Wastewater-based epidemiology for COVID-19 using dynamic artificial neural networks.

Global efforts in vaccination have led to a decrease in COVID-19 mortality but a high circulation of SARS-CoV-2 is still observed in several countries, resulting in some cases of severe lockdowns. In this sense, wastewater-based epidemiology remains a powerful tool for supporting regional health administrations in assessing risk levels and acting accordingly. In this work, a dynamic artificial neural network (DANN) has been developed for predicting the number of COVID-19 hospitalized patients in hospitals in Valladolid (Spain). This model takes as inputs a wastewater epidemiology indicator for COVID-19 (concentration of RNA from SARS-CoV-2 N1 gene reported from Valladolid Wastewater Treatment Plant), vaccination coverage, and past data of hospitalizations. The model considered both the instantaneous values of these variables and their historical evolution. Two study periods were selected (from May 2021 until September 2022 and from September 2022 to July 2023). During the first period, accurate predictions of hospitalizations (with an overall range between 6 and 171) were favored by the correlation of this indicator with N1 concentrations in wastewater (r = 0.43, p < 0.05), showing accurate forecasting for 1 day ahead and 5 days ahead. The second period's retraining strategy maintained the overall accuracy of the model despite lower hospitalizations. Furthermore, risk levels were assigned to each 1 day ahead prediction during the first and second periods, showing agreement with the level measured and reported by regional health authorities in 95 % and 93 % of cases, respectively. These results evidenced the potential of this novel DANN model for predicting COVID-19 hospitalizations based on SARS-CoV-2 wastewater concentrations at a regional scale. The model architecture herein developed can support regional health authorities in COVID-19 risk management based on wastewater-based epidemiology.

Airborne SARS-CoV-2 is more frequently detected in environments related to children and elderly but likely non-infectious, Norway, 2022.

This study investigates the presence of SARS-CoV-2 in indoor and outdoor environments in two cities in Norway between April and May 2022. With the lifting of COVID-19 restrictions in the country and a focus on vaccination, this research aims to shed light on the potential for virus transmission in various settings. Air sampling was conducted in healthcare and non-healthcare facilities, covering locations frequented by individuals across different age groups. The study found that out of 31 air samples, only four showed the presence of SARS-CoV-2 RNA by RT-qPCR, with no viable virus detected after RNAse pre-treatment. These positive samples were primarily associated with environments involving children and the elderly. Notably, sequencing revealed mutations associated with increased infectivity in one of the samples. The results highlight the importance of considering children as potential sources of virus transmission, especially in settings with prolonged indoor exposure. As vaccination coverage increases globally, and with children still representing a substantial unvaccinated population, the study emphasizes the need to re-implement mask-wearing mandates indoors and in public transport to reduce virus transmission. The findings have implications for public health strategies to control COVID-19, particularly in the face of new variants and the potential for increased transmission during the autumn and winter seasons.

Resilience and robustness of alphaproteobacterial methanotrophs upon methane feast-famine scenarios.

Most of methane (CH4) emissions contain low CH4 concentrations and typically occur at irregular intervals, which hinders the implementation and performance of methane abatement processes. This study aimed at understanding the metabolic mechanisms that allow methane oxidizing bacteria (MOB) to survive for long periods of time under methane starvation. To this aim, we used an omics-approach and studied the diversity and metabolism of MOB and non-MOB in bioreactors exposed to low CH4 concentrations under feast-famine cycles of 5 days and supplied with nutrient-rich broth. The 16S rRNA and the pmoA transcripts revealed that the most abundant and active MOB during feast and famine conditions belonged to the alphaproteobacterial genus Methylocystis (91-65%). The closest Methylocystis species were M. parvus and M. echinoides. Nitrifiers and denitrifiers were the most representative non-MOB communities, which likely acted as detoxifiers of the system. During starvation periods, the induced activity of CH4 oxidation was not lost, with the particulate methane monooxygenase of alphaproteobacterial MOB playing a key role in energy production. The polyhydroxyalkanoate and nitrification metabolisms of MOB had also an important role during feast-famine cycles, maintaining cell viability when CH4 concentrations were negligible. This research shows that there is an emergence and resilience of conventional alphaproteobacterial MOB, being the genus Methylocystis a centrepiece in environments exposed to dilute and intermittent methane emissions. This knowledge can be applied to the operation of bioreactors subjected to the treatment of dilute and discontinuous emissions via controlled bioaugmentation.

