Biomass Biorefinery Research Articles

Pretreatment of Wheat Straw with Phosphoric Acid and Hydrogen Peroxide to Simultaneously Facilitate Cellulose Digestibility and Modify Lignin as Adsorbents.

Effective valorization of lignin is crucial to achieve a sustainable, economic and competitive biorefinery of lignocellulosic biomass. In this work, an integrated process was proposed based on a concentrated phosphoric acid plus hydrogen peroxide (PHP) pretreatment to simultaneously facilitate cellulose digestibility and modify lignin as adsorbent. As a dominant constitutor of PHP pretreatment, H2O2 input and its influence on the overall fractionation/lignin modification performance was thoroughly investigated. Results indicated that wheat straw was fractionated more efficiently by increasing the H2O2 input. H2O2 input had a significant influence on the digestibility of the obtained cellulose-rich fraction whereby almost 100.0% cellulose-glucose conversion can be achieved even with only 0.88% H2O2 input. Besides, the adsorption capacity of lignin on MB was improved (74.3 to 210.1 mg g−1) due to the oxidative-modification in PHP pretreatment with H2O2 inputs. Regression analysis indicated that –COOH groups mainly governed the lignin adsorption (R2 = 0.946), which displayed the considerable adsorption capacities for typical cationic substances. This work shows a promising way to integrate the lignin modification concept into the emerging PHP pretreatment process with the dual goal of both cellulose utilization and lignin valorization.

Transcription Factor Atf1 Regulates Expression of Cellulase and Xylanase Genes during Solid-State Fermentation of Ascomycetes.

Transcriptional regulation of cellulolytic and xylolytic genes in ascomycete fungi is controlled by specific carbon sources in different external environments. Here, comparative transcriptomic analyses of Penicillium oxalicum grown on wheat bran (WB), WB plus rice straw (WR), or WB plus Avicel (WA) as the sole carbon source under solid-state fermentation (SSF) revealed that most of the differentially expressed genes (DEGs) were involved in metabolism, specifically, carbohydrate metabolism. Of the DEGs, the basic core carbohydrate-active enzyme-encoding genes which responded to the plant biomass resources were identified in P. oxalicum, and their transcriptional levels changed to various extents depending on the different carbon sources. Moreover, this study found that three deletion mutants of genes encoding putative transcription factors showed significant alterations in filter paper cellulase production compared with that of a parental P. oxalicum strain with a deletion of Ku70 (ΔPoxKu70 strain) when grown on WR under SSF. Importantly, the ΔPoxAtf1 mutant (with a deletion of P. oxalicumAtf1, also called POX03016) displayed 46.1 to 183.2% more cellulase and xylanase production than a ΔPoxKu70 mutant after 2 days of growth on WR. RNA sequencing and quantitative reverse transcription-PCR revealed that PoxAtf1 dynamically regulated the expression of major cellulase and xylanase genes under SSF. PoxAtf1 bound to the promoter regions of the key cellulase and xylanase genes in vitro This study provides novel insights into the regulatory mechanism of fungal cellulase and xylanase gene expression under SSF.IMPORTANCE The transition to a more environmentally friendly economy encourages studies involving the high-value-added utilization of lignocellulosic biomass. Solid-state fermentation (SSF), that simulates the natural habitat of soil microorganisms, is used for a variety of applications such as biomass biorefinery. Prior to the current study, our understanding of genome-wide gene expression and of the regulation of gene expression of lignocellulose-degrading enzymes in ascomycete fungi during SSF was limited. Here, we employed RNA sequencing and genetic analyses to investigate transcriptomes of Penicillium oxalicum strain EU2101 cultured on medium containing different carbon sources and to identify and characterize transcription factors for regulating the expression of cellulase and xylanase genes during SSF. The results generated will provide novel insights into genetic engineering of filamentous fungi to further increase enzyme production.

