Yeast extracts from different manufacturers and supplementation of amino acids and micro elements reveal a remarkable impact on alginate production by A. vinelandii ATCC9046.

BACKGROUND: In research and production, reproducibility is a key factor, to meet high quality and safety standards and maintain productivity. For microbial fermentations, complex substrates and media components are often used. The complex media components can vary in composition, depending on the lot and manufacturing process. These variations can have an immense impact on the results of biological cultivations. The aim of this work was to investigate and characterize the influence of the complex media component yeast extract on cultivations of Azotobacter vinelandii under microaerobic conditions. Under these conditions, the organism produces the biopolymer alginate. The focus of the investigation was on the respiration activity, cell growth and alginate production. RESULTS: Yeast extracts from 6 different manufacturers and 2 different lots from one manufacturer were evaluated. Significant differences on respiratory activity, growth and production were observed. Concentration variations of three different yeast extracts showed that the performance of poorly performing yeast extracts can be improved by simply increasing their concentration. On the other hand, the results with well-performing yeast extracts seem to reach a saturation, when their concentration is increased. Cultivations with poorly performing yeast extract were supplemented with grouped amino acids, single amino acids and micro elements. Beneficial results were obtained with the supplementation of copper sulphate, cysteine or a combination of both. Furthermore, a correlation between the accumulated oxygen transfer and the final viscosity (as a key performance indicator), was established. CONCLUSION: The choice of yeast extract is crucial for A. vinelandii cultivations, to maintain reproducibility and comparability between cultivations. The proper use of specific yeast extracts allows the cultivation results to be specifically optimised. In addition, supplements can be applied to modify and improve the properties of the alginate. The results only scratch the surface of the underlying mechanisms, as they are not providing explanations on a molecular level. However, the findings show the potential of optimising media containing yeast extract for alginate production with A. vinelandii, as well as the potential of targeted supplementation of the media.

[Production of mucic acid from pectin-derived D-galacturonic acid: a review].

Mucic acid is a hexaric acid that can be biosynthesized by oxidation of D-galacturonic acid, which is the main constituent of pectin. The structure and properties of mucic acid are similar to that of glucaric acid, and can be widely applied in the preparation of important platform compounds, polymers and macromolecular materials. Pectin is a cheap and abundant renewable biomass resource, thus developing a process enabling production of mucic acid from pectin would be of important economic value and environmental significance. This review summarized the structure and hydrolysis of pectin, the catabolism and regulation of D-galacturonic acid in microorganisms, and the strategy for mucic acid production based on engineering of corresponding pathways. The future application of mucic acid are prospected, and future directions for the preparation of mucic acid by biological method are also proposed.

Effect of Extraction Procedures with Ultrasound and Cellulolytic Enzymes on the Structural and Functional Properties of Citrus grandis Osbeck Seed Mucilage.

The structural and functional properties of Citrus grandis Osbeck (CGO) seed mucilage by different extraction practices, including conventional citrate buffer, ultrasonic-assisted (UAE), enzymatic-assisted extraction (EAE) with cellulase or Celluclast® 1.5 L and various ultrasonic-assisted enzymatic extraction (UAEE) procedures were investigated. It was found that CGO seed from agricultural and processing byproducts is an excellent new source of high methoxyl pectin with quite high intrinsic viscosity (about 108.64 dL/g) and molecular weight (about 1.9 × 106) as compared with other pectin sources. UAEE with Celluclast® 1.5 L enhanced the extraction yield most pronouncedly (about 2.3 times). Moreover, the monosaccharide composition of CGO seed mucilage is least affected by EAE with Celluclast® 1.5 L. In contrast, EAE with cellulase dramatically reduces the galacturonic acid (GalA) content to less than 60 molar%, and increases the glucose (Glc) content pronouncedly (to about 40 molar%), which may be considered as an adverse effect in terms of pectin purity. Though extraction procedures involved with ultrasound and cellulolytic enzymes generally show a decrease in GalA contents, weight average molar mass and intrinsic viscosity, EAE with Celluclast® 1.5 L is least affected, followed by UAE and UAEE with Celluclast® 1.5 L. These features can be leveraged in favor of diversified applications.

