Pretreated sugarcane bagasse matches performance of synthetic media for lipid production with Yarrowia lipolytica.

Engineered strains of Yarrowia lipolytica with modified lipid profiles and other desirable properties for microbial oil production are widely reported but are almost exclusively characterized in synthetic laboratory-grade media. Ensuring translatable performance between synthetic media and industrially scalable lignocellulosic feedstocks is a critical challenge. Yarrowia lipolytica growth and lipid production were characterized in media derived from two-step acid-catalyzed glycerol pretreatment of sugarcane bagasse. Fermentation performance was benchmarked against laboratory-grade synthetic growth media, including detailed characterization of media composition, nitrogen utilization, biomass and lipid production, and fatty acid product profile. A Yarrowia lipolytica strain modified to enable xylose consumption consumed all sugars, glycerol, and acetic acid, accumulating lipids to 34-44 % of cell dry weight. Growth and lipid content when grown in sugarcane bagasse-derived media were equivalent to or better than that observed with synthetic media. These sugarcane bagasse-derived media are suitable for transferable development of Yarrowia lipolytica fermentations from synthetic media.

Establishment of efficient system for bagasse bargaining: Combining fractionation of saccharides, recycling of high-viscosity solvent and dismantling.

Sugarcane bagasse (SCB) has a recalcitrant structure, which hinders its component dismantling and subsequent high value utilization. Some organic solvents are favorable to dismantle lignocellulose, but their high viscosity prevents separation of components and reuse of solvents. Herein, ethylene glycol phenyl ether (EGPE)-acid system is used as an example to develop green and efficient methods to dismantle SCB, purify polysaccharides and lignin, and reuse solvents. Results show that dismantling SCB at 130 °C, 0.5 % H2SO4, and 100 min can obtain 85.5 % cellulose recovery, 94.1 % hemicellulose removal and 83.7 % lignin removal. Different molecular weight saccharides are separated by membranes filtration and centrifugation, and lignin recovered by antisolvent precipitation. The solvent recovered by distillation, achieving high dismantling efficiency of 89.2 % cellulose recovery, 94.1 % hemicellulose removal and 94.4 % lignin removal after four recycles. Results show a promising approach for the closed-loop process of dismantling lignocellulose, fractionating saccharides, and reusing solvents in high-viscosity systems.

The OsNAC41-RoLe1-OsAGAP module promotes root development and drought resistance in upland rice.

Drought is a major environmental stress limiting crop yields worldwide. Upland rice (Oryza sativa) has evolved complex genetic mechanisms for adaptative growth under drought stress. However, few genetic variants that mediate drought resistance in upland rice have been identified, and little is known about the evolution of this trait during rice domestication. In this study, using a genome-wide association study we identified ROOT LENGTH 1 (RoLe1) that controls rice root length and drought resistance. We found that a G-to-T polymorphism in the RoLe1 promoter causes increased binding of the transcription factor OsNAC41 and thereby enhanced expression of RoLe1. We further showed that RoLe1 interacts with OsAGAP, an ARF-GTPase activating protein involved in auxin-dependent root development, and interferes with its function to modulate root development. Interestingly, RoLe1 could enhance crop yield by increasing the seed-setting rate under moderate drought conditions. Genomic evolutionary analysis revealed that a newly arisen favorable allelic variant, proRoLe1-526T, originated from the midwest Asia and was retained in upland rice during domestication. Collectively, our study identifies an OsNAC41-RoLe1-OsAGAP module that promotes upland rice root development and drought resistance, providing promising genetic targets for molecular breeding of drought-resistant rice varieties.

Natural variation in the Tn1a promoter regulates tillering in rice.

Rice tillering is an important agronomic trait that influences plant architecture and ultimately affects yield. This can be genetically improved by mining favourable variations in genes associated with tillering. Based on a previous study on dynamic tiller number, we cloned the gene Tiller number 1a (Tn1a), which encodes a membrane-localised protein containing the C2 domain that negatively regulates tillering in rice. A 272 bp insertion/deletion at 387 bp upstream of the start codon in the Tn1a promoter confers a differential transcriptional response and results in a change in tiller number. Moreover, the TCP family transcription factors Tb2 and TCP21 repress the Tn1a promoter activity by binding to the TCP recognition site within the 272 bp indel. In addition, we identified that Tn1a may affect the intracellular K+ content by interacting with a cation-chloride cotransporter (OsCCC1), thereby affecting the expression of downstream tillering-related genes. The Tn1a+272 bp allele, associated with high tillering, might have been preferably preserved in rice varieties in potassium-poor regions during domestication. The discovery of Tn1a is of great significance for further elucidating the genetic basis of tillering characteristics in rice and provides a new and favourable allele for promoting the geographic adaptation of rice to soil potassium.

