Current paradigms and future challenges in harnessing gut bacterial symbionts of insects for biodegradation of plastic wastes.

The ubiquitous incorporation of plastics into daily life, coupled with inefficient recycling practices, has resulted in the accumulation of millions of metric tons of plastic waste, that poses a serious threat to the Earth's sustainability. Plastic pollution, a global problem, disrupts the ecological balance and endangers various life forms. Efforts to combat plastic pollution are underway, with a promising avenue being biological degradation facilitated by certain insects and their symbiotic gut microorganisms, particularly bacteria. This review consolidates existing knowledge on plastic degradation by insects and their influence on gut microbiota. Additionally, it delves into the potential mechanisms employed by insects in symbiosis with gut bacteria, exploring the bioconversion of waste plastics into value-added biodegradable polymers through mineralization. These insights hold significant promise for the bio-upcycling of plastic waste, opening new horizons for future biomanufacturing of high-value chemicals from plastic-derived compounds. Finally, we weigh the pros and cons of future research endeavors related to the bioprospection of plastic-degrading bacteria from underexplored insect species. We also underscore the importance of bioengineering depolymerases with novel characteristics, aiming for their application in the remediation and valorization of waste plastics.

Fermentative Production of Ergothioneine by Exploring Novel Biosynthetic Pathway and Remodulating Precursor Synthesis Pathways.

Ergothioneine (EGT) is a naturally occurring derivative of histidine with diverse applications in the medicine, cosmetic, and food industries. Nevertheless, its sustainable biosynthesis faces hurdles due to the limited biosynthetic pathways, complex metabolic network of precursors, and high cost associated with fermentation. Herein, efforts were made to address these limitations first by reconstructing a novel EGT biosynthetic pathway from Methylobacterium aquaticum in Escherichia coli and optimizing it through plasmid copy number. Subsequently, the supply of precursor amino acids was promoted by engineering the global regulator, recruiting mutant resistant to feedback inhibition, and blocking competitive pathways. These metabolic modifications resulted in a significant improvement in EGT production, increasing from 35 to 130 mg/L, representing a remarkable increase of 271.4%. Furthermore, an economical medium was developed by replacing yeast extract with corn steep liquor, a byproduct of wet milling of corn. Finally, the production of EGT reached 595 mg/L with a productivity of 8.2 mg/L/h by exploiting fed-batch fermentation in a 10 L bioreactor. This study paves the way for exploring and modulating a de novo biosynthetic pathway for efficient and low-cost fermentative production of EGT.

Efficient Fermentative Production of ß-Alanine from Glucose through Multidimensional Engineering of Escherichia coli.

ß-Alanine, a valuable ß-type amino acid, is experiencing increased demand due to its multifaceted applications in food flavoring, nutritional supplements, pharmaceuticals, and the chemical industry. Nevertheless, the sustainable biosynthesis of ß-alanine currently faces challenges due to the scarcity of robust strains, attributed to the complexities of modulating multiple genes and the inherent physiological constraints. Here, systems metabolic engineering was implemented in Escherichia coli to overcome these limitations. First, an efficient l-aspartate-α-decarboxylase (ADC) was recruited for ß-alanine biosynthesis. To conserve phosphoenolpyruvate flux, we subsequently modified the endogenous glucose assimilation system by inactivating the phosphotransferase system (PTS) and introducing an alternative non-PTS system, which increased ß-alanine production to 1.70 g/L. The supply of key precursors, oxaloacetate and l-aspartate, was synergistically improved through comprehensive modulation, including strengthening main flux and blocking bypass metabolism, which significantly increased the ß-alanine titer to 3.43 g/L. Next, the expression of ADC was optimized by promoter and untranslated region (UTR) engineering. Further transport engineering, which involved disrupting ß-alanine importer CycA and heterologously expressing ß-alanine exporter NCgI0580, improved ß-alanine production to 8.48 g/L. Additionally, corn steep liquor was used to develop a cost-effective medium. The final strain produced 74.03 g/L ß-alanine with a yield of 0.57 mol/mol glucose during fed-batch fermentation.

Random mutagenesis and transcriptomics-guided rational engineering in Zygosaccharomyces rouxii for elevating D-arabitol biosynthesis.