Production of mucic acid from pectin-derived D-galacturonic acid: a review / 生物工程学报

Mucic acid is a hexaric acid that can be biosynthesized by oxidation of D-galacturonic acid, which is the main constituent of pectin. The structure and properties of mucic acid are similar to that of glucaric acid, and can be widely applied in the preparation of important platform compounds, polymers and macromolecular materials. Pectin is a cheap and abundant renewable biomass resource, thus developing a process enabling production of mucic acid from pectin would be of important economic value and environmental significance. This review summarized the structure and hydrolysis of pectin, the catabolism and regulation of D-galacturonic acid in microorganisms, and the strategy for mucic acid production based on engineering of corresponding pathways. The future application of mucic acid are prospected, and future directions for the preparation of mucic acid by biological method are also proposed.

Metabolic engineering of Bacillus subtilis with an endopolygalacturonase gene isolated from Pectobacterium. carotovorum; a plant pathogenic bacterial strain.

Pectinolytic enzymes or pectinases are synthesized naturally by numerous microbes and plants. These enzymes degrade various kinds of pectin which exist as the major component of the cell wall in plants. A pectinase gene encoding endo-polygalacturonase (endo-PGase) enzyme was isolated from Pectobacterium carotovorum a plant pathogenic strain of bacteria and successfully cloned into a secretion vector pHT43 having σA-dependent promoter for heterologous expression in Bacillus subtilis (WB800N).The desired PCR product was 1209bp which encoded an open reading frame of 402 amino acids. Recombinant proteins showed an estimated molecular weight of 48 kDa confirmed by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. Transformed B. subtilis competent cells harbouring the engineered pHT43 vector with the foreign endo-PGase gene were cultured in 2X-yeast extract tryptone medium and subsequently screened for enzyme activity at various temperatures and pH ranges. Optimal activity of recombinant endo-PGase was found at 40°C and pH 5.0. To assay the catalytic effect of metal ions, the recombinant enzyme was incubated with 1 mM concentration of various metal ions. Potassium chloride increased the enzyme activity while EDTA, Zn++ and Ca++, strongly inhibited the activity. The chromatographic analysis of enzymatic hydrolysates of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed tri and tetra-galacturonates as the end products of recombinant endo-PGase hydrolysis. Conclusively, endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis for the first time using pHT43 expression vector and could be assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safer to escape endotoxins for commercial enzyme production as compared to yeast and fungi. Additionally, the hydrolysis products generated by the recombinant endo-PGase activity offer their useful applications in food and beverage industry for quality products.

Structural model and functional properties of an exo-polygalacturonase from Neosartorya glabra.

A purified exo-polygalacturonase of Neosartorya glabra (EplNg) was successfully characterized. EplNg native presented 68.2 kDa, with 32% carbohydrate content. The deglycosylated form showed 46.3 kDa and isoelectric point of 5.4. The identity of EplNg was confirmed as an exo-polygalacturonase class I (EC 3.2.1.67) using mass spectrometry and Western-Blotting. Capillary electrophoresis indicated that only galacturonic acid was released by the action of EplNg on sodium polypectate, confirming an exoenzyme character. The structural model confers that EplNg has a core formed by twisted parallel ß-sheets structure. Among twelve putative cysteines, ten were predicted to form disulfide bridges. The catalytic triad predicted is composed of Asp223, Asp245, and Asp246 aligned along with a distance in 4-5 Å, suggesting that EplNg probably does not perform the standard inverting catalytic mechanism described for the GH28 family. EplNg was active from 30 to 90 °C, with maximum activity at 65 °C, pH 5.0. The Km and Vmax determined using sodium polypectate were 6.9 mg·mL-1 and Vmax 690 µmol·min-1.mg-1, respectively. EplNg was active and stable over a wide range of pH values and temperatures, confirming the interesting properties EplNg and provide a basis for the development of the enzyme in different biotechnological processes.