NOX4-mediated astrocyte ferroptosis in Alzheimer's disease.

This study investigates NADPH oxidase 4 (NOX4) involvement in iron-mediated astrocyte cell death in Alzheimer's Disease (AD) using single-cell sequencing data and transcriptomes. We analyzed AD single-cell RNA sequencing data, identified astrocyte marker genes, and explored biological processes in astrocytes. We integrated AD-related chip data with ferroptosis-related genes, highlighting NOX4. We validated NOX4's role in ferroptosis and AD in vitro and in vivo. Astrocyte marker genes were enriched in AD, emphasizing their role. NOX4 emerged as a crucial player in astrocytic ferroptosis in AD. Silencing NOX4 mitigated ferroptosis, improved cognition, reduced Aß and p-Tau levels, and alleviated mitochondrial abnormalities. NOX4 promotes astrocytic ferroptosis, underscoring its significance in AD progression.

QTL detection and candidate gene identification of qCTB1 for cold tolerance in the Yunnan plateau landrace rice.

Cold stress is one of the main abiotic stresses that affects rice growth and production worldwide. Dissection of the genetic basis is important for genetic improvement of cold tolerance in rice. In this study, a new source of cold-tolerant accession from the Yunnan plateau, Lijiangxiaoheigu, was used as the donor parent and crossed with a cold-sensitive cultivar, Deyou17, to develop recombinant inbred lines (RILs) for quantitative trait locus (QTL) analysis for cold tolerance at the early seedling and booting stages in rice. In total, three QTLs for cold tolerance at the early seedling stage on chromosomes 2 and 7, and four QTLs at the booting stage on chromosomes 1, 3, 5, and 7, were identified. Haplotype and linear regression analyses showed that QTL pyramiding based on the additive effect of these favorable loci has good potential for cold tolerance breeding. Effect assessment in the RIL and BC3F3 populations demonstrated that qCTB1 had a stable effect on cold tolerance at the booting stage in the genetic segregation populations. Under different cold stress conditions, qCTB1 was fine-mapped to a 341-kb interval between markers M3 and M4. Through the combination of parental sequence comparison, candidate gene-based association analysis, and tissue and cold-induced expression analyses, eight important candidate genes for qCTB1 were identified. This study will provide genetic resources for molecular breeding and gene cloning to improve cold tolerance in rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01488-3.

The wall-associated kinase GWN1 controls grain weight and grain number in rice.

Grain size is a crucial agronomic trait that determines grain weight and final yield. Although several genes have been reported to regulate grain size in rice (Oryza sativa), the function of Wall-Associated Kinase family genes affecting grain size is still largely unknown. In this study, we identified GRAIN WEIGHT AND NUMBER 1 (GWN1) using map-based cloning. GWN1 encodes the OsWAK74 protein kinase, which is conserved in plants. GWN1 negatively regulates grain length and weight by regulating cell proliferation in spikelet hulls. We also found that GWN1 negatively influenced grain number by influencing secondary branch numbers and finally increased plant grain yield. The GWN1 gene was highly expressed in inflorescences and its encoded protein is located at the cell membrane and cell wall. Moreover, we identified three haplotypes of GWN1 in the germplasm. GWN1hap1 showing longer grain, has not been widely utilized in modern rice varieties. In summary, GWN1 played a very important role in regulating grain length, weight and number, thereby exhibiting application potential in molecular breeding for longer grain and higher yield.

Natural variation in MORE GRAINS 1 regulates grain number and grain weight in rice.

Grain yield is determined mainly by grain number and grain weight. In this study, we identified and characterized MORE GRAINS1 (MOG1), a gene associated with grain number and grain weight in rice (Oryza sativa L.), through map-based cloning. Overexpression of MOG1 increased grain yield by 18.6%-22.3% under field conditions. We determined that MOG1, a bHLH transcription factor, interacts with OsbHLH107 and directly activates the expression of LONELY GUY (LOG), which encodes a cytokinin-activating enzyme and the cell expansion gene EXPANSIN-LIKE1 (EXPLA1), positively regulating grain number per panicle and grain weight. Natural variations in the promoter and coding regions of MOG1 between Hap-LNW and Hap-HNW alleles resulted in changes in MOG1 expression level and transcriptional activation, leading to functional differences. Haplotype analysis revealed that Hap-HNW, which results in a greater number and heavier grains, has undergone strong selection but has been poorly utilized in modern lowland rice breeding. In summary, the MOG1-OsbHLH107 complex activates LOG and EXPLA1 expression to promote cell expansion and division of young panicles through the cytokinin pathway, thereby increasing grain number and grain weight. These findings suggest that Hap-HNW could be used in strategies to breed high-yielding temperate japonica lowland rice.

Enhanced enzymatic sugar production from corn stover by combination of water extraction and glycerol-assisted instant catapult steam explosion.

Glycerol-assisted instant catapult steam explosion (ICSE) of lignocellulose is an effective pretreatment method for enhancing sugar production compared to glycerol-free ICSE. In this study, glycerol-assisted ICSE of corn stover was studied in order to understand the reaction mechanisms and further optimize the process. Results showed that water extraction of corn stover prior to ICSE reduced pseudo-lignin formation. The combination of water extraction and glycerol-assisted ICSE led to the formation of lignin with a lower molecular weight (Mw) of 2851 g/mol than 3521 g/mole of that from the combination of water extraction and glycerol-free ICSE. 1H-13C NMR analysis revealed that glycerol likely reacted with lignin carboxylic OHs through esterification while etherification of aliphatic OHs was not observed in ICSE. These lignin analyses indicated that glycerol protected lignin from condensation/repolymerization during glycerol-assisted ICSE. Enzymatic hydrolysis results showed that without water extraction increasing glycerol usage from 0.2 kg/kg stover to 0.4 kg/kg stover improved glucan digestibility to 78% but further increase to 0.5 kg/kg stover reduced glucan digestibility. In addition, at the glycerol usage of 0.2-0.4 kg/kg stover, washing of pretreated stover for removal of glycerol and other biomass-derived compounds did not improve glucan digestibility compared to unwashed ones. Combination of water extraction and glycerol-assisted ICSE led to a high glucan digestibility of 89.7% and a total glucose yield of 25.5 g glucose/100 g stover, which were 30.1% and 7.5 g/100 g stover higher than those derived from glycerol-free ICSE of stover, respectively. Since glycerol is a low-cost carbon source, the resulting enzymatic hydrolysate that contained both glucose and glycerol may be directly used to produce bioproducts by microbial fermentation.

Upland rice genomic signatures of adaptation to drought resistance and navigation to molecular design breeding.

Upland rice is a distinctive drought-aerobic ecotype of cultivated rice highly resistant to drought stress. However, the genetic and genomic basis for the drought-aerobic adaptation of upland rice remains largely unclear due to the lack of genomic resources. In this study, we identified 25 typical upland rice accessions and assembled a high-quality genome of one of the typical upland rice varieties, IRAT109, comprising 384 Mb with a contig N50 of 19.6 Mb. Phylogenetic analysis revealed upland and lowland rice have distinct ecotype differentiation within the japonica subgroup. Comparative genomic analyses revealed that adaptive differentiation of lowland and upland rice is likely attributable to the natural variation of many genes in promoter regions, formation of specific genes in upland rice, and expansion of gene families. We revealed differentiated gene expression patterns in the leaves and roots of the two ecotypes and found that lignin synthesis mediated by the phenylpropane pathway plays an important role in the adaptive differentiation of upland and lowland rice. We identified 28 selective sweeps that occurred during domestication and validated that the qRT9 gene in selective regions can positively regulate drought resistance in rice. Eighty key genes closely associated with drought resistance were appraised for their appreciable potential in drought resistance breeding. Our study enhances the understanding of the adaptation of upland rice and provides a genome navigation map of drought resistance breeding, which will facilitate the breeding of drought-resistant rice and the "blue revolution" in agriculture.

Low-temperature pretreatment by AlCl3-catalyzed 1,4-butanediol solution for producing 'ideal' lignin with super-high content of ß-O-4 linkages.

