Production of oxalic acid and use in co 2-sequestering cement

Abstract

Methods of manufacturing oxalic acid from lignocellulosic biomass and use of the manufactured oxalic acid the acidic activator in a new-concept, acid-base cement (ABC) are disclosed.

Description

PRODUCTION OF OXALIC ACID AND USE IN CO2- SEQUESTERING CEMENTCROSS-REFERENCE TO RELATED APPLICATION

[0001] This invention claims priority to U.S. Provisional Application Serial No. 63 / 515,205, filed July 24, 2023, the disclosure of which is herein incorporated by reference in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under EE0008256 and EE0008910 awarded by the Department of Energy. The government has certain rights in the invention.BACKGROUND OF THE DISCLOSURE

[0003] The present disclosure relates generally to the manufacture and utilization of oxalic acid, derived from lignocellulosic biomass. In one particularly suitable embodiment, the oxalic acid is manufactured and then used as the acidic activator in a new-concept, acid-base cement (ABC) made exclusively from abundant but under-utilized base materials obtained from local industrial byproducts or solid wastes. The methods of the present disclosure allow for reduction in atmospheric CO2 by transforming cement manufacturing into a calcination-free (i.e. , low embodied energy) and CCh-negative process by avoiding energy- and CCh-intensive (~1 t CCh / t cement), thermal decomposition of CaCCh and kiln-processing of other raw materials. Recent advances in preprocessing of biomass into high concentration slurries provides a unique feedstock for cost-effective production of oxalic acid, which can then be used as a cement additive, thereby sequestering atmospheric CO2, initially captured in biomass, in the concrete manufacturing process.

[0004] Annual global consumption of concrete is 30 Gt and 4.5 Gt in the US. Concrete is a ubiquitous component of roads, building, and global infrastructure. The US Department of Energy has identified the cement manufacturing sector as one of the most impactful areas to target for emissions and energy reduction since it contributes to nearly 10% of the anthropogenic CO2 emissions. The dominant cementin the US, ordinary Portland cement (PC), generates between 0.70 to 0.95 tons CO2 per ton. A significant fraction of the >4 Gt CO2 emitted from manufacturing of ~4.5 Gt cement per year can potentially be turned into negative carbon emissions. Traditionally, the term “cement” refers to the hydraulic binders of concretes that are formulated from Portland cement (PC) clinker. The major challenges of reducing CO2 load due to the Clinker (PC) are related to CaCCh decomposition and calcination at 1450°C that converts CaO and other oxides into clinker. In total, this leads to emission of nearly It CO2 from It PC. The U.S. industry is promoting Type IL cement, which uses up to 15% ground limestone to replace PC clinker, and thus, reduces CO2 emissions associated with cement by about 10%. Decade-long research in Europe on limestone calcined clay cement (LC3) where limestone and calcined clay replaces up to 45% of PC clinker (LC3, 2014) has resulted in commercial products that claim 30% lower CO2 emissions. However, further improvements in the eco- efficiency of cement by this approach are limited. This has resulted in the need for new opportunities for attaining a carbon-neutral / negative goal.

[0005] Other attractive niche solutions - such as alkali-activated materials and related approaches by recent XPRIZE winners (XPRIZE, 2021) - do not yet achieve the combined performance and eco- and economic-efficiencies needed for larger scale application. Electrochemical approaches to CaO conversion may reduce a portion of emission from fuel burning, but have little impact on emissions due to decomposition and conversion of CaO into clinker.

[0006] Numerous studies and large-scale projects have begun to address scalable methods for decreasing release of carbon from agricultural production, and conversely utilizing agriculture to capture and sequester atmospheric carbon. The present disclosure provides solutions to the above by utilizing oxalic acid, derived from lignocellulosic biomass as the acidic activator in a new-concept, acid-base cement (ABC) made exclusively from abundant, but utilized base materials, obtained from local industrial byproducts. Examples of these base materials include copper slag in Arizona; steel slag in Pennsylvania and Indiana; mine tailings; waste rock of matic / ultramatic nature; and globally abundant concrete recycling fines (CRF). Unlike traditional Portland cement (PC) that requires manufacturing temperatures of 1450°C and results in release of 0.8-ton CO2 / ton cement, the new-concept ABC avoids kilnprocessing and decreases CO2 emissions by 80%, while maintaining desired concrete properties. ABC thus represents a novel carbon capture, utilization, and storage (CCUS) strategy that sequesters CO2 in mineralized form in concrete that when successfully developed, is applicable at a Gt scale.BRIEF DESCRIPTION OF THE DISCLOSURE