D-arabitol, a versatile compound with applications in food, pharmaceutical, and biochemical industries, faces challenges in biomanufacturing due to poor chassis performance and unclear synthesis mechanisms. This study aimed to enhance the performance of Zygosaccharomyces rouxii to improve D-arabitol production. Firstly, a mutant strain Z. rouxii M075 obtained via atmospheric and room temperature plasma-mediated mutagenesis yielded 42.0 g/L of D-arabitol at 96 h, with about 50 % increase. Transcriptome-guided metabolic engineering of pathway key enzymes co-expression produced strain ZR-M3, reaching 48.9 g/L D-arabitol after 96 h fermentation. Finally, under optimized conditions, fed-batch fermentation of ZR-M3 in a 5 L bioreactor yielded an impressive D-arabitol titer of 152.8 g/L at 192 h, with a productivity of 0.8 g/L/h. This study highlights promising advancements in enhancing D-arabitol production, offering potential for more efficient biomanufacturing processes and wider industrial applications.

Metabolic Remodulation of Chassis and Corn Stover Bioprocessing to Unlock 3-Hydroxypropionic Acid Biosynthesis from Agrowaste-Derived Substrates.

Embracing the principles of sustainable development, the valorization of agrowastes into value-added chemicals has nowadays received significant attention worldwide. Herein, Escherichia coli was metabolically rewired to convert cellulosic hydrolysate of corn stover into a key platform chemical, namely, 3-hydroxypropionic acid (3-HP). First, the heterologous pathways were introduced into E. coli by coexpressing glycerol-3-P dehydrogenase and glycerol-3-P phosphatase in both single and fusion (gpdp12) forms, making the strain capable of synthesizing glycerol from glucose. Subsequently, a glycerol dehydratase (DhaB123-gdrAB) and an aldehyde dehydrogenase (GabD4) were overexpressed to convert glycerol into 3-HP. A fine-tuning between glycerol synthesis and its conversion into 3-HP was successfully established by 5'-untranslated region engineering of gpdp12 and dhaB123-gdrAB. The strain was further metabolically modulated to successfully prevent glycerol flux outside the cell and into the central metabolism. The finally remodulated chassis produced 32.91 g/L 3-HP from the cellulosic hydrolysate of stover during fed-batch fermentation.

Sustainable bio-manufacturing of D-arabitol through combinatorial engineering of Zygosaccharomyces rouxii, bioprocess optimization and downstream separation.

Biosynthesis of D-arabitol, a high value-added platform chemical, from renewable carbon sources provides a sustainable and eco-friendly alternative to the chemical industry. Here, a robust brewing yeast, Zygosaccharomyces rouxii, capable of naturally producing D-arabitol was rewired through genome sequencing-based metabolic engineering. The recombinant Z. rouxii obtained by reinforcing the native D-xylulose pathway, improving reductive power of the rate-limiting step, and inhibiting the shunt pathway, produced 73.61% higher D-arabitol than the parent strain. Subsequently, optimization of the fermentation medium composition for the engineered strain provided 137.36 g/L D-arabitol, with a productivity of 0.64 g/L/h in a fed-batch experiment. Finally, the downstream separation of D-arabitol from the complex fermentation broth using an ethanol precipitation method provided a purity of 96.53%. This study highlights the importance of D-xylulose pathway modification in D-arabitol biosynthesis, and pave a complete and efficient way for the sustainable manufacturing of this value-added compound from biosynthesis to preparation.

A comprehensive review of recent advances in the characterization of L-rhamnose isomerase for the biocatalytic production of D-allose from D-allulose.

D-Allose and D-allulose are two important rare natural monosaccharides found in meager amounts. They are considered to be the ideal substitutes for table sugar (sucrose) for, their significantly lower calorie content with around 80 % and 70 % of the sweetness of sucrose, respectively. Additionally, both monosaccharides have gained much attention due to their remarkable physiological properties and excellent health benefits. Nevertheless, D-allose and D-allulose are rare in nature and difficult to produce by chemical methods. Consequently, scientists are exploring bioconversion methods to convert D-allulose into D-allose, with a key enzyme, L-rhamnose isomerase (L-RhIse), playing a remarkable role in this process. This review provides an in-depth analysis of the extractions, physiological functions and applications of D-allose from D-allulose. Specifically, it provides a detailed description of all documented L-RhIse, encompassing their biochemical properties including, pH, temperature, stabilities, half-lives, metal ion dependence, molecular weight, kinetic parameters, specific activities and specificities of the substrates, conversion ratio, crystal structure, catalytic mechanism as well as their wide-ranging applications across diverse fields. So far, L-RhIses have been discovered and characterized experimentally by numerous mesophilic and thermophilic bacteria. Furthermore, the crystal forms of L-RhIses from E. coli and Stutzerimonas/Pseudomonas stutzeri have been previously cracked, together with their catalytic mechanism. However, there is room for further exploration, particularly the molecular modification of L-RhIse for enhancing its catalytic performance and thermostability through the directed evolution or site-directed mutagenesis.