Extraction, purification, hypoglycemic and antioxidant activities of red clover (Trifolium pratense L.) polysaccharides.

Hot water extraction was applied to extract red clover (Trifolium pratense L.) polysaccharides (RCP) and the extraction conditions were optimized using the response surface methodology (RSM). An RCP yield of 12.72 ± 0.14% was achieved under the optimum extraction conditions: extracting time of 95 min, extracting temperature of 93 °C, and solvent-material ratio of 21 mL/g. A component named RCP-1.1 with the molecular weight of 7528.81 kDa was purified from RCP. RCP-1.1 was composed of glucose, galacturonic acid, arabinose, and galactose, with molar percentages of 52.54, 1.04, 16.31, and 30.11%, respectively. At the determination concentration of 10 mg/mL, the α-glucosidase inhibition ability of RCP-1.1 reached 86.72% of that of acarbose. The scavenging rates of RCP-1.1 (3.0 mg/mL) for DPPH and ABTS radicals reached 91.82% and 98.95% of that of ascorbic acid (3.0 mg/mL), respectively. Based on these results, RCP-1.1 possesses the potential to be used as a natural hypoglycemic agent or an antioxidant.

Comparison of Biological and Chemical Pretreatment on Coproduction of Pectin and Fermentable Sugars from Apple Pomace.

Apple pomace, an abundant accessible source of carbohydrate platform chemicals, is refractory to cellulase degradation because of the main barrier problem of pectin constitute. A rapid and portable method for the coproduction of pectin and fermentable sugars was developed using the pretreatment of acetic acid, followed by enzymatic hydrolysis. Compared with pectinase, acetic acid pretreatment provided the highest pectin yield of 19.1% and the highest enzymatic hydrolysis yield from apple pomace. The acidic pretreated apple pomace cellulose was easily and completely hydrolyzed into fermentable sugars. More than 98.2% conversion of cellulose was achieved in a batch hydrolysis using a cellulase loading of 25 FPU/g cellulose and 10% total solids without any special strategies. A mass balance analysis showed that 95.5 g pectin and 110.2 g fermentable sugars were produced from 500-g oven-dried apple pomace. The integrated process is suggestive of environment-friendly and recyclable methods for the industrial utilization of apple pomace.

Production pectin oligosaccharides using Humicola insolens Y1-derived unusual pectate lyase.

The economical production of pectin oligosaccharides with a specific degree of polymerization and structure from agro-food waste is an industrially important process. This study identified a novel pectate lyase gene (plhy1) from the thermophilic cellulolytic fungus H. insolens Y1 and tested its ability to produce pectin oligosaccharides. The recombinant PLHY1 produced in Pichia pastoris was superior to other similar enzymes due to its high thermal and pH stability. PLHY1 demonstrated optimal enzymatic activity at 55°C and pH 10.0 in the presence of 0.4 mM Ca2+, and preferred methyl esterified substrates for digestion. High performance anion exchange chromatography-pulsed amperometric detector and ultra high performance liquid chromatography in combination with electrospray ionization tandem mass spectrometry analysis showed that galacturonic acid-oligosaccharides with a small degree of polymerization (4-6) were the major hydrolysates produced by the degradation of apple peel pectin by PLHY1. The properties of PLHY1 make it valuable for application in the agro-food industry for the production of pectin oligosaccharides.

Structural, rheological and functional properties of galactose-rich pectic polysaccharide fraction from leek.