The challenge of converting biomass polysaccharides into levulinic acid through heterogeneous catalytic processes

AbstractThe differences between a biorefinery and an oil refinery are determined by the higher oxygen content of the biorefinery's biomass, its high degree of functionalization, its low thermal stability, its polar components, which are mostly acidic, its highly heterogeneous structure, and its quality variation as result of genotypic and phenotypic characteristics. Levulinic acid (LA) is one of the main high value‐added chemicals that can be produced from lignocellulosic biomass as raw material. The main challenges for the conversion of lignocellulosic biomass to levulinic acid are related to the improvement of the technologies to obtain a pure and cost‐competitive product, the design and use of efficient heterogeneous catalysts, and the improvements in the selectivity and useful life of the catalyst. This is an up‐to‐date review of the state of knowledge about the heterogeneous catalytic conversion of biomass into LA, addressing the technical hurdles that impede the attainment of high yields. This work outlines the chemistry of LA synthesis and discusses in detail the influence of the lignocellulosic raw material, reaction time, temperature, solvent according to the chemical pathway, and efficiency of the chosen Lewis and Brønsted solid acid catalysts. © 2019 Society of Chemical Industry and John Wiley & Sons, Ltd.

Sequential Fractionation of Lignocellulosic Biomass Using CO2‐Assisted Hydrolysis Combined with γ‐Valerolactone Treatment

A biomass biorefinery strategy for the selective fractionation of poplar into its three main compositions (hemicellulose sugars, lignin, and high‐purity cellulose) with a two‐step process is developed. The first step consists of the selective hydrolysis of hemicellulose at mild conditions in a CO2/H2O system. This leads to various hemicellulose sugars and other intermediates in the water‐soluble fraction, with more than 87.90% of the hemicellulose being extracted from the original poplar (180 °C, 2.0 MPa CO2, 10 min). The pretreated sample is subsequently used for extracting lignin in a γ‐valerolactone (GVL)/H2O with acid catalysts for the next step. Meanwhile, four acid catalysts, including sulfuric acid and solid heteropoly acids (phosphomolybdic acid, silicotungstic acid, and phosphotungstic acid), are studied for the selective delignification in the GVL/H2O system, resulting in more than 91.35% of the original lignin being removed in the cellulose‐rich substrates (140 °C, 30 mM silicotungstic acid, 3 h). Overall, the biorefinery approach fractions the poplar wood into three primary components: 1) various oligosaccharides, monosaccharides, and other intermediates derived from hemicellulose; 2) cellulose‐rich substrate with a purity of cellulose as high as 90.76%, which has great potential as biochemicals or biomaterials; and 3) high‐purity and proportion lignin recovered from biomass.

Bioremoval of heavy metals from metal mine tailings water using microalgae biomass

The copper industry generates large quantities of mine tailings water. Thus, this study aims to select a microalgae species that is both tolerant and capable of the bioremoval of heavy metals from metal mine tailings water as well as the potential biorefinery of this microalgae biomass. Chlorella vulgaris and Scenedesmus spinosus were tested for tolerance to metal mine tailings water (MTW) in northern Chile and synthetic treatments of Cu and Mo (0.1 and 0.5 mg/L). Additionally, the biomass generated was characterized to evaluate its potential applications. The main results showed greater tolerance of C. vulgaris cultured in MTW treatment. In fact, high removal efficiency of Cu and Mo was detected for this microalgae in MTW: 64.7% and 99.9%, respectively. Similarly, Cu (55%) and Mo (80.3%) removal was observed at 0.5 mg/L synthetic concentration treatments after 72 h. However, cell wall fluorescence and chlorophyll parameters were mainly affected by 0.5 mg/L Cu synthetic concentration, where the mean fluorescence intensity (MFI) was 878, compared with the other treatments (≥1800). Morphological cell changes in the MTW treatment were observed using SEM images. The presence of Mo on the microalgae surface was detected by 0.47% and 0.82% in both Mo synthetic treatments exposed. Additionally, the characterization of microalgae biomass exposed to MTW showed a higher protein content and a minor difference of lipid content compared with the control treatment, which could be used in biorefinery processes. This study reveals the capability of C. vulgaris to remove heavy metals from this mine tailings water and the effect that occurs in microalgae cells.

Enhancement of high-solids enzymatic hydrolysis efficiency of alkali pretreated sugarcane bagasse at low cellulase dosage by fed-batch strategy based on optimized accessory enzymes and additives

Obtaining higher amount of final sugars with low cellulase dosage has great economic benefits for the industrial biorefinery of lignocellulosic biomass. The optimization of accessory enzymes and additives were performed using single factor and orthogonal experiment firstly, after that, fed-batch strategy was applied to enhance the high-solids enzymatic hydrolysis efficiency of alkali pretreated sugarcane bagasse (SCB). A novel enzymatic hydrolysis procedure with 22% (w/v) substrate content and cellulase dosage of only 4 FPU/g dry biomass (DM) was developed, after digested for 48 h, the achieved glucose titer, yield and productivity were 122 g/L, 80% and 2.54 g L−1 h−1, respectively. Results obtained in this study indicated a potential finding for the industrial application of lignocellulosic biomass.