Botanical filters for the abatement of indoor air pollutants.

Nowadays, people spend 80-90% of their time indoors, while recent policies on energy efficient and safe buildings require reduced building ventilation rates and locked windows. These facts have raised a growing concern on indoor air quality, which is currently receiving even more attention than outdoors pollution. Prevention is the first and most cost-effective strategy to improve indoor air quality, but once pollution is generated, a battery of physicochemical technologies is typically implemented to improve air quality with a questionable efficiency and at high operating costs. Biotechnologies have emerged as promising alternatives to abate indoor air pollutants, but current bioreactor configurations and the low concentrations of indoor air pollutants limit their widespread implementation in homes, offices and public buildings. In this context, recent investigations have shown that potted plants can aid in the removal of a wide range of indoor air pollutants, especially volatile organic compounds (VOCs), and can be engineered in aesthetically attractive configurations. The original investigations conducted by NASA, along with recent advances in technology and design, have resulted in a new generation of botanical biofilters with the potential to effectively mitigate indoor air pollution, with increasing public aesthetics acceptance. This article presents a review of the research on active botanical filters as sustainable alternatives to purify indoor air.

Ectoines production from biogas in pilot bubble column bioreactors and their subsequent extraction via bio-milking.

Despite the potential of biogas from waste/wastewater treatment as a renewable energy source, the presence of pollutants and the rapid decrease in the levelized cost of solar and wind power constrain the use of biogas for energy generation. Biogas conversion into ectoine, one of the most valuable bioproducts (1000 €/kg), constitutes a new strategy to promote a competitive biogas market. The potential for a stand-alone 20 L bubble column bioreactor operating at 6% NaCl and two 10 L interconnected bioreactors (at 0 and 6% NaCl, respectively) for ectoine production from biogas was comparatively assessed. The stand-alone reactor supported the best process performance due to its highest robustness and efficiency for ectoine accumulation (20-52 mgectoine/gVSS) and CH4 degradation (up to 84%). The increase in N availability and internal gas recirculation did not enhance ectoine synthesis. However, a 2-fold increase in the internal gas recirculation resulted in an approximately 1.3-fold increase in CH4 removal efficiency. Finally, the recovery of ectoine through bacterial bio-milking resulted in efficiencies of >70% without any negative impact of methanotrophic cell recycling to the bioreactors on CH4 biodegradation or ectoine synthesis.

Quantification and Whole Genome Characterization of SARS-CoV-2 RNA in Wastewater and Air Samples.

Wastewater-based epidemiology has emerged as a promising and efficacious surveillance system for SARS-CoV-2 and other infectious diseases in many nations. The process typically involves wastewater concentration, nucleic acid extraction, amplification of selected genomic segments, and detection and quantification of the amplified genomic segment. This methodology can similarly be leveraged to detect and quantify infectious agents, such as SARS-CoV-2, in air samples. Initially, SARS-CoV-2 was presumed to spread primarily through close personal contact with droplets generated by an infected individual while speaking, sneezing, coughing, singing, or breathing. However, a growing number of studies have reported the presence of SARS-CoV-2 RNA in the air of healthcare facilities, establishing airborne transmission as a viable route for the virus. This study presents a composite of established protocols to facilitate environmental detection, quantification, and sequencing of viruses from both wastewater and air samples.

Environmental Dissemination of SARS-CoV-2 in a University Hospital during the COVID-19 5th Wave Delta Variant Peak in Castile-León, Spain.

The dominant SARS-CoV-2 Delta variant (B.1.617.2) became the main circulating variant among countries by mid 2021. Attention was raised to the increased risk of airborne transmission, leading to nosocomial outbreaks even among vaccinated individuals. Considering the increased number of COVID-19 hospital admissions fueled by the spread of the variant, with Spain showing the highest COVID-19 rates in mainland Europe by July 2021, the aim of this study was to assess SARS-CoV-2 environmental contamination in different areas of a University Hospital in the region of Castile-León, Spain, during the peak of the 5th wave of COVID-19 in the country (July 2021). Air samples were collected from sixteen different areas of the Hospital using a Coriolis® µ air sampler. Surface samples were collected in these same areas using sterile flocked plastic swabs. RNA extraction followed by a one-step RT-qPCR were performed for detection of SARS-CoV-2 RNA. Of the 21 air samples, only one was positive for SARS-CoV-2 RNA, from the emergency waiting room. Of the 40 surface samples, 2 were positive for SARS-CoV-2 RNA, both from the microbiology laboratory. These results may be relevant for risk assessment of nosocomial infection within healthcare facilities, thus helping prevent and minimize healthcare staff's exposure to SARS-CoV-2, reinforcing the importance of always wearing appropriate and well-fit masks at all times and proper PPE when in contact with infected patients.