Rewiring Bacillus subtilis and bioprocess optimization for oxidoreductive reaction-mediated biosynthesis of D-tagatose.

D-tagatose holds significant importance as a functional monosaccharide with diverse applications in food, medicine, and other fields. This study aimed to explore the oxidoreductive pathway for D-tagatose production, surpassing the contemporary isomerization-mediated biosynthesis approach in order to enhance the thermodynamic equilibrium of the reactions. Initially, a novel galactitol dehydrogenase was discovered through biochemical and bioinformatics analyses. By co-expressing the galactitol dehydrogenase and xylose reductase, the oxidoreductive pathway for D-tagatose synthesis was successfully established in Bacillus subtilis. Subsequently, pathway fine-tuning was achieved via promoter regulation and dehydrogenase-mediated cofactor regeneration, resulting in 6.75-fold higher D-tagatose compared to that produced by the strain containing the unmodified promoter. Finally, optimization of fermentation conditions and medium composition produced 39.57 g/L D-tagatose in a fed-batch experiment, with a productivity of 0.33 g/L/h and a yield of 0.55 mol/mol D-galactose. These findings highlight the potential of the constructed redox pathway as an effective approach for D-tagatose production.

Efficient biosynthesis of 3-hydroxypropionic acid from glucose through multidimensional engineering of Escherichia coli.

3-Hydroxypropionic acid (3-HP) is a top value-added chemical with multifaceted application in chemical, material, and food field. However, limited availability of robust strains and elevated fermentation costs currently impose constraints on sustainable biosynthesis of 3-HP. Herein, transporter engineering, metabolic dynamic modulation, and enzyme engineering were combined to address above limitations. First, a glucose-utilizing 3-HP biosynthetic pathway was constructed in Escherichia coli, followed by recruiting alternative glucose transport system to overcome center metabolism overflow. Next, the Cra (a transcription factor)-dependent switch was applied to autonomously fine-tune carbon flux, which alleviated growth retardation and improved the 3-HP production. Subsequently, inactivation of glycerol facilitator (GlpF) increased intracellular glycerol levels and boosted 3-HP biosynthesis, but caused toxic intermediate 3-hydroxypropionaldehyde (3-HPA) accumulation. Furthermore, semi-rational design of aldehyde dehydrogenase (YdcW) increased its activity and eliminated 3-HPA accumulation. Finally, fed-batch fermentation of the final strain resulted in 52.73 g/L 3-HP, with a yield of 0.59 mol/mol glucose.

Enhanced fermentable sugar production in lignocellulosic biorefinery by exploring a novel corn stover and configuring high-solid pretreatment conditions.

This study aimed to produce enhanced fermentable sugars from a novel stover system through the bioprocessing of its soluble sugars and insoluble carbohydrates. The pretreatment conditions were optimized for this high sugar-containing stover (HSS) to control inhibitor formation and obtain enhanced fermentable sugar concentrations. The optimum temperature, acid loading, and reaction time for the pretreatment were 155 °C, 0.5%, and 30 min, respectively, providing up to 97.15% sugar yield and 76.51 g/L total sugars at 10% solid-load. Sugar concentration further increased to 126.9 g/L at 20% solid-load, generating 3.89 g/L acetate, 0.92 g/L 5-hydroxymethyl furfural, 0.82 g/L furfural, and 3.75 g/L total phenolics as inhibitors. To determine the effects of soluble sugars in HSS on fermentable sugar yield and inhibitor formation, sugar-removed HSS was further studied under the optimum conditions. Although prior removal of sugars exhibited a reduction in inhibitor generation, it also decreased total fermentable sugar production to 115.45 g/L.