[0007] Generally, the present disclosure is directed to methods of producing oxalic acid by fermenting a carbohydrate source with a microbial to form a fermentation broth and then isolating oxalic acid from the fermentation broth.

[0008] The present disclosure is further directed to methods of manufacturing CCh-sequestering cement. The methods provide for the manufacture and utilization of oxalic acid, derived from lignocellulosic biomass, as the acidic activator in a new- concept, acid-base cement (ABC). The methods of the present disclosure allow for reduction in atmospheric CO2 by transforming cement manufacturing into a calcination-free (i.e., low embodied energy) and C Ch-negative process by avoiding energy- and CCh-intensive (~1 1 CCh / t cement), thermal decomposition of CaCCh and kiln-processing of other raw materials. In one embodiment, the method comprises: fermenting a carbohydrate source with a microbial to form a fermentation broth; isolating oxalic acid from the fermentation broth; mixing oxalic acid with at least one material comprising silicate or oxide to produce the CCh-sequestering cement.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The disclosure will be better understood, and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:

[0010] FIG. 1 depicts the combination of biomass derived carboxylic acid (i.e., oxalic acid) that sequesters CO2 within a new' concept acid-base (ABC) cement as described in the present disclosure.

[0011] FIG. 2A depicts traditional cement (i.e., Portland cement (PC) manufacturing paradigm and sources of CO2 emission.

[0012] FIG. 2B depicts future manufacturing paradigm of the carbon-negative OxCem and sources of CO2 emission.

[0013] FIG. 3 shows a standard curve for mixed organic acids that was diluted accordingly from the stock solution as described in Example 1.

[0014] FIG. 4 depicts comparison of the oxalic acid peak in the samples and the standards as analyzed in Example 1.

[0015] FIG. 5 depicts yield stress of 300 g / L decreased from > 6000 (not shown) to < 200 (liquefied slurry) as analyzed in Example 2.

[0016] FIG. 6 depicts oxalic acid (OA) precipitation with CaC'E from fermentation broth. Day 0 and 10, ImL of sample in 30 mL of 10% (w / v) CaCh as analyzed in Examples 2 and 3.

[0017] FIG. 7 depicts rheology of liquefied 300 g / L biomass as described in Example 3.DETAILED DESCRIPTION

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

[0019] The present disclosure is directed to the manufacture and utilization of oxalic acid, derived from lignocellulosic biomass, as the acidic activator in a new- concept, acid-base cement (ABC) made exclusively from abundant but under-utilized base materials obtained from local industrial byproducts or solid wastes. These materials may include copper and steel slags and mine tailings abundant in certain regions, and globally abundant concrete recycling fines (CRF). Unlike traditional Portland Cement (PC), ABC does not require energy-intensive manufacturing processes and 1450°C temperatures that generates nearly 1-ton CO2 per ton of cement. The new-concept ABC avoids kiln processing and CO2 emissions due to limestone decomposition, maintains desired concrete properties, and, in addition, sequesters CO2 when biomass-derived oxalic acid is used.

[0020] Biomass itself (for example com stalks or sugarcane bagasse) integrates CO2 into its structure and thereby represents a form of sequestered carbon. Sustainable sources of lignocellulosic biomass are available at the meaningful scalerequired for the proposed approach with 1 to 1.5 gigaton (Gt) generated annually and available for bioconversion according to a recent Department of Energy (DOE) study (reported at BETO Peer Review, April 2023). Carbon integrated into biomass remains sequestered when the cellulosic fraction is fermented to oxalic acid (C2O4H2). This results in long-term carbon sequestration when concrete is formed from ABC.Separately, researchers have shown oxalic acid is an effective curing agent that improves the properties of cement / concrete, and is more effective than concentrated CO2, which also has been tested for this purpose.