High contents of internal ß-O-4 linkages in lignin are critical for high-yield production of high-value aromatic monomers by depolymerization. However, it remains great challenge due to lack of suitable protection strategy. In this work, a very effective lignin-first strategy was developed to produce ideal lignin with a super high content of ß-O-4 linkages (up to 72 %) from poplar, in which the pretreatment was undertaken at low temperatures of 90-130 °C with the use of AlCl3-catalyzed 1, 4-butanediol solution. 2D-HSQC NMR spectra revealed that lignin ß-O-4 linkages were protected from etherification of the OH group by 1, 4-butanediol at the α position of lignin aliphatic chains. Besides, the OH groups at the γ position of lignin was also etherified, leading the formation of a structure of Ph-CH=CHCH2O(CH2)4OH. Interestingly, structure protection facilitated the formation of lignin nanoparticles via self-assembly (<100 nm). In addition, it was observed from pyrolysis results that addition of 1, 4-butanediol remarkably protected the structure of lignin by avoiding condensation, promoting the production of aromatics. The cellulose-rich fraction possessed a high cellulose digestibility of 91.64 % by enzymatic hydrolysis at a cellulase dosage of 15 FPU/g cellulose, approximately 6-fold untreated poplar (15.91 %). This low-temperature lignin-first strategy was of great importance for multi-products biorefining lignocellulose because it leads to the production of both lignin with super high content of ß-O-4 linkages for depolymerization and highly digestible cellulose for sugar production.

Adsorption of NO2, SO2, H2S, and NH3 on Os-Doped WSe2 Monolayers: A First-Principles Study.

In this study, DFT calculations are used to analyze the adsorption of industrial waste gases (NO2, SO2, H2S, and NH3) on WSe2 monolayers. The adsorption energy, energy band, density of states, charge transfer, and recovery time of the adsorption structures between the target gas molecules and the Os-doped WSe2 are studied. Compared with pure WSe2 monolayer, Os surface bonding doping WSe2 (Os-modified WSe2) and Os doping with Se vacancy of WSe2 (Os-embedded WSe2) exhibit improved gas molecule adsorption ability. Among them, the adsorption energy of the Os-modified WSe2 monolayer on NO2, SO2, H2S, and NH3 is greater than that of the WSe2 monolayer. At the same time, it is proved that the Os-embedded WSe2 can be used as a gas sensor for H2S and NH3 gas molecules at a high temperature.

Genome-Wide Association Study Reveals the Genetic Basis of Total Flavonoid Content in Brown Rice.

Flavonoids have anti-inflammatory, antioxidative, and anticarcinogenic effects. Breeding rice varieties rich in flavonoids can prevent chronic diseases such as cancer and cardio-cerebrovascular diseases. However, most of the genes reported are known to regulate flavonoid content in leaves or seedlings. To further elucidate the genetic basis of flavonoid content in rice grains and identify germplasm rich in flavonoids in grains, a set of rice core collections containing 633 accessions from 32 countries was used to determine total flavonoid content (TFC) in brown rice. We identified ten excellent germplasms with TFC exceeding 300 mg/100 g. Using a compressed mixed linear model, a total of 53 quantitative trait loci (QTLs) were detected through a genome-wide association study (GWAS). By combining linkage disequilibrium (LD) analysis, location of significant single nucleotide polymorphisms (SNPs), gene expression, and haplotype analysis, eight candidate genes were identified from two important QTLs (qTFC1-6 and qTFC9-7), among which LOC_Os01g59440 and LOC_Os09g24260 are the most likely candidate genes. We also analyzed the geographic distribution and breeding utilization of favorable haplotypes of the two genes. Our findings provide insights into the genetic basis of TFC in brown rice and could facilitate the breeding of flavonoid-rich varieties, which may be a prevention and adjuvant treatment for cancer and cardio-cerebrovascular diseases.

Genetic-based dissection of resistance to bacterial leaf streak in rice by GWAS.