Biocatalytic gateway to convert glycerol into 3-hydroxypropionic acid in waste-based biorefineries: Fundamentals, limitations, and potential research strategies.

Microbial conversion of bioenergy-derived waste glycerol into value-added chemicals has emerged as an important bioprocessing technology due to its eco-friendliness, feasible technoeconomics, and potential to provide sustainability in biodiesel and bioethanol production. Glycerol is an abundant liquid waste from bioenergy plants with a projected volume of 6 million tons by 2025, accounting for about 10% of biodiesel and 2.5% of bioethanol yields. 3-Hydroxypropionic acid (3-HP) is a major product of glycerol bioconversion, which is the third largest biobased platform compound with expected market size and value of 3.6 million tons/year and USD 10 billion/year, respectively. Despite these biorefinery values, 3-HP biosynthesis from glycerol is still at an immature stage of commercial exploitation. The main challenges behind this immaturity are the toxic effects of 3-HPA on cells, the distribution of carbon flux to undesirable pathways, low tolerance of cells to glycerol and 3-HP, co-factor dependence of enzymes, low enzyme activity and stability, and the problems of substrate inhibition and specificity of enzymes. To address these challenges, it is necessary to understand the fundamentals of glycerol bioconversion and 3-HP production in terms of metabolic pathways, related enzymes, cell factories, midstream process configurations, and downstream 3-HP recovery, as discussed in this review critically and comprehensively. It is equally important to know the current challenges and limitations in 3-HP production, which are discussed in detail along with recent research efforts and remaining gaps. Finally, possible research strategies are outlined considering the recent technological advances in microbial biosynthesis, aiming to attract further research efforts to achieve a sustainable and industrially exploitable 3-HP production technology. By discussing the use of advanced tools and strategies to overcome the existing challenges in 3-HP biosynthesis, this review will attract researchers from many other similar biosynthesis technologies and provide a common gateway for their further development.

d-Arabitol Ameliorates Obesity and Metabolic Disorders via the Gut Microbiota-SCFAs-WAT Browning Axis.

d-Arabitol, which is typically found in mushrooms, lichens, and higher fungi, might play an effective role in alleviating visceral fat accumulation and insulin resistance particularly for its low calorie and glycemic index. However, the regulatory mechanisms of d-arabitol for alleviating obesity and associated metabolic disorders remain poorly understood. This study aimed to investigate and analyze the underlying relationship between d-arabitol-mediated gut microbiota and obesity. The results showed that d-arabitol dramatically ameliorated body weight gain, fat accumulation, and insulin resistance in HFD-fed rats. Likewise, d-arabitol remarkably increased the relative abundance of the genera Blautia, Anaerostipes, and Phascolarctobacterium and decreased the genera Romboutsia and Clostridium_sensu_stricto_1. Furthermore, these alterations in gut microflora increased SCFAs, which in turn indirectly promoted AMPK-PGC-1α-related white adipose tissue (WAT) browning. Therefore, d-arabitol would have the potential to alleviate obesity through the gut microbiota-SCFAs-WAT browning axis. It could be considered as a sugar substitute for the obese population and diabetic patients.

High-level co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol: Metabolic engineering and process optimization.

3-Hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO) are value-added chemicals with versatile applications in the chemical, pharmaceutical, and food industries. Nevertheless, sustainable production of 3-HP and 1,3-PDO is often limited by the lack of efficient strains and suitable fermentation configurations. Herein, attempts have been made to improve the co-production of both metabolites through metabolic engineering of Escherichia coli and process optimization. First, the 3-HP and 1,3-PDO co-biosynthetic pathways were recruited and optimized in E. coli, followed by coupling the pathways to the transhydrogenase-mediated cofactor regeneration systems that increased cofactor availability and product synthesis. Next, pathway rebalancing and block of by-product formation significantly improved 3-HP and 1,3-PDO net titer. Subsequently, glycerol flux toward 3-HP and 1,3-PDO synthesis was maximized by removing metabolic repression and fine-tuning the glycerol oxidation pathway. Lastly, the combined fermentation process optimization and two-stage pH-controlled fed-batch fermentation co-produced 140.50 g/L 3-HP and 1,3-PDO, with 0.85 mol/mol net yield.