[0021] Biomass-derived Oxalic Acid. Traditional methods to produce oxalic acid face several issues including: high cost, energy-intensity, and pollution (due to heavy usage of strong acids). Further, energy-efficient synthesis of oxalic acid from biomass or CO2, as envisioned by EU’s OCCEAN project (EU, "Oxalic acid from CO2 using electrochemistry at demonstration scale," SPIRE 2030, 2018. [Online], Available: https: / / www.spire2030.eu / ocean; E. Schuler, M. Demetriou, N. R. Shiju and G. Gruter, "Towards sustainable oxalic acid from CO2 and biomass," ChemSusChem, vol. 14. pp. 3636-3664. 2021), utilizes renewable resources (i.e.. biomass) or energy (e.g., solar thermal energy and electricity for electrochemical reduction). However, electrochemical reduction of CO2 is associated with high electricity consumption that competes with other manufacturing sectors.

[0022] The invention described here utilizes oxalic acid from biomass via fermentation pathways.

[0023] Feedstock for oxalic acid manufacture: Biomass slurries are formed using a recently developed approach and achieve up to 300 g / L lignocellulose in a pumpable fluid (i.e,. a shear thinning slurry with a low yield stress) based on a results with a 1 liter, agitated fed batch bioreactor containing low levels of endoglucanase enzyme (Szeto et al, Biotech Prog 37(6), e3216)). The formation of high solids slurries is key to achieving plant operability7and having the possibility7of containing product at sufficiently high concentration to be practical (i.e., between 10 and 200 g / L).

[0024] Through the fermentation process described herein, it is foreseeable to obtain pumpable and mixable slurries initially containing up to 300 g / L carbohydrate source and corresponding target yield stresses < 200 Pa (corresponds to slurries thatmay be pumped). These slurries are the basic feedstock for production of oxalic acid based on fermentation with microbes such as Aspergillus species.[0025 J More particularly, the fermentation used in the methods of the present disclosure include the use of carbohydrate sources such as glucose from starch in com, sucrose from sugarcane or sugar beet, sugars from food processing wastes, as well as glucose derived from lignocellulosic materials including crop residues (including com stover, wheat straw, soybean hulls, sugar cane bagasse, hardwood chips), as well as purposely grown cellulosic crops including grasses and energy cane. The latter category7of materials, i.e., lignocellulosics, are processed to glucose, xylose, and other monosaccharides through the hydrolysis of the structural carbohydrates in these biomass sources. The derived monosaccharides are fermentable to oxalic acid and other bio products.

[0026] While starch is broken down to glucose by amylases and glucoamylase enzy mes, lignocellulosic materials are hydrolyzed to sugars when acids in aqueous media are used at temperatures above 140°C, or by enzymes that represent a consortium of hydrolytic enzymes (cellulose enzyme mixtures that include endocellulases, cellobiohydrolases, and beta- glucosidases) that carry out hydrolysis at about 50°C and pH 4.5 to 5.5.

[0027] The amount of carbohydrate (i.e. lignocellulosic) required for the fermentation step is in the range of 100 to 300 g / L solids initially, at an enzyme loading of 1 to 20 FPU / g solids (equivalent to about 2 to 40 mg enzyme protein per g solids). Hydrolysis to sugars that may be fermented to oxalic acid by a separate microorganism (or the same microorganisms in cases where the microbe generates its own cellulases) is carried out at pH 4 to 6 (depending on type of enzyme that is used), 50°C, mild agitation, for 24 to 96 hours. Once the hydrolysis (which may be accompanied by simultaneous fermentation to the desired product, oxalic acid, but also to fuels and other bioproducts) occurs, the total undissolved solids will decrease and sugars that are fermentable by Aspergillus or yeasts or other microbes as described herein are generated at equivalent concentrations of between 30 and 180 g / L.

[0028] Suitable microbes for use in the fermentation process includes fungi strains of Aspergillus, Trichoderma and Penicillium species. The fungi produce amixture of oxalic acid, citric acid and gluconic acid, as a media acidification mechanism to prevent competitive microorganisms' growth. With the goal of biomass fermentation for oxalic acid production, it is fundamental that the fungus is able to produce cellulolytic enzymes in order to be able to break down cellulose into sugars.

[0029] Most of the research available in the literature report the use of modified Aspergillus niger strains, with overexpression of oxaloacetate hydrolase enzyme and / or lacking glucose oxidase. However, OA titers up to 21 g / L have also been reported in fermentations with wild-type A. niger strain, in periods of 10 to 12 days. Accordingly, particularly suitable strains include P. stiptica (BRFM1148), G. weberianum (BRFM1548), A. brasiliensis (BRFM103), A. niger (BRFM420), A. niger (BRFM421).A. niger (BRFM422), A. niger (BRFM431). A. niger (BRFM434), A. niger 12, A. niger 3T5B8, A. oryzae P27C3, T. reesei Rut-C30, A. niger A12, mutant A. niger WU-2223L, mutant A. niger WU-2223L, and mutant A. carbonarius.

[0030] The combination of high solids concentrations, low yield stress, and fermentation to oxalic acid opens up a billion-ton potential for utilizing agriculture to provide large volumes of oxalic acid that capture CO2 when the biomass-derived oxalic acid is sequestered in the cement product, which itself has a dramatically decreased carbon footprint, resulting in a CO2 negative cement.

[0031] Recovery of the oxalic acid product is based on a prosaic approach. The same ability to form hydrates when oxalic acid is mixed with sinters, will be used in recovering fermentation derived carboxylic acid by adding Ca or other divalent ions to filtered fermentation broth. Filtration before precipitation of the product removes residual biomass particles and other large particulates. Filtration or centrifugation after precipitation recovers the oxalate precipitate. Washing of the precipitate removes adhering impurities. Partial drying gives oxalic acid suitable for cement formulation

[0032] Further, in one embodiment, removal may be achieved by addition of small amounts of cement to the clarified filtration broth.

[0033] Basics of ABC. The obtained oxalic acid is the added to components for use in making ABC cements. Acid-base cements (ABCs) are special alternative cements formulated by a base (e.g., MgO and FeO) and an acidic component (e.g., hydrophosphates). Magnesium phosphate cement (MPC) is the most widely-studiedABC because of its performance merits. MPC sets quickly even at low temperatures (<-10°C). and produces high strength concrete with little shrinkage and superior durability as compared to Portland cement (PC) concrete. Carbonation-hardening cements (CHCs) are another class of ABCs, which use reactive MgO or wollastonite (CaO SiO2) as base, and CO2 as the acidic component.

[0034] Due to the need of CO2 curing, CHCs are limited to pre-fabrication (attractive in the carbon capture, utilization, and storage (CCUS) market). Despite the merits, wide-spread applications of ABCs still need to develop. Apart from raw materials scarcity and need for calcination to produce oxide bases, technical concerns include: (1) difficulty to control setting; (2) strength reduction upon long exposure to water because of mild solubility of reaction products; and (3) poor steel protectiveness - anecdotally - because of the intrinsically low pH (8-10). The UN report cautions that the goal of substantive CO2 reduction from cement industry is too rigorous to be addressed by existing approaches. This goal must rely on innovations (e.g., alternative cements) that minimize decomposition of CaCOs and combustion of fuels; enable CCUS; and maintain performance comparable to PC.