An acid-extracted polysaccharide from alchohol-insoluble solids of leek was obtained. The sugar composition indicated that galactose and galacturonic acid were the major sugars, followed by small amounts of rhamnose and arabinose. The fraction contained a relatively high methyl-esterified homogalacturonan next to rhamnogalacturonan type I decorated with galactose-rich side chains. The fraction consisted of three high Mw populations, covering the range of 10-100 kDa. Enzymatic fingerprinting was performed with HG/RG-I degrading enzymes to elucidate the structure. The oligomers were analysed using LC-HILIC-MS, HPAEC, and MALDI-TOF MS. The data revealed the presence of GalA sequences, having different patterns of methyl-esterification, RG-I composed of unbranched segments and segments heavily substituted with ß-(1→4)-linked galactan chains of varying length. The rheological study showed the shear-thinning, weak thixotropic, anti-thixotropic, and non-Newtonian behavior of the polysaccharide. The pectin exhibited higher water holding capacity than oil-holding capacity and the fraction did form stable foams at high concentration.

Diet-derived galacturonic acid regulates virulence and intestinal colonization in enterohaemorrhagic Escherichia coli and Citrobacter rodentium.

Enteric pathogens sense the complex chemistry within the gastrointestinal tract to efficiently compete with the resident microbiota and establish a colonization niche. Here, we show that enterohaemorrhagic Escherichia coli and Citrobacter rodentium, its surrogate in a mouse infection model, sense galacturonic acid to initiate a multi-layered program towards successful mammalian infection. Galacturonic acid utilization as a carbon source aids the initial pathogen expansion. The main source of galacturonic acid is dietary pectin, which is converted to galacturonic acid by the prominent member of the microbiota, Bacteroides thetaiotamicron. This is regulated by the ExuR transcription factor. However, galacturonic acid is also sensed as a signal through ExuR to modulate the expression of the genes encoding a molecular syringe known as a type III secretion system, leading to infectious colitis and inflammation. Galacturonic acid acts as both a nutrient and a signal directing the exquisite microbiota-pathogen relationships within the gastrointestinal tract. This work highlights that differential dietary sugar availability influences the relationship between the microbiota and enteric pathogens, as well as disease outcomes.

Sustainable Galactarate-Based Polymers: Multi-Enzymatic Production of Pectin-Derived Polyesters.

Large amounts of agricultural wastes are rich in pectins that, in many cases, disrupt the processing of food residues due to gelation. Despite pectins being a promising sustainable feedstock for bio-based chemical production, the current pathways to produce platform molecules from this polysaccharide are hazardous and entail the use of strong acids. The present work describes a sequence of biocatalyzed reactions that involves 1) the extraction of pectin from sugar beet pulp and enzymatic recovery of galacturonic acid (GalA), followed by 2) the enzymatic oxidation of the GalA aldehyde and the recovery of galactaric acid (GA), and 3) the biocatalyzed polycondensation of GA to obtain fully bio-based polyesters carrying lateral hydroxy functionalities. The acid-free pectin extraction is optimized using enzymes and microwave technology. The conditions for enzymatic oxidation of GalA allow the separation of the GA produced by a simple centrifugation step that leads to the enzyme-catalyzed polycondensation reactions.

Oligogalacturonide production upon Arabidopsis thaliana-Botrytis cinerea interaction.

Despite an ever-increasing interest for the use of pectin-derived oligogalacturonides (OGs) as biological control agents in agriculture, very little information exists-mainly for technical reasons-on the nature and activity of the OGs that accumulate during pathogen infection. Here we developed a sensitive OG profiling method, which revealed unsuspected features of the OGs generated during infection of Arabidopsis thaliana with the fungus Botrytis cinerea Indeed, in contrast to previous reports, most OGs were acetyl- and methylesterified, and 80% of them were produced by fungal pectin lyases, not by polygalacturonases. Polygalacturonase products did not accumulate as larger size OGs but were converted into oxidized GalA dimers. Finally, the comparison of the OGs and transcriptomes of leaves infected with B. cinerea mutants with reduced pectinolytic activity but with decreased or increased virulence, respectively, identified candidate OG elicitors. In conclusion, OG analysis provides insights into the enzymatic arms race between plant and pathogen and facilitates the identification of defense elicitors.

Does long-term cadmium exposure influence the composition of pectic polysaccharides in the cell wall of Medicago sativa stems?