BACKGROUND: Rice is the second-largest food crop in the world and vulnerable to bacterial leaf streak disease. A thorough comprehension of the genetic foundation of agronomic traits was essential for effective implementation of molecular marker-assisted selection. RESULTS: Our study aimed to evaluate the vulnerability of rice to bacterial leaf streak disease (BLS) induced by the gram-negative bacterium Xanthomonas oryzae pv. oryzicola (Xoc). In order to accomplish this, we first analyzed the population structure of 747 accessions and subsequently assessed their phenotypes 20 days after inoculation with a strain of Xoc, GX01. We conducted genome-wide association studies (GWAS) on a population of 747 rice accessions, consisting of both indica and japonica subpopulations, utilizing phenotypic data on resistance to bacterial leaf streak (RBLS) and sequence data. We identified a total of 20 QTLs associated with RBLS in our analysis. Through the integration of linkage mapping, sequence analysis, haplotype analysis, and transcriptome analysis, we were able to identify five potential candidate genes (OsRBLS1-OsRBLS5) that possess the potential to regulate RBLS in rice. In order to gain a more comprehensive understanding of the genetic mechanism behind resistance to bacterial leaf streak, we conducted tests on these genes in both the indica and japonica subpopulations, ultimately identifying superior haplotypes that suggest the potential utilization of these genes in breeding disease-resistant rice varieties. CONCLUSIONS: The findings of our study broaden our comprehension of the genetic mechanisms underlying RBLS in rice and offer significant insights that can be applied towards genetic improvement and breeding of disease-resistant rice in rapidly evolving environmental conditions.

RRS1 shapes robust root system to enhance drought resistance in rice.

A strong root system facilitates the absorption of water and nutrients from the soil, to improve the growth of crops. However, to date, there are still very few root development regulatory genes that can be used in crop breeding for agriculture. In this study, we cloned a negative regulator gene of root development, Robust Root System 1 (RRS1), which encodes an R2R3-type MYB family transcription factor. RRS1 knockout plants showed enhanced root growth, including longer root length, longer lateral root length, and larger lateral root density. RRS1 represses root development by directly activating the expression of OsIAA3 which is involved in the auxin signaling pathway. A natural variation in the coding region of RRS1 changes the transcriptional activity of its protein. RRS1T allele, originating from wild rice, possibly increases root length by means of weakening regulation of OsIAA3. Knockout of RRS1 enhances drought resistance by promoting water absorption and improving water use efficiency. This study provides a new gene resource for improving root systems and cultivating drought-resistant rice varieties with important values in agricultural applications.

Different Doping of VSe2 Monolayers as Adsorbent and Gas Sensing Material for Scavenging and Detecting SF6 Decomposed Species.

The application of intrinsic and transition metals (TM)-doped VSe2 monolayers for the detection of faulty gases in SF6 electrical insulated equipment is investigated based on first-principles calculations. The electron density difference, density of state, and adsorption energy are analyzed to further clarify the reaction mechanism. The results show that the intrinsic VSe2 monolayer has weak adsorption performance for SO2 and SOF2 molecules, but the adsorption properties of the system are significantly improved after doping TM atoms. Among them, the TM-doped VSe2 monolayer has better sensing performance for SO2 than for SOF2 molecules. Furthermore, the modulating effect of biaxial strain on the gas-sensitive properties of TM-doped VSe2 system is also analyzed. Finally, the recovery time of the gas molecules on the solid adsorbent is evaluated. The results confirm that the TM-doped VSe2 monolayer can be used as a novel sensing material or scavenger to ensure the normal operation of SF6 electrical insulated equipment. This will provide a prospective insight for experimenters to implement VSe2-based sensing materials or scavengers.

Adsorption properties of small gas molecules on SnSe2 monolayer supported with transition metal: first-principles calculations.

The adsorption properties of CH4, H2S, SO2, CO, H2O and NO molecules on transition metal-supported SnSe2 surface are investigated by the first-principles method. The calculation results show that the transition metal (TM = Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu) has the lowest adsorption energy when supporting at the Sn site of SnSe2, indicating the system is relatively stable. Also, we find that CH4, SO2 and H2O molecules tend to adsorb on Sc-supported SnSe2 surface, H2S and NO molecules prefer to adsorb on V-supported SnSe2 surface, while CO molecule and Fe-supported SnSe2 surfaces have strong interaction. And, CH4, H2S and H2O molecules act as donors to provide electrons to the substrate, while SO2, CO and NO molecules act as acceptors to gain electrons from the substrate. An analysis of charge difference density and density of states reveals that the adsorption energies of gas molecules are related to charge transfer and orbital hybridization. We hope that this work not only provides a promising sensor material, but also provides a new idea for the rational design of two-dimensional materials.