Current Concentrations of Zn, Cu, and As in Piggery Wastewater Compromise Nutrient Removals in Microalgae-Bacteria Photobioreactors Due to Altered Microbial Communities.

The treatment of pig manure is a major environmental issue, and photobioreactors containing consortia of microalgae and bacteria have proven to be a promising and sustainable treatment alternative. This work studies the effect of Cu, Zn and As, three toxic elements frequently present in piggery wastewater, on the performance and microbiome of photobioreactors. After dopage with Zn (100 mg/L), Cu (100 mg/L), and As (500 µg/L), the high biomass uptake of Zn (69-81%) and Cu (81-83%) decreased the carbon removal in the photobioreactors, inhibited the growth of Chlorella sp., and affected heterotrophic bacterial populations. The biomass As uptake result was low (19%) and actually promoted microalgae growth. The presence of Cu and As decreased nitrogen removal, reducing the abundance of denitrifying bacterial populations. The results showed that metal(loid)s significantly affected 24 bacterial genera and that they did not recover after exposure. Therefore, this study makes an important contribution on the impact of the presence of metal(loid)s in piggery wastewater that compromises the overall performance of PBRs, and so, the environmental and health impact of treated effluents.

Centralized and decentralized wastewater-based epidemiology to infer COVID-19 transmission - A brief review.

Wastewater-based epidemiology has shown to be a promising and innovative approach to measure a wide variety of illicit drugs that are consumed in the communities. In the same way as for illicit drugs, wastewater-based epidemiology is a promising approach to understand the prevalence of viruses in a community-level. The ongoing coronavirus disease 2019 (COVID-19) pandemic created an unprecedented burden on public health and diagnostic laboratories all over the world because of the need for massive laboratory testing. Many studies have shown the applicability of a centralized wastewater-based epidemiology (WBE) approach, where samples are collected at WWTPs. A more recent concept is a decentralized approach for WBE where samples are collected at different points of the sewer system and at polluted water bodies. The second being particularly important in countries where there are insufficient connections from houses to municipal sewage pipelines and thus untreated wastewater is discharged directly in environmental waters. A decentralized approach can be used to focus the value of diagnostic tests in what we call targeted-WBE, by monitoring wastewater in parts of the population where an outbreak is likely to happen, such as student dorms, retirement homes and hospitals. A combination of centralized and decentralized WBE should be considered for an affordable, sustainable, and successful WBE implementation in high-, middle- and low-income countries.

Elucidating the key environmental parameters during the production of ectoines from biogas by mixed methanotrophic consortia.

Anaerobic digestion (AD) is a robust biotechnology for the valorisation of organic waste into biogas. However, the rapid decrease in renewable electricity prices requires alternative uses of biogas. In this context, the engineering of innovative platforms for the bio-production of chemicals from CH4 has recently emerged. The extremolyte and osmoprotectant ectoine, with a market price of ~1000€/Kg, is the industrial flagship of CH4-based bio-chemicals. This work aimed at optimizing the accumulation of ectoines using mixed microbial consortia enriched from saline environments (a salt lagoon and a salt river) and activated sludge, and biogas as feedstock. The influence of NaCl (0, 3, 6, 9 and 12 %) and Na2WO4 (0, 35 and 70 µg L-1) concentrations and incubation temperature (15, 25 and 35 °C) on the stoichiometry and kinetics of the methanotrophic consortia was investigated. Consortia enriched from activated sludge at 15 °C accumulated the highest yields of ectoine and hydroxyectoine at 6 % NaCl (105.0 ± 27.2 and 24.2 ± 5.4 mgextremolyte gbiomass-1, respectively). The consortia enriched from the salt lagoon accumulated the highest yield of ectoine and hydroxyectoine at 9 % NaCl (56.6 ± 2.5 and 51.0 ± 2.0 mgextremolyte gbiomass-1, respectively) at 25 °C. The supplementation of tungsten to the cultivation medium did not impact on the accumulation of ectoines in any of the consortia. A molecular characterization of the enrichments revealed a relative abundance of ectoine-accumulating methanotrophs of 7-16 %, with Methylomicrobium buryatense and Methylomicrobium japanense as the main players in the bioconversion of methane into ectoine.