BACKGROUND: The heavy metal cadmium (Cd) accumulates in the environment due to anthropogenic influences. It is unessential and harmful to all life forms. The plant cell wall forms a physical barrier against environmental stress and changes in the cell wall structure have been observed upon Cd exposure. In the current study, changes in the cell wall composition and structure of Medicago sativa stems were investigated after long-term exposure to Cd. Liquid chromatography coupled to mass spectrometry (LC-MS) for quantitative protein analysis was complemented with targeted gene expression analysis and combined with analyses of the cell wall composition. RESULTS: Several proteins determining for the cell wall structure changed in abundance. Structural changes mainly appeared in the composition of pectic polysaccharides and data indicate an increased presence of xylogalacturonan in response to Cd. Although a higher abundance and enzymatic activity of pectin methylesterase was detected, the total pectin methylation was not affected. CONCLUSIONS: An increased abundance of xylogalacturonan might hinder Cd binding in the cell wall due to the methylation of its galacturonic acid backbone. Probably, the exclusion of Cd from the cell wall and apoplast limits the entry of the heavy metal into the symplast and is an important factor during tolerance acquisition.

Biphasic Scaffolds from Marine Collagens for Regeneration of Osteochondral Defects.

BACKGROUND: Collagens of marine origin are applied increasingly as alternatives to mammalian collagens in tissue engineering. The aim of the present study was to develop a biphasic scaffold from exclusively marine collagens supporting both osteogenic and chondrogenic differentiation and to find a suitable setup for in vitro chondrogenic and osteogenic differentiation of human mesenchymal stroma cells (hMSC). METHODS: Biphasic scaffolds from biomimetically mineralized salmon collagen and fibrillized jellyfish collagen were fabricated by joint freeze-drying and crosslinking. Different experiments were performed to analyze the influence of cell density and TGF-ß on osteogenic differentiation of the cells in the scaffolds. Gene expression analysis and analysis of cartilage extracellular matrix components were performed and activity of alkaline phosphatase was determined. Furthermore, histological sections of differentiated cells in the biphasic scaffolds were analyzed. RESULTS: Stable biphasic scaffolds from two different marine collagens were prepared. An in vitro setup for osteochondral differentiation was developed involving (1) different seeding densities in the phases; (2) additional application of alginate hydrogel in the chondral part; (3) pre-differentiation and sequential seeding of the scaffolds and (4) osteochondral medium. Spatially separated osteogenic and chondrogenic differentiation of hMSC was achieved in this setup, while osteochondral medium in combination with the biphasic scaffolds alone was not sufficient to reach this ambition. CONCLUSIONS: Biphasic, but monolithic scaffolds from exclusively marine collagens are suitable for the development of osteochondral constructs.

An Alginate/Cyclodextrin Spray Drying Matrix to Improve Shelf Life and Antioxidant Efficiency of a Blood Orange By-Product Extract Rich in Polyphenols: MMPs Inhibition and Antiglycation Activity in Dysmetabolic Diseases.

Alginate and ß-cyclodextrin were used to produce easily dosable and spray-dried microsystems of a dried blood orange extract with antidysmetabolic properties, obtained from a by-product fluid extract. The spray-dried applied conditions were able to obtain a concentrate dried extract without the loss of AOA and with TPC and TMA values of 35-40% higher than that of the starting material. They were also effective in producing microparticles with 80-100% of encapsulation efficiency. The 2% sodium alginate was capable of improving the extract shelf life, while the beta-cyclodextrin (1 : 1 molar ratio with dried extract) prolonged the extract antioxidant efficiency by 6 hours. The good inhibition effect of the dried extract on the AGE formation and the MMP-2 and MMP-9 activity is presumably due to a synergic effect exerted by both anthocyanin and bioflavonoid extract compounds and was improved by the use of alginate and cyclodextrin.

Combinatorial control of gene expression in Aspergillus niger grown on sugar beet pectin.

Aspