, respectively. Batch tests also showed that when 0.5 mM GB was added, the removal efficiency increased mostly. High-throughput sequencing illustrated that the Anammox bacteria abundance decreased, while the abundance of Thiobacillus increased with increasing substrate concentration. The abundance of Candidatus Kuenenia and Candidatus Brocadia in the upper of UASB reactor was significantly higher than that in the bottom. The main functional bacteria in the bottom of UASB reactor was Thiobacillus.

Optimization, purification and characterization of laccase from lignocellulolytic fungi Dichotomopilus funicola NFCCI 4534 and Alternaria padwickii NFCCI 4535

In the present study, lignocellulolytic fungi were isolated by lignocellulolytic waste (Banana waste) soil from the Bhilai-Durg region of Chhattisgarh, India, which was capable to produce laccase enzyme. The laccase producing potent organisms was identified as Dichotomopilus funicola NFCCI 4534 and Alternaria padwickii NFCCI 4535 using molecular characterization by Internal Transcribed Spacer (ITS). For the better production of laccase enzyme physical (incubation period, temperature and pH) and nutritional (carbon, nitrogen and vitamin source) parameters optimized by one factor-at-a time and response surface methodology approach were used. Laccase enzyme was partially purified by ammonium sulphate precipitation and dialysis. The partially purified laccase was characterized by the effect of substrate concentration, temperature, pH, metal ions and inhibitors. Results showed that out of 5 positive cultures only 2 cultures showed the best laccase production after the quantitative screening. ITS-rDNA gene sequencing and BLAST analysis were used to characterize the isolated fungal species and they were found to be D. funicola NFCCI 4534 and A. padwickii NFCCI 4535. The temperature 24 °C and 32 °C and pH 6.0 and 6.5 were suitable for production of D. funicola and A. padwickii, respectively. It was found that Ferric chloride and ferrous sulphate exhibited maximum laccase activity as compared to other metal ions. However, EDTA and l-arginine inhibited D. funicola and A. padwickii, respectively.

Inorganic phosphate self-sufficient whole-cell biocatalysts containing two co-expressed phosphorylases facilitate cellobiose production.

Cellobiose, a natural disaccharide, attracts extensive attention as a potential functional food/feed additive. In this study, we present an inorganic phosphate (Pi) self-sufficient biotransformation system to produce cellobiose by co-expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP). The Bifidobacterium adolescentis SP (BASP) and Cellvibrio gilvus CBP (CGCBP) were co-expressed in Escherichia coli. Escherichia coli cells containing BASP and CGCBP were used as whole-cell catalysts to convert sucrose and glucose to cellobiose. The effects of reaction pH, temperature, Pi concentration, and substrate concentration were investigated. In the optimum biotransformation conditions, 800 mM cellobiose was produced from 1.0 M sucrose, 1.0 M glucose, and 50 mM Pi, within 12 hr. The by-product fructose and residual substrate (sucrose and glucose) were efficiently removed by treatment with yeast, to help purify the product cellobiose. The wider applicability of this Pi self-sufficiency strategy was demonstrated in the production of laminaribiose by co-expressing SP and laminaribiose phosphorylase. This study suggests that the Pi self-sufficiency strategy through co-expressing two phosphorylases has the advantage of great flexibility for enhanced production of cellobiose (or laminaribiose).

First report of collagenase production by Trichosporon sp. strain isolated from pollen of Amazonian bee (Melipona seminigra seminigra)

Trichosporon yeasts are widely employed to produce lipids, lipases, and aspartic peptidases, but there are no previous studies on collagenase production. This work aimed to select the best collagenase producing Amazonian Trichosporon strains. Moreover, a 23-full factorial design (FFD) and a 22-central composite design combined with Response Surface Methodology were applied to optimize production and find the best conditions for hydrolysis of type I bovine collagen. Most of the studied strains had some collagenolytic activity, but the selected one achieved the highest value (44.02 U) and a biomass concentration of 2.31 g/L. The best collagenase production conditions were 160 rpm of agitation, pH 5.5 and a substrate concentration of 4.0 g/L. The former experimental design showed that substrate concentration was the only statistically significant factor on both biomass concentration and collagenase activity, while the latter showed simultaneous effects of substrate concentration and pH on collagenolytic activity, which peaked at pH 5.5–6.4 and substrate concentration of 3.0–3.4 g/L. An additional 2³-FFD was finally used to optimize the conditions collagen hydrolysis, and pH 6, 25 °C and a substrate concentration of 7.5 (g/L) ensured the highest hydrolysis degree. This study is the first that describes optimized conditions of collagenase production by Trichosporon strains.