Evaluation of pretreatments for solubilisation of components and recovery of fermentable monosaccharides from microalgae biomass grown in piggery wastewater.

Microalgae-bacteria biomass cultured in wastewater is an interesting renewable material capable of metabolising nutrients from wastes into carbohydrates, proteins, and lipids through photosynthesis. Despite the interest in the valorisation of this biomass to improve the viability of microalgae-based wastewater treatment processes, very scarce research has been devoted to the fractional recovery of its components. This work evaluates the effect of different pretreatments coupled with enzymatic hydrolysis on the solubilisation of biomass components and on the recovery of fermentable monosaccharides (glucose and xylose) from Scenedesmaceae based biomass grown in a thin layer reactor feed with piggery wastewater. Chemical pretreatments generated high concentrations of byproducts, mainly organic acids. No bacterial DNA was found in these pretreated biomasses. The acid pretreatment provided the highest carbohydrate solubilisation (98%) and monosaccharide recovery (81%). Enzymatic hydrolysis coupled with alkaline NaOH 2 M pretreatment achieved almost complete solubilisation of the biomass components, but high carbohydrate losses. Physical pretreatments remarkably increased the solubilisation of the biomass components during the enzymatic hydrolysis step, especially bead milling, which achieved solubilisation yields of 83% of carbohydrates, 43% of proteins, and 60% of lipids. The presence of viable bacteria in these pretreated biomasses could be related to the high carbohydrate losses and the generation of methanol and ethanol in addition to organic acids as byproducts.

A systematic comparison of ectoine production from upgraded biogas using Methylomicrobium alcaliphilum and a mixed haloalkaliphilic consortium.

Biogas is the byproduct of anaerobic digestion with the highest valorization potential, however its full exploitation is limited by the lack of tax incentives and the inherent presence of pollutants. The development of technologies for biogas conversion into added-value products is crucial in order to ensure the competitiveness of this bioresource. This study constitutes the first proof of concept of upgraded biogas bioconversion into the high profit margin product ectoine. Ectoine represents the most expensive product synthesized by microorganisms with a retail value of 1000 $ kg-1 and a yearly increasing demand that currently entails a total market opportunity of 15000 M€. First, the production of ectoine from upgraded biogas was assessed in batch bioreactors. The presence of H2S did not exert a negative effect on the growth of the haloalkaliphilic ectoine producers, and ectoine yields up to 49 mg g biomass-1 were obtained. A second experiment conducted in continuous bubble column bioreactors confirmed the feasibility of the process under continuous mode (with ectoine yields of 109 mg g biomass-1). Finally, this study revealed that the removal of toxic compounds (i.e. medium dilution rate of 0.5 day-1) and process operation with a consortium composed of methylotrophic/non-methylotrophic ectoine producers enhanced upgraded biogas bioconversion. This research discloses the basis for the application of this innovative technology and could boost the economic performance of anaerobic digestion.

Technology validation of photosynthetic biogas upgrading in a semi-industrial scale algal-bacterial photobioreactor.

The performance of photosynthetic biogas upgrading coupled to wastewater treatment was evaluated in an outdoors high rate algal pond (HRAP) interconnected to an absorption column at semi-industrial scale. The influence of biogas flowrate (274, 370 and 459 L h-1), liquid to biogas ratio (L/G = 1.2, 2.1 and 3.5), type of wastewater (domestic versus centrate) and hydraulic retention time in the HRAP (HRT) on the quality of the biomethane produced was assessed. The highest CO2 and H2S removal efficiencies (REs) were recorded at the largest L/G due to the higher biogas-liquid mass transfer at increasing liquid flowrates. No significant influence of the biogas flowrate on process performance was observed, while the type of wastewater was identified as a key operational parameter. CO2 and H2S-REs of 99% and 100% at a L/Gmax = 3.5 were recorded using centrate. The maximum CH4 content in the biomethane (90%) was limited by N2 and O2 desorption.

Removal of contaminants of emerging concern from urban wastewater in novel algal-bacterial photobioreactors.

This work evaluates the removal of five pharmaceuticals and personal care products, i.e., ibuprofen, naproxen, salicylic acid, triclosan and propylparaben, from urban wastewater under two novel algal-bacterial photobioreactor settings. The first configuration (phase A) consisted of an anoxic-aerobic photobioreactor operating at a hydraulic retention time (HRT) of 2d at different concentrations of total organic carbon (TOC) (90mgL-1-200mgL-1). In the second configuration (phase B) an anaerobic step was introduced before the anoxic tank to set a photosynthetic A2O process. In this phase, the HRT varied between 3 and 4d and the TOC was kept constant at 200mgL-1. In addition, the impact of external aeration in the aerobic photobioreactor was assessed. The maximum removals for ibuprofen, naproxen, salicylic acid, triclosan and propylparaben (94±1%, 52±43%, 98±2%, 100±0%, 100±0%, respectively) were recorded during phase B. In phase A, low TOC concentrations triggered higher ibuprofen and naproxen removals likely due to the high contribution of biological oxidation on their removal. In phase B, total or very high removal efficiencies were observed for ibuprofen, propylparaben and triclosan independently on the operating conditions. In contrast, the removal efficiency of naproxen and salicylic acid decreased when the HRT dropped from 4 to 3d in the absence of external aeration, which suggests that biodegradation played a key role in their removal. In addition, sorption might have contributed to the elimination of triclosan and propylparaben from the wastewater.

Novel haloalkaliphilic methanotrophic bacteria: An attempt for enhancing methane bio-refinery.

Methane bioconversion into products with a high market value, such as ectoine or hydroxyectoine, can be optimized via isolation of more efficient novel methanotrophic bacteria. The research here presented focused on the enrichment of methanotrophic consortia able to co-produce different ectoines during CH4 metabolism. Four different enrichments (Cow3, Slu3, Cow6 and Slu6) were carried out in basal media supplemented with 3 and 6% NaCl, and using methane as the sole carbon and energy source. The highest ectoine accumulation (∼20 mg ectoine g biomass-1) was recorded in the two consortia enriched at 6% NaCl (Cow6 and Slu6). Moreover, hydroxyectoine was detected for the first time using methane as a feedstock in Cow6 and Slu6 (∼5 mg g biomass-1). The majority of the haloalkaliphilic bacteria identified by 16S rRNA community profiling in both consortia have not been previously described as methanotrophs. From these enrichments, two novel strains (representing novel species) capable of using methane as the sole carbon and energy source were isolated: Alishewanella sp. strain RM1 and Halomonas sp. strain PGE1. Halomonas sp. strain PGE1 showed higher ectoine yields (70-92 mg ectoine g biomass-1) than those previously described for other methanotrophs under continuous cultivation mode (∼37-70 mg ectoine g biomass-1). The results here obtained highlight the potential of isolating novel methanotrophs in order to boost the competitiveness of industrial CH4-based ectoine production.

Bio-conversion of methane into high profit margin compounds: an innovative, environmentally friendly and cost-effective platform for methane abatement.

Despite the environmental relevance of CH4 and forthcoming stricter regulations, the development of cost-efficient and environmentally friendly technologies for CH4 abatement is still limited. To date, one of the most promising solutions for the mitigation of this important GHG consists of the bioconversion of CH4 into bioproducts with a high profit margin. In this context, methanotrophs have been already proven as cell-factories of some of the most expensive products synthesized by microorganisms. In the case of ectoine (1000 $ kg-1), already described methanotrophic genera such as Methylomicrobium can accumulate up to 20% (ectoine wt-1) using methane as the only carbon source. Moreover, α-methanotrophs, such as Methylosynus and Methylocystis, are able to store bioplastic concentrations up to 50-60% of their total cell content. More than that, methanotrophs are one of the greatest potential producers of methanol and exopolysaccharides. Although this methanotrophic factory could be enhanced throughout metabolic engineering, the valorization of CH4 into valuable metabolites has been already consistently demonstrated under continuous and discontinuous mode, producing more than one compound in the same bioprocess, and using both, single strains and specific consortia. This review states the state-of-the-art of this innovative biotechnological platform by assessing its potential and current limitations.

Quantitative analysis of methane monooxygenase (MMO) explains process robustness in continuous and feast-famine bioreactors treating methane.

The ability of methanotrophs to rapidly respond to intentional or accidental stress conditions caused by operational failures or process fluctuations is of utmost importance to guarantee the robustness of CH4 abatement biotechnologies. In this study, the performance of a continuous and two feast-famine (5:5 days feast-famine cycles) stirred tank reactors treating diluted CH4 emissions (4-5% v/v) was comparatively assessed for 149 days. The robustness of the three bioreactors towards a 5 days CH4 deprivation episode was thoroughly evaluated at a molecular level (pmoA gene expression level) and correlated to macroscopic process performance. The bioreactors recovered their steady-state abatement performance (in terms of CH4 elimination capacity and CO2 production rate) within 1.5-2 h following CH4 supply resumption concomitantly with a maximum in pmoA gene expression, regardless of the previous operational mode. However, while methanotrophs from the continuous unit maintained higher basal levels of pmoA expression as a strategy for a rapid CH4 metabolism initiation, the strategy of the feast-famine adapted-methanotrophs consisted on a more accurate regulation of their pmoA transcripts levels along with a higher and/or more rapid induction of the pmoA gene by CH4 availability.

Multiresidue analytical method for pharmaceuticals and personal care products in sewage and sewage sludge by online direct immersion SPME on-fiber derivatization - GCMS.

The work here presented aimed at developing an analytical method for the simultaneous determination of 22 pharmaceuticals and personal care products, including 3 transformation products, in sewage and sludge. A meticulous method optimization, involving an experimental design, was carried out. The developed method was fully automated and consisted of the online extraction of 17 mL of water sample by Direct Immersion Solid Phase MicroExtraction followed by On-fiber Derivatization coupled to Gas Chromatography - Mass Spectrometry (DI-SPME - On-fiber Derivatization - GC - MS). This methodology was validated for 12 of the initial compounds as a reliable (relative recoveries above 90% for sewage and 70% for sludge; repeatability as %RSD below 10% in all cases), sensitive (LODs below 20 ng L-1 in sewage and 10 ng g-1 in sludge), versatile (sewage and sewage-sludge samples up to 15,000 ng L-1 and 900 ng g-1, respectively) and green analytical alternative for many medium-tech routine laboratories around the world to keep up with both current and forecast environmental regulations requirements. The remaining 10 analytes initially considered showed insufficient suitability to be included in the final method. The methodology was successfully applied to real samples generated in a pilot scale sewage treatment reactor.

Effect of pretreatments on biogas production from microalgae biomass grown in pig manure treatment plants.

Methane production from pretreated and raw mixed microalgae biomass grown in pig manure was evaluated. Acid and basic pretreatments provided the highest volatile solids solubilisation (up to 81%) followed by alkaline-peroxide and ultrasounds (23%). Bead milling and steam explosion remarkably increased the methane production rate, although the highest yield (377 mL CH4/g SV) was achieved by alkali pretreatment. Nevertheless, some pretreatments inhibited biogas production and resulted in lag phases of 7-9 days. Hence, experiments using only the pretreated solid phase were performed, which resulted in a decrease in the lag phase to 2-3 days for the alkali pretreatment and slightly increased biomass biodegradability of few samples. The limiting step during the BMP test (hydrolysis or microbial inhibition) for each pretreatment was elucidated using the goodness of fitting to a first order or a Gompertz model. Finally, the use of digestate as biofertilizer was evaluated applying a biorefinery concept.

Technologies for the bioconversion of methane into more valuable products.

Methane, with a global warming potential twenty five times higher than that of CO2 is the second most important greenhouse gas emitted nowadays. Its bioconversion into microbial molecules with a high retail value in the industry offers a potential cost-efficient and environmentally friendly solution for mitigating anthropogenic diluted CH4-laden streams. Methane bio-refinery for the production of different compounds such as ectoine, feed proteins, biofuels, bioplastics and polysaccharides, apart from new bioproducts characteristic of methanotrophic bacteria, has been recently tested in discontinuous and continuous bioreactors with promising results. This review constitutes a critical discussion about the state-of-the-art of the potential and research niches of biotechnologies applied in a CH4 biorefinery approach.