[0035] The methods of the present disclosure seek to revolutionize cement manufacturing by avoiding calcination; leveraging abundant but overlooked raw materials with low / negative carbon-embodiment; enabling carbon-negative manufacturing to result in precipitated oxalic acid, enabling carbon sequestration by added lignocellulosic biomass derived OA to cement, to serve as a curing agent for concrete formation and simultaneously sequestering OA.EXAMPLEEXAMPLE 1

[0036] In this Example, oxalic acid is produced from com stover using the methods of the present disclosure.Materials and MethodsPre-culture preparation

[0037] The growth medium for amplifying Aspergillus niger contained 1.4 g of (NH4)2SO4, 2 g of KH2PO4, 0.3 g of CaCh, 0.3 g of urea, 5 g of Soy peptone, 2 of gyeast extract, 1 ml of Tween 80, 1 ml of lx salt solution, 0.2g of MgSO^YFbO, and 30 g of Glucose.[0038J A lx salt solution contained 5 mg FeSO4-7H2O, 1.6 mg MnSCh-FhO, 1.4 mg ZnSO4-7H2O, and 2 mg CaCh per liter. Because of the difficulty in measuring small amounts of chemicals above, the salt solution used was prepared as 10 times concentrated and 0. 1 ml if the 1 Ox salt solution was inoculated in growth medium. The pH of the medium was 5.04 initially and adjusted to 4.55 for 4. niger to grow by adding NaOH. The grow th medium and containers were autoclaved.

[0039] Aspergillus niger spores were collected and counted under microscope. Around 2 * 109spores were inoculated into growth medium to make a total of 200 ml mixture (107spores / ml). Then, the fungi were incubated at 32 °C and 200 rpm for 50 hours as a pre-culture.Liquefaction of com stover

[0040] Around 7.5 g of 30% solid com stover pellets were added into 25 ml of water with 400 pl of Celluclast 1.5 enzyme. The mixture was placed in the incubator for 6 hours at 50 °C. After incubation, the slurry (in 2 tubes) was combined into one to make 50 ml liquified material.Inoculum

[0041] A total of 11 flasks were prepared. Each flask contained 50 ml of liquified biomass, 38 ml of lx salt solution, and 10 ml pre-culture. The pH of these mixtures fell in the range from 4.09 to 4.21. In flask 1, 2 ml of IM NaOH was added and raised pH to 7.58. Because the liquified biomass solution has a buffer capacity, the pH decreased to 7. 17 after 5 minutes. Final decisions were made to add 2 ml of the NaOH in every flask to make a 100 ml reaction volume and a pH around 7. pH adjustment

[0042] The pH of the samples w as measured every day from day 2 to day 12. The sample w ith pH that is low er than 6 was raised above 6 by adding 5M kOH.Sample collection

[0043] Samples were collected from day 0, 2, 4. 6, 8, 10 and 12. Biomass and fungi mixtures were collected in falcon tubes and centrifuged at 6800 rpm for 10 minutes. The liquid then, was filtered through 90 mm filter paper. Collected filtrate and leftover solid were kept in freezer for further analysis.HPLC DetectionStandards[0044J A stock solution containing 10 g / L oxalic acid, 2 g / L citric acid and 1 g / L formic acid was first prepared. Data points on standard curve ware tested by diluting the stock solution to 1.5, 1, 0.75, 0.5, 0.25, 0.1 g / L oxalic acid concentration (FIG. 3). The areas under the curve were calculated using the embedded integration function in EMPOWER (HPLC software). Then, the areas under the oxalic acid peak were plotted with their concentration and fitted into a linear model so that an empirical relationship was determined. The model showed a reasonable R square value.HPLC samples and quantification

[0045] After unfreezing the samples, 1.5 ml filtrate was filtered by a 0.2 pm syringe filter and injected into 2 ml vial. Oxalic acid measurement used column #87, flow speed at 0.6 ml / min, temperature at 30 °C, and 0.005M H2SO4 as mobile phase. Results were collected by RI and UV detector. RI detector recorded all the detectable peaks including sugar and organic acids. UV detector at 210 nm to 218 nm range (214 nm were commonly used) detected oxalic acid. However, UV detection result should be under 1 AU to be useful. So, samples were diluted 12 times to fall in the suitable range. The results were shown in Figure 3. The area under the peak was then determined by the integration function in EMPOWER. For DO and D2. the function had trouble identifying the start point and end point for oxalic acid peak. The decision was made to manually set the start point at 6.43 mins and end at 7.28 mins. The estimated concentration, then, was calculated by the function modeled in FIG. 4. Results are shown in Table 1.Table 1 Estimation of the Oxalic acid concentration in fermented samplesTitrimetric method for oxalic acid quantification

[0046] Samples from fermented broth were analyzed by reduction with KMnO4. Oxalic acid from fermented broth was precipitated with 20ml of 10% CaCf at boiling temperature. The precipitated calcium oxalate was separated by centrifuging and w ashed with 100 ml of distilled water. Centrifuged pellet was then dried in the vacuum oven to obtain the dry matter. The dried precipitate was dissolved in HSO4, and the liberated oxalic acid was determined by titration with KMnO4. The same procedure was carried for the oxalic acid standard of lOmg / ml oxalic acid.EXAMPLE 2

[0047] In this Example, the energy-efficient pathway of producing cementforming activator from biomass (e g., cornstalk) will be analyzed.Carbon (CO2 in the air) is captured by the biomass and utilized by fungal fermentation for organic acid production. A convergent approach that combines biomass derived acid activators, and particularly oxalic acid, in acid-base cement (ABC) is proposed, thereby providing a pathway to sequester biologically fixed carbon in ABC cement. Initially, microbial selection is started with a search of culture collections for Aspergillus spp (key candidates in Table 2). Directed evolution and application of molecular or synthetic biology will be applied to enhance rates, yields, and concentrations of oxalic production and possibly other carboxylic acids identified below.Table 2: Kobayashi; Yang et al., 2014. Yoshoika et al., 2020

[0048] The proposed research plan may at first appear to be counter-intuitive since acids may be corrosive to formed concrete and since the emphasis on directing evolution of industrial Aspergillus strains has been on suppressing oxalic acid formation in order to maximize citric acid production for a range of metallurgical and food uses. Citric acid is the world's largest consumed organic acid, and has been produced by A. niger for over 50 years. When oxalic acid is incorporated as part of the concrete curing process, and forms oxalate hydrates of Ca, Mg, or Fe, the effect is the opposite. The concrete is protected. The carboxylate hydrates become an integral part of the concrete structure and inhibit corrosion of concrete and imbedded iron rebar. For development of suitable^, niger strains, the approach will maximize oxalic acid while minimizing citric acid (co-product of the citric acid cycle). In order to enhance rates, yields, and titer of oxalic acid, high glucose concentrations and anacidic pH (at 4 to 6), known to lead to synthesis of oxalic acid over citric acid, will be used. High glucose concentrations will require high slurry concentrations of lignocellulosic biomass, which in itself has proven challenging, but has been addressed.

[0049] Research in the laboratory has demonstrated that high slurry' concentrations may be achieved by mixing com stover pellets with minimal amounts of cellulase enzyme to achieve 300 g / L slurries (i. e. , liquefied biomass) with yield stresses < 200 Pa (FIG. 3) (dos Santos et al., 2021; Szeto et al, 2021). Hence, the slurries are readily pumped and agitated which in turn enables efficient, submerged fermentations of high initial concentrations of lignocellulosic substrates, which is a necessary condition for the high product titers (i.e., 100 g / L) that allow efficient recovery of oxalic acid by precipitation. A slurry fermentation is carried out to obtain oxalic with an in-house strain of A. niger (A-12), and then detect the product by adding fermentation broth to CaCl2(FIG. 4). These conditions are not practical, but demonstrate the concept of production of oxalic acid directly from cellulosic slurry prepared using enzyme, water, and agitation without heating or pretreatment.

[0050] In order to achieve high yields of biomass derived acid activator and retain the inherent low7carbon footprint associated with lignocellulosic biomass, efficient wild-type microorganisms, in addition to the ones that have already been tested, will be identified from known carboxylic acid producers. In order to improve yields, strain mutagenesis of A. niger can be achieved randomly and / or be predicted and modelled in fermentation performance using genome-scale metabolic models (Upton et al., 2017, 2020). Advances in A. niger CRISPR / Cas9 genome editing for citric acid production by CRISPR / Cas9 is well developed, leading to a yield of 0.95 g citric acid / g of glucose after fermentation optimization (Tong, 2019; Pappagiani, 2007). Succinic acid production in Aspergillus has been increased via ribonucleoprotein-based CRISPR-Cas9 system, including studies of fermentation from biomass (beet molasses and wheat straw) (Y ang et al., 2020).

[0051] Fungal strains of the filamentous fungi Aspergillus niger, Trichoderma reesei, and Phanerochaete chrysosporium have been reported as producers of acetic, citric, and succinic acids (among others), with acetic acid inducing succinic acid production (Alcantara, 2021). Hundreds of filamentous fungi have completelysequenced genomes (Kuck, 2010), and over 66 strains have been already been as organic acids producers. Among them, Aspergilli species has shown to be the most robust, acid- and thermo-tolerant, and able to grow in wide pH and temperature ranges (Schuster et al., 2002). Aspergillus niger is also capable of producing cellulolytic enzymes, which are key for lignocellulose bioconversion and more recently liquefaction of lignocellulosic biomass (Cunha et al, 2014; Liaud et al., 2014). Advances in A. niger CRISPR / Cas9 genome editing for citric acid production by CRISPR / Cas9 is very well developed, leading to a yield of 0.95 g citric acid / g of glucose after fermentation optimization (Tong, 2019; Pappagiani, 2007). These methods will be applied to maximize another product of the citric acid cycle, i.e.. oxalic acid. Succinic acid production in Aspergillus has been increased via ribonucleoprotein-based CRISPR-Cas9 system, including studies of fermentation from biomass (beet molasses and wheat straw) (Yang et al., 2020). These will form the starting point of research for Example 3.EXAMPLE 3

[0052] In this Example, biological processes for converting biomass into organic acids will be based on the enzyme catalyzed liquefaction of lignocellulosic biomass resulting in high biomass concentrations with rheology that ensures sufficient rates and yields when fermented in an agitated bioreactor. The input of energy / chemicals that other processes use to obtain fermentable feedstocks will be minimized or avoided altogether so that the carbon benefit of the biomass-to- chemicals strategy is maximal. It is hypothesized that selected or engineered microorganisms will enable synthesis of organic acids directly from high concentration lignocellulosic biomass slurries. Oxalic acid will be the focus of the first biomass conversion project. Achieving high slurry concentrations of biomass resulting in high glucose concentrations, that together with low pH, enhance the rate, yield, and titer of oxalic acids, has been recently addressed. Specifically, 300 g / L slurries (with yield stresses <200 Pa) liquefied from com stover pellets using minimal amounts of cellulase enzyme have been obtained (FIG. 6). This type of slurry has been subsequently been used, initially at 150 g / L, to cany' out baseline tests with Aspergillus to obtain oxalic acid. Microbial selection and development will beaddressed by the team starting with a search for filamentous fungi that have been reported as organic acid producers. Hundreds of such fungi have completely sequenced genomes, among which Aspergillus niger has shown to be the most robust and resilient for producing cellulolytic enzymes and converting lignocellulose. For citric acid production, A. niger CRISPR / Cas9 genome editing has been applied to maximize its production while suppressing other byproducts, leading to a yield of 0.95 g citric acid / g of glucose after fermentation optimization. The same type of technique will be applied, except to maximize another product of the citric acid cycle, i.e. oxalic acid. Methods include strain mutagenesis of A. niger to maximize the yield of oxalic acid while suppressing citric acid and other byproducts. Production of oxalic acid will also be maximized by using high substrate concentration and low fermentation pH. Preliminary success has been achieved with an niger fermentation of thick com stover slurry, with first runs yielding oxalic acid recovered by precipitation with CaCL (FIG. 5).EXAMPLE 4

[0053] In this Example, cement and alternative concrete properties of CO2 sequestering ABC cement (i.e., OxCem) will be tested at the kg scale. The cement will be formulated with oxalic acid initially produced from liquefied lignocellulosic com stover in 100 to 200 mL shaker-flask and scaled to 1 to 10 L agitated bioreactor fermentations. These results will be compared against ordinary Portland Cement. Oxalic acid manufactured from petroleum feedstocks does not incorporate the benefits of the low carbon footprint product from lignocellulosic biomass obtained through fermentation or possibly other conversion pathways.

[0054] Initially biomass-derived oxalic acid will be separated and recovered from the fermentation broth by methanol + HC1 precipitation and then added to various formulations of the modified cement for testing. As research progresses, a “breadboard'’ will be assembled that scales fermentation to 10 L and employs OA recovery by precipitation with Ca++ or other divalent cations. Sufficient OA will result to be able to formulate and test 2 to 5 kg cement samples at environmentally realistic conditions. This will culminate in a prototy pe demonstration, accompanied with a full economic analysis, assessment of Life Cycle Assessment (LCA)parameters and GHG impact based on bench-scale measurements. Models that relate cement properties to carbon sequestration potential will be developed and validated against experimental measurements, resulting in a scalable protype, i.e., cement formulations for alternative concrete that are positioned for development of manufacturing accompanied by large scale testing.

Claims

CLAIMSWhat is claimed is:

1. A method of producing oxalic acid, the method comprising: fermenting a carbohydrate source with a microbial to form a fermentation broth; isolating oxalic acid from the fermentation broth.

2. The method according to claim 1, wherein the carbohydrate source is selected from the group consisting of com, wheat, sugarcane, sugar beet, bagasse, hardwood, grasses, and lignocellulosics.

3. The method according to claim 2, wherein the carbohydrate source provides a carbohydrate selected from the group consisting of glucose, sucrose, xylose, starch and other monosaccharides.

4. The method according to claim 1 comprising fermenting from about 100 g / L to about 300 g / L carbohydrate source with the microbial.

5. The method according to claim 1, wherein the microbial is selected from the group consisting of P. stiptica (BRFM1148), G. weberianum (BRFM1548), A. brasiliensis (BRFM103). A. niger (BRFM420), A. niger (BRFM421), A. niger (BRFM422), A. niger (BRFM431), A. niger (BRFM434), A. niger 12. A. niger 3T5B8, A. oryzae P27C3, T. reesei Rut-C30, A. niger A12, mutant A. niger WU- 2223L, mutant A. niger WU-2223L, and mutant A. carbonarius .

6. The method according to claim 1, wherein fermenting occurs at a pH of from about 4 to about 6.

7. The method according to claim 1, wherein fermenting occurs for a period of from about 24 hours to about 96 hours.

8. The method according to claim 1 further comprising hydrolyzing the carbohydrate source prior to fermenting the carbohydrate source with the microbial to form the fermentation broth.

9. The method according to claim 8 comprising hydrolyzing the carbohydrate source with an acid in aqueous media at a temperature above 140°C.

10. The method according to claim 8 comprising hydrolyzing the carbohydrate source with an enzy me at a temperature of about 50°C and a pH of from about 4.5 to about 5.5.

11. The method according to claim 10, wherein enzyme is selected from the group consisting of cellulose, endo-cellulase, amylase, glycoamylase, cellobiohydrolase and beta-glucosidases.

12. The method according to claim 10 comprising hydrolyzing the carbohydrate source with from about 1 FPU / g to about 20 FPU / g enzyme.

13. The method according to claim 8 comprising fermenting from about 30 g / L to about 180 g / L carbohydrate source with the microbial.

14. The method according to claim 1 comprising isolating the oxalic acid from the fermentation broth by filtration and precipitation.

15. The method according to claim 14, wherein divalent ions are added to filtered fermentation broth to precipitate oxalic acid.

16. The method according to claim 15, wherein the divalent ions are calcium ions.

17. The method according to claim 14 further comprising washing the precipitate from the fermentation broth.

18. The method according to claim 14 further comprising drying the precipitate from the fermentation broth.

19. A method of manufacturing CCh-sequestering cement with the oxalic acid produced according to claim 1, comprising mixing the produced oxalic acid with at least one material comprising silicate or oxide to produce the CCh-sequestering cement20. The method according to claim 19, wherein the at least one material is an oxide selected from the group consisting of MgO and FeO to produce the CO2- sequestering cement.

21. The method according to claim 19 further comprising mixing a hydrophospate with the oxalic acid and at least one material.

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