Theoretical and Numerical Analysis of Nonlinear Processes in Amperometric Enzyme Electrodes with Cyclic Substrate Conversion

A theoretical model of amperometric enzyme electrodes has been developed in which chemical amplification occurs in a single enzyme membrane via cyclic substrate conversion. The system is based on non-stationary diffusion equations with a nonlinear factor related to the Michaelis–Menten kinetics of the enzymatic reaction. By solving the nonlinear equations using the AGM technique, simple analytical expressions of concentration substrate, product, and amperometric current response are derived. Further, biosensor sensitivity, resistance, and gain are obtained from the current. MATLAB programming was used to carry out the digital simulation. The analytical results are validated with the numerical results. The effect of substrate concentration, maximum enzymatic rate, and membrane thickness on biosensor response was evaluated.

Lignin-based solid acid catalyst for cellulose residue conversion into levulinic acid in biphasic system

Phosphoric acid-activated lignin-based solid acid catalysts (PLSA) were prepared from lignosulfonate by phosphoric acid activation, carbonation, and sulfonation. The physicochemical properties of catalysts were determined by SEM, N2 adsorption-desorption, FT-IR, XRD, XPS and element analysis. Masses of phosphorous-containing group were grafted on catalyst surface during phosphoric acid activation and carbonation to provide sites for subsequent -SO3H substitution. PLSA could efficiently convert cellulose residue into levulinic acid. The effects of reaction temperature, residue time, catalyst loading, substrate concentration, solvent and catalyst stability were investigated and optimized. Using PLSA as catalyst, 67.9 mol% LA yield was obtained from 4 g cellulosic residue at 190 ℃ in 150 min in MIBK/H2O-NaCl. Overall, this work provides a valuable basis for the application of lignin-based solid acid in biomass valorization.

Hydrogen Production from Biomass and Organic Waste Using Dark Fermentation: An Analysis of Literature Data on the Effect of Operating Parameters on Process Performance

In the context of hydrogen production from biomass or organic waste with dark fermentation, this study analysed 55 studies (339 experiments) in the literature looking for the effect of operating parameters on the process performance of dark fermentation. The effect of substrate concentration, pH, temperature, and residence time on hydrogen yield, productivity, and content in the biogas was analysed. In addition, a linear regression model was developed to also account for the effect of nature and pretreatment of the substrate, inhibition of methanogenesis, and continuous or batch operating mode. The analysis showed that the hydrogen yield was mainly affected by pH and residence time, with the highest yields obtained for low pH and short residence time. High hydrogen productivity was favoured by high feed concentration, short residence time, and low pH. More modest was the effect on the hydrogen content. The mean values of hydrogen yield, productivity, and content were, respectively, 6.49% COD COD−1, 135 mg L−1 d−1, 51% v/v, while 10% of the considered experiments obtained yield, productivity, and content of or higher than 15.55% COD COD−1, 305.16 mg L−1 d−1, 64% v/v. Overall, this study provides insight into how to select the optimum operating conditions to obtain the desired hydrogen production.

Kinetic Study on the Effect of Substrate and Micronutrient Inhibition during Anaerobic Fermentation of Biohydrogen

There are several factors that influence the production of biohydrogen by dark fermentation including inoculum seeds, type and concentration of substrate, pH, temperature, presence of micronutrient and reactor configuration. Previous research has proven that the concentration of substrate and the presence of micronutrient will influence the yield and productivity of biohydrogen production. However, improvement of yield and productivity of the process can only be achieved once the system is under the optimum amount of substrate and micronutrient. Therefore, the best way to determine the effect of substrate concentration and presence of micronutrient is through kinetic study that was done using Monod model along with Andrews model. Besides that, the substrate inhibition effect also will be evaluated to determine the maximum substrate that needs to be supplied for maximum hydrogen production, and thus supplied the information for economic feasibility for fermentation process. In the meantime, the inhibition effect of adding the iron nanoparticles also had been evaluated in order to understand the interaction effect between iron nanoparticles and bacteria in term of catabolism reaction. It was found that increasing the substrate concentration more than 10 g/l will cause the inhibition to the system, in which it will slow down the reaction process and reduced the production of hydrogen. While the presence of iron NPs more than its optimum value (200 mg/l) will inhibit the bacterial growth and hence, affect the hydrogen production. For both cases, when the inhibition occurred at the respective concentration, it was found that the metabolic pathway was shifted to produce more hydrogen-consuming metabolite such as propionate acid, and thus, dropped the hydrogen production.