Natural variation in Tiller Number 1 affects its interaction with TIF1 to regulate tillering in rice.

Tiller number per plant-a cardinal component of ideal plant architecture-affects grain yield potential. Thus, alleles positively affecting tillering must be mined to promote genetic improvement. Here, we report a Tiller Number 1 (TN1) protein harbouring a bromo-adjacent homology domain and RNA recognition motifs, identified through genome-wide association study of tiller numbers. Natural variation in TN1 affects its interaction with TIF1 (TN1 interaction factor 1) to affect DWARF14 expression and negatively regulate tiller number in rice. Further analysis of variations in TN1 among indica genotypes according to geographical distribution revealed that low-tillering varieties with TN1-hapL are concentrated in Southeast Asia and East Asia, whereas high-tillering varieties with TN1-hapH are concentrated in South Asia. Taken together, these results indicate that TN1 is a tillering regulatory factor whose alleles present apparent preferential utilization across geographical regions. Our findings advance the molecular understanding of tiller development.

Variations in OsSPL10 confer drought tolerance by directly regulating OsNAC2 expression and ROS production in rice.

Drought is a major factor restricting the production of rice (Oryza sativa L.). The identification of natural variants for drought stress-related genes is an important step toward developing genetically improved rice varieties. Here, we characterized a member of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family, OsSPL10, as a transcription factor involved in the regulation of drought tolerance in rice. OsSPL10 appears to play a vital role in drought tolerance by controlling reactive oxygen species (ROS) production and stomatal movements. Haplotype and allele frequency analyses of OsSPL10 indicated that most upland rice and improved lowland rice varieties harbor the OsSPL10Hap1 allele, whereas the OsSPL10Hap2 allele was mainly present in lowland and landrace rice varieties. Importantly, we demonstrated that the varieties with the OsSPL10Hap1 allele showed low expression levels of OsSPL10 and its downstream gene, OsNAC2, which decreases the expression of OsAP37 and increases the expression of OsCOX11, thus preventing ROS accumulation and programmed cell death (PCD). Furthermore, the knockdown or knockout of OsSPL10 induced fast stomatal closure and prevented water loss, thereby improving drought tolerance in rice. Based on these observations, we propose that OsSPL10 confers drought tolerance by regulating OsNAC2 expression and that OsSPL10Hap1 could be a valuable haplotype for the genetic improvement of drought tolerance in rice.

Effects of lignocellulosic biomass type on the economics of hydrothermal treatment of digested sludge for solid fuel and soil amendment applications.

Digested sludge is a waste stream from anaerobic digestion (AD) in wastewater treatment plants. Hydrothermal treatment (HTT) of sludge mixed with lignocellulosic biomass is an attractive approach to improve sludge dewaterability and generate value-added products. However, process economics has not been well understood. In this study, firstly, the effect of biomass type on the energy properties of hydrochars was studied. Secondly, two scenarios were simulated to evaluate the effects of biomass type on the economics (processing 50,000 tonnes of sludge per year) of HTT of digested sludge for solid fuel and soil amendment applications. The two HTT scenarios included sludge alone and sludge-biomass mixtures (four cases for four biomass feedstocks) at 180 °C for 60 min. In both scenarios, HTT liquids were returned to existing AD facilities for biomethane production to offset the energy cost of the HTT process. The results showed that the higher heating value significantly increased from 16.0-17.0 MJ kg-1 in the sludge alone case to 18.0-23.0 MJ kg-1 in sludge-biomass mixtures (except for rice husk). With the use of saved transport cost as a revenue source, HTT of sludge-biomass led to a net present value (NPV) range of AU$ 9.9-20.3 million (20 years) and an internal rate of return (IRR) range of 25.0 %-45.2 % for solid fuel application of resulting hydrochar compared to an NPV of AU$ 18.4 million and an IRR of 55.0 % from HTT of sludge alone scenario. HTT of sludge-biomass led to a NPV range of AU$ 4.5-14.5 million and an IRR range of 17.2 %-35.7 % for soil amendment application while the hydrochar from HTT of sludge alone was not recommended for soil application due to the high contents of heavy metals. This study provides useful and critical information for process scale-up and commercialization for integration into wastewater treatment plants.