Sequential treatment with pressurized fluid processing and ultrasonication for biorefinery of canola straw towards lignocellulosic nanofiber production

Lignocellulosic nanofibers (LCNF) were obtained via pressurized fluid processing and ultrasonication from canola straw. Pressurized aqueous ethanol (PAE) treatment at 140–220 °C, 50–100 bar and 0–20% (v/v) ethanol was evaluated for removal of non-cellulosic material from canola straw. PAE treatment at 180 °C, 50 bar and 20% (v/v) ethanol removed most of the hemicelluloses, leading to a treated fiber mainly composed of cellulose (63%) and lignin (20%). The removal of hemicellulose was confirmed by the functional groups change, increased thermal stability from 326 to 369 °C and crystallinity from 30 to 46% of the PAE treated fiber. Then, ultrasonication at theoretical specific energy (TSE) of 4–20 kJ/g was studied to obtain LCNF from the PAE treated fiber. Ultrasonication at TSE of 20 kJ/g resulted in fibrillation yield of 36 wt.% and LCNF with an average diameter of 21 nm. The sequential treatment with PAE and ultrasonication has great potential for biorefinery of biomass towards LCNF production.

Green Process for Extraction of Lignin by the Microwave-Assisted Ionic Liquid Approach: Toward Biomass Biorefinery and Lignin Characterization

A facile and efficient strategy for the extraction of lignin and lignocellulosic biomass processing is crucial for sustainability of current biorefineries. In this work, lignin was extracted from e...

Pre-treatment of sugarcane bagasse with aqueous ammonia–glycerol mixtures to enhance enzymatic saccharification and recovery of ammonia

In this work, an efficient aqueous ammonia with glycerol (AAWG) method to improve the digestibility of sugarcane bagasse (SCB) was developed. Response surface methodology was utilized to optimize the AAWG parameters to achieve the maximum total fermentable sugar concentration (TFSC) and total fermentable sugar yield (TFSY). Under optimal AAWG conditions, 13.59 g/L TFSC (9.25% ammonia, 1.86 h, 180 °C) and 0.4449 g/g TFSY (9.51% ammonia, 1.78 h, 180 °C) were achieved, with delignification of 77.81% and 70.91%, respectively. Compared to pretreatment with glycerol or aqueous ammonia, the AAWG method significantly enhanced the enzymatic efficiency of SCB. The ammonia was recovered from the pretreatment liquid by distillation, and about one-third of the ammonia was retained. The overall results indicate that AAWG is effectively used as a pretreatment method for recovering ammonia, which would largely contribute to the economic benefits of biomass biorefinery.

Oil Palm Biomass Biorefinery for Sustainable Production of Renewable Materials.

Oil palm biomass is widely known for its potential as a renewable resource for various value-added products due to its lignocellulosic content and availability. Oil palm biomass biorefinery is an industry that comes with sociopolitical benefits through job opportunities, as well as potential environmental benefits. Many studies have been conducted on the technological advancements of oil-palm biomass-derived renewable materials, which are discussed comprehensively in this review. Recent technological developments have made it possible to bring new and innovative technologies to commercialization, such as compost, biocharcoal, biocomposites, and bioplastics.

A unique model of gravity assisted solvent free microwave based extraction of essential oil from mentha leaves ensuring biorefinery of leftover waste biomass for extraction of nutraceuticals: Towards cleaner and greener technology

Abstract: The rising discourse over concerns on climate change due to non environmental compatibility of traditional industrial processes have fuelled the development of digitized, high performing, ecofriendly technologies which are in tandem with environment. In the context of extraction of essential oil, drawbacks of classical hydrodistillation such as thermal degradation, high energy consumption, time consuming, low yield have made way for more greener approach. The novelty in the proposed work lies in the development of a rapid, solvent free, gravity assisted microwave based extraction model. The model ensures complete bio-refinery of the residual biomass for extraction of other non-volatile principles with special emphasis to nutraceuticals. The developed working protocol is based on a novel approach of delivering a high power microwave surge concept. An initial high power microwave surge at 60% power level was applied for 2 min, followed by a surge at 40% power level for 2 min and finally followed by sustained microwave firing at 20% power level for 6 min. The above conditions resulted in extraction of 10 mL (dehydrated) of oil from 60 g fresh mentha leaves. The yield of oil was 5 times higher than conventional hydrodistillation method for 6 h. The results of extraction yield were found to be in tandem with the oil quality (volatile principles and biological activity). The biomass obtained after extraction of oil retained the phenolics, flavonoids and other nutraceutical principles thus making it suitable for re-extraction.