High-level production of d-arabitol by Zygosaccharomyces rouxii from glucose: Metabolic engineering and process optimization.

d-Arabitol is a top value-added compound with wide applications in the food, pharmaceutical and biochemical industries. Nevertheless, sustainable biosynthesis of d-arabitol is limited by lack of efficient strains and suitable fermentation process. Herein, metabolic engineering and process optimization were performed in Zygosaccharomyces rouxii to overcoming these limitations. Adopting systems metabolic engineering include enhancement of innate biosynthetic pathway, supply of precursor substrate d-ribulose-5P and cofactors regeneration, a novel recombinant strain ZR-5A with good performance was obtained, which boosted d-arabitol production up to 29.01 g/L, 59.31 % higher than the parent strain. Further with the optimum medium composition and fed-batch fermentation, the strain ZR-5A finally produced 149.10 g/L d-arabitol with the productivity of 1.04 g/L/h, which was the highest titer ever reported by Z.rouxii system. This is the first report on the use of metabolic engineering to construct Z. rouxii chassis for the sustainable production of d-arabitol.

Enhancing biomass and lipid productivity of a green microalga Parachlorella kessleri for biodiesel production using rapid mutation of atmospheric and room temperature plasma.

BACKGROUND: Microalgae, with their high adaptability to various stress conditions and rapid growth, are considered excellent biomass resources for lipid production and biodiesel feedstocks. However, lipid yield and productivity of the natural strains are common bottlenecks in their large-scale use for lipid production, which can be overcome by evolving new strains using conventional and advanced mutagenic techniques. It is challenging to generate microalgae strains capable of high lipid synthesis through natural selection. As a result, random mutagenesis is currently considered a viable option in many scenarios. The objective of this study was to explore atmospheric and room temperature plasma (ARTP) as a random mutagenesis technique to obtain high lipid-accumulating mutants of a green microalga for improved biodiesel production. RESULTS: A green microalgal species was isolated from the Chinese Yellow Sea and identified as Parachlorella kessleri (OM758328). The isolated microalga was subsequently mutated by ARTP to obtain high lipid-accumulating mutants. Based on the growth rate and lipid content, 5 mutants (named M1, M2, M4, M5, and M8) were selected from 15 pre-selected mutants. These five mutants varied in their growth rate from 0.33 to 0.68 day-1, with the lipid content varying between 0.25 g/L in M2 to 0.30 g/L in M8 at 10th day of cultivation. Among the mutants, M8 showed the maximum biomass productivity (0.046 g/L/day) and lipid productivity (20.19 mg/L/day), which were 75% and 44% higher than the wild strain, respectively. The triglyceride (TAG) content of M8 was found to be 0.56 g/L at 16th day of cultivation, which was 1.77-fold higher than that of the wild strain. Furthermore, M8 had the highest saturated fatty acids (C16-18) with the lowermost polyunsaturated fatty acid content, which are favorable properties of a biodiesel feedstock according to international standards. CONCLUSION: The mutant strain of P. kessleri developed by the ARTP technique exhibited significant improvements in biomass productivity, lipid content, and biodiesel quality. Therefore, the biomass of this mutant microalga could be a potential feedstock for biodiesel production.

Improving tolerance and 1,3-propanediol production of Clostridium butyricum using physical mutagenesis, adaptive evolution and genome shuffling.

Bioconversion efficiency of glycerol to 1,3-propanediol (1,3-PD) by Clostridium butyricum is bottlenecked by its low tolerance to various stressors, especially glycerol as the substrate, 1,3-PD as the end product, and butyric acid as a by-product, which eventually decreases 1,3-PD yield. This study aimed at improving the tolerance and 1,3-PD production capability of C. butyricum using random mutagenesis and evolutionary techniques. Mutagenesis of wild strain by atmospheric room temperature plasma (ARTP) provided the first population with maximum tolerance to 160 g/L glycerol, while microbial microdroplet culture system (MMC)-mediated adaptive laboratory evolution (ALE) generated the second population with tolerance to 100 g/L 1,3-PD. Subsequently, genome shuffling of both populations yielded a final strain, GJH-418, which generated 60.12 g/L1,3-PD with a productivity of 1.72 g/L/h. The transcript analysis of the mutant and wild strains revealed the possible involvement of 8 genes in high tolerance and high 1,3-PD production through either up- or down-regulation.