A semi-closed loop microalgal biomass production-platform for ethanol from renewable sources of nitrogen and phosphorous

Production of microalgal biomass for feed and fuels demands unsustainable large amounts of fertilizers. The most broadly considered alternative sources of nutrients/fertilizer for microalgae are wastewater and internal recycling in closed-loop production platforms. However, these strategies largely disable co-production of feed and fuel in biomass biorefineries for an increased economic and environmental feasibility.In this study, we aimed at providing proof-of-concept for a semi-closed loop microalgal production-platform and biomass biorefinery for ethanol and feed from renewable resources of N and P. Atmospheric N2 was assimilated into a N2-fixing cyanobacterial biomass, which sustained growth of a microalga that accumulated high levels of carbohydrates (up to 60% (w/w)) as a sole source of fertilizer. The microalgal biomass was efficiently saccharified with H2SO4, which was recycled to release soluble PO43- from bone meal as a renewable source of P. Fermenting these P-enriched preparations with yeasts quantitatively produced ethanol at theoretical yields, a concentration of up to 50 g ethanol. L−1 and a yield of 0.25 g ethanol. g biomass−1. Calculations suggested a potential yield from 7600 to 10,800 L ethanol. ha−1. year−1, under Buenos Aires environmental conditions, which would be higher than that currently obtained from maize feedstocks. The residual fermentation vinasse, supplemented with P and containing other downstream-process reagents, was recycled as a sole source of macronutrients for the cultivation of the N2-fixing cyanobacterium to close the production cycle. Water recycling and co-production of residual biomass enriched in fat and protein as potential feed are also shown. This semi-closed loop biomass production-platform reconciles the concepts of microalgal biomass biorefineries for the co-production of feedstocks for biofuels and feed and nutrients recycling in closed-loop systems that largely minimizes production of waste.

Ethanol and protein production from minimally processed biomass of a genetically-modified cyanobacterium over-accumulating sucrose

One of the main bottlenecks of a microalgal or cyanobacterial biomass biorefinery is the separation of different useful fractions using simple, low energy-consuming, cost-effective, and scalable separation processes. Although the carbohydrates-rich biomass of these microorganisms presents clear advantages over conventional terrestrial crops as feedstocks for ethanol, it still requires acid and/or enzymatic hydrolysis for efficient fermentation. Here, we show the genetic modification of carbohydrates partitioning in a filamentous cyanobacterium towards the accumulation of sucrose up to 10% (w/w) as a readily fermentable feedstock. We optimized two methods for the preparation of concentrated sucrose syrups, which were efficiently converted into ethanol by yeasts, without the need of additional pretreatments. Biomass drying and milling, followed by aqueous extraction of sugars and proteins, and the recovery of proteins by short pulses of heat, kept the value of sugars as a feedstock for ethanol and protein for feed supplements within a cost-effective biomass biorefinery.

CLR-4, a novel conserved transcription factor for cellulase gene expression in ascomycete fungi.

Fungal degradation of lignocellulosic biomass requires various (hemi-)cellulases and is an important part of the natural carbon cycle. Although induction of cellulases has been described for some saprobic filamentous fungi, the regulation of cellulase transcription is complex and many aspects are still poorly understood. Here, we identified and characterized the novel cellulase regulation factor NcCLR-4 in Neurospora crassa and its ortholog MtCLR-4 in Myceliophthora thermophila. Deletion of CLR-4 resulted in similarly defective cellulolytic enzyme production and activities. Transcriptome analyses of ΔNcclr-4/ΔMtclr-4 revealed the down-regulation of genes encoding (hemi-)cellulases and pivotal regulators (clr-1, clr-2 and xyr-1) and key genes in the cAMP signaling pathway such as adenylate cyclase Nccr-1. Intracellular cAMP levels were markedly lower in ΔNcclr-4/ΔMtclr-4 than in wild-type during cellulose utilization. In electrophoretic mobility shift (EMSA) and DNase I footprinting assays, NcCLR-4/MtCLR-4 can directly bound to the promoters of Nccr-1/Mtcr-1 (encoding adenylyl cyclase). EMSAs also demonstrated that NcCLR-4/MtCLR-4 could directly bound to clr-1 (encoding a key cellulase regulator), Mtclr-2 and Mtxyr-1 (encoding biomass deconstruction regulators). These findings about the novel cellulase expression regulators NcCLR-4 and MtCLR-4 enrich our understanding of how cellulose degradation is regulated and provide new targets for engineering fungi to deconstruct plant biomass in biorefineries.