Biocatalytic conversion of a lactose-rich dairy waste into D-tagatose, D-arabitol and galactitol using sequential whole cell and fermentation technologies.

Dairy industry waste has been explored as a cheap and attractive raw material to produce various commercially important rare sugars. In this study, a lactose-rich dairy byproduct, namely cheese whey powder (CWP), was microbially converted into three low caloric sweeteners using whole-cell and fermentation technologies. Firstly, the simultaneous lactose hydrolysis and isomerization of lactose-derived D-galactose into D-tagatose was performed by an engineered Escherichia coli strain co-expressing ß-galactosidase and L-arabinose isomerase, which eventually produced 68.35 g/L D-tagatose during sequential feeding of CWP. Subsequently, the mixed syrup containing lactose-derived D-glucose and residual D-galactose was subjected to fermentation by Metschnikowia pulcherrima E1, which produced 60.12 g/L D-arabitol and 28.26 g/L galactitol. The net titer of the three rare sugars was 156.73 g/L from 300 g/L lactose (equivalent to 428.57 g/L CWP), which was equivalent to 1.12 mol product/mol lactose and 52.24% conversion efficiency in terms of lactose.

Enrichment and balancing of nutrients for improved methane production using three compositionally different agro-livestock wastes: Process performance and microbial community analysis.

Balanced nutrition is important for maximizing anaerobic digestion (AD) performance. Herein, the strategy of balancing sugar-fiber-nitrogen nutrients was first established for improved methane production by co-digesting two agricultural and one livestock wastes with complementary compositional properties, such as banana pseudo-stem (BPS), sugarcane baggage (SCB), and chicken manure (CM) having high sugar, fiber and nitrogen contents, respectively. The maximum methane yield was 186.5 mL/g VSadded with a mixture of 45.7% BPS, 26.2% SCB and 28.1% CM (with 1: 11.3: 0.3 of sugar to fiber to nitrogen ratio), increasing by 16.1%, 53.3%, 122.6% than those of mono- BPS, SCB, and CM, respectively. The co-digestion process remained stable under an organic load of 4 g VS/(L·day), which was attributed to the predominant presence of Bacteroidetes, Proteobacteria, Thauera, uncultured_bacterium_p_Aegiribacteria, and hydrogenotrophic methanogens. This study provides a deeper understanding of the co-digestion with agricultural and livestock wastes from the perspective of nutrient balance.

Enhanced Biosynthesis of D-Arabitol by Metschnikowia reukaufii Through Optimizing Medium Composition and Fermentation Conditions.

D-Arabitol is an important functional sugar alcohol, which can be used in the preparation of foods, chemicals, and medicines. Despite biological production of D-arabitol from low-cost substrates has recently been the focus of research, low yield of this technology has limited its large-scale exploitation. Optimization of this bioprocess could be a promising option to improve the yield of D-arabitol. In this study, one-factor-at-a-time (OFAT) strategy and Box-Behnken design (BBD) were used to increase D-arabitol production by Metschnikowia reukaufii CICC 31,858 through optimizing the fermentation conditions and medium composition. The OFAT optimization provided the optimal conditions for temperature, agitation speed, and fermentation time of 30℃, 220 rpm, and 6 days, respectively. Likewise, the optimum concentrations of peptone, ammonium sulfate, KH2PO4, MgSO4·7H2O, and fumaric acid in the fermentation medium were (g/L) 7.5, 1, 2, 0.5, and 7.5, respectively. Under these optimum conditions, 80.43 g/L of D-arabitol was produced from 200 g/L of glucose, with a productivity of 0.56 g/L/h. The BBD optimization with three important components of fermentation medium (KH2PO4, MgSO4·7H2O, and fumaric acid) showed that the predicted titer of D-arabitol varied from 47.21 to 89.27 g/L, and the actual titer of D-arabitol ranged from 47.36 to 89.83 g/L. The optimum concentrations (g/L) of KH2PO4, MgSO4·7H2O, and fumaric acid in the fermentation medium were found to be 1.0, 0.5, and 4.7 g/L, respectively. Under the optimum conditions, 92.45 g/L of D-arabitol was finally produced with the yield and productivity of 0.46 g/g and 0.64 g/L/h, respectively.