Novel Kinetic Models of Xylan Dissolution and Degradation during Ethanol Based Auto-Catalyzed Organosolv Pretreatment of Bamboo.

Due to the invalidity of traditional models, pretreatment conditions dependent parameter of susceptible dissolution degree of xylan (dX) was introduced into the kinetic models. After the introduction of dX, the dissolution of xylan, and the formation of xylo-oligosaccharides and xylose during ethanol based auto-catalyzed organosolv (EACO) pretreatments of bamboo were well predicted by the pseudo first-order kinetic models (R2 > 97%). The parameter of dX was verified to be a variable dependent of EACO pretreatment conditions (such as solvent content in pretreatment liquor and pretreatment temperature). Based on the established kinetic models of xylan dissolution, the dissolution of glucan and the formation of degradation products (furfural and acetic acid) could also be empirically modeled (R2 > 97%). In addition, the relationship between xylan and lignin removal can provide guidance for alleviating the depositions of lignin or pseudo-lignin. The parameter of dX derived novel kinetic models can not only be used to reveal the multi-step reaction mechanisms of xylan, but also control the final removal of main components in bamboo during EACO pretreatments, indicating scientific and practical significance for governing the biorefinery of woody biomass.

Fractionation and Structural Characterization of Hemicellulose from Steam-Exploded Banana Rachis

Banana production in tropical countries generates significant quantity of waste. Biorefinery of food waste biomass into cellulose, hemicellulose, lignin or pectin macromolecules have grown interest in recent scientific literature. In this paper, hemicellulose from banana rachis (Musa cavendish) was extracted by steam explosion at three severity levels (2.97, 3.57 and 3.78) and was further fractionated by graded ethanol precipitation method (15%, 60% and 80%). The recovered hemicelluloses sub-fractions (H1, H2 and H3) were characterized for their chemical composition and structural features by HPSEC, TGA/DTG, FTIR, 1H and 2D NMR techniques. The hemicellulose extraction yield increased with the severity level and treatment duration. The average molecular weight of the extracted hemicellulose macromolecules decreased from H1-60% ethanol hemicellulose sub-fraction with 143 790 g/mol, followed by H3-60% ethanol hemicellulose sub-fraction with 110 841 g/mol and finally H2-60% ethanol hemicellulose sub-fraction with 61 404 g/mol. The H1-60% ethanol hemicellulose sub-fraction extracted during the steam explosion at the lowest severity level showed the largest molecular weight and exhibited rather a high arabinose/xylose ratio and uronic acid content. Structural analysis revealed that hemicellulose from the 60%-ethanol hemicellulose sub-fractions were mainly arabino-glucuronoxylan (AGX). However, chemical analysis also revealed significant contents of co-extracted residual lignin. Although the ethanol fractionation helped at lowering the lignin content in the 60%-ethanol hemicellulose sub-fractions (20.1% in H2-60%, 24.0% in H1-60% and 28.0 in H3-60%) relatively to 80%-ethanol hemicellulose sub-fractions, additional purification step was still required to improve the quality of the extracted hemicellulose sub-fractions (purity and coloration). Nevertheless these results proved that steam explosion was an effective technique for the extraction of high molecular mass AGX hemicellulose macromolecules from banana rachis residues.

Green biorefinery of larch wood biomass to obtain the bioactive compounds, functional polymers and nanoporous materials

The first green biorefinery of larch wood based on the fractionation of biomass into dihydroquercetin (DHQ), arabinogalactan (AG), microcrystalline cellulose (MCC) and soluble lignin (SL) is reported. The new green method of one-step isolation of DHQ and AG from larch wood by ethanol–water solution was developed. The first results of kinetic studies and optimization of the process of extracted larch wood peroxide fractionation into MCC and SL in acetic acid–water medium in the presence of green TiO2 catalyst are described. The products obtained from larch wood were characterized by FTIR, NMR, XRD, AFM and chemical methods. The scheme of larch wood biorefinery is suggested which integrates the developed processes of woody biomass fractionation into DHQ, AG, MCC and SL. All developed methods use non-toxic and less-toxic reagents, such as water, ethanol, hydrogen peroxide and acetic acid.

Environmental performance of bioethanol production from oil palm frond petiole sugars in an integrated palm biomass biorefinery

Generation of biofuels from renewable resources such as lignocellulosic biomass is a promising approach to reduce the sole reliability on the depleting fossil fuel. The aim of this work is to assess the potential environmental impact of bioethanol production from oil palm frond in a conceptual oil palm based biorefinery model, utilizing wet disc milling as a pretreatment method. A cradle-to-gate approach was selected, beginning with the harvesting and transportation of the frond petiole from the plantation, followed by production of oil palm frond petiole sugars via pretreatment and saccharification prior to bioethanol fermentation, and finally purification of the fermentation products to obtain anhydrous bioethanol. A life cycle assessment was performed using CML 2 baseline 2000 method (SimaPro v8.0), where ten impact categories were evaluated. It was found that the most significant environmental impact was from sugar recovery process with contribution of more than 90 %. This is mainly due to high power consumption by wet disc milling during pretreatment. Apart from that, production of enzyme and chemicals which were used during saccharification consumes high energy thus contributing major problems to the surrounding. Finding of this study helps to identify the hotspot which can be improved to establish a more energy efficient and greener system for bioethanol production from oil palm frond petiole sugars.

Fractionation of lignocellulosic biopolymers from sugarcane bagasse using formic acid-catalyzed organosolv process.

A one-step formic acid-catalyzed organosolv process using a low-boiling point acid-solvent system was studied for fractionation of sugarcane bagasse. Compared to H2SO4, the use of formic acid as a promoter resulted in higher efficiency and selectivity on removals of hemicellulose and lignin with increased enzymatic digestibility of the cellulose-enriched solid fraction. The optimal condition from central composite design analysis was determined as 40min residence time at 159°C using water/ethanol/ethyl acetate/formic acid in the respective ratios of 43:20:16:21%v/v. Under this condition, a 94.6% recovery of cellulose was obtained in the solid with 80.2% cellulose content while 91.4 and 80.4% of hemicellulose and lignin were removed to the aqueous-alcohol-acid and ethyl acetate phases, respectively. Enzymatic hydrolysis of the solid yielded 84.5% glucose recovery compared to available glucan in the raw material. Physicochemical analysis revealed intact cellulose fibers with decreased crystallinity while the hemicellulose was partially recovered as mono- and oligomeric sugars. High-purity organosolv lignin with < 1% sugar cross-contamination was obtained with no major structural modification according to Fourier-transform infrared spectroscopy. The work represents an alternative process for efficient fractionation of lignocellulosic biomass in biorefineries.

A Novel 3,6-anhydro-L-galactose Dehydrogenase Produced by a Newly Isolated Raoultella ornithinolytica B6-JMP12

Despite an increasing potential of red algal biomass as a feedstock, biological conversion of red algal biomass has been limited by lack of feasible microorganisms which can convert structured AHG, which is a main component of red algal carbohydrate, into a common metabolite. In the AHG uptake pathway, AHG dehydrogenase (AHGD) is known to be a key step, therefore it is important to find an efficient dehydrogenase to break down 3,6- anhydro-L-galactose (AHG) for practical use of red macroalgae biomass in biorefineries requires. In this study, we isolate a novel AHG dehydrogenase (AHGD) with high activity produced by a newly isolated bacteria strain, Raoultella ornithinolytica B6–JMP12. The stability and compatibility of the enzyme were evaluated under various conditions to achieve high enzyme production. The AHGD was partially purified using conventional protein purification techniques such as ammonium sulfate precipitation and ion exchange followed by gel filtration chromatography, 37.24 fold with a final specific activity of 5.47 U/mg of protein with 32% yield recovery. SDS-PAGE was used to determine the molecular weight of the partially purified AHGD and its molecular weight was found to be around ~34 kDa. The optimal pH and temperature for the partially purified AHGD were 7.0 and 35°C, respectively. The Km and Vmax for 3,6-anhydro-L-galactose are 0.63 mg/mL and 0.38 μM/mL/min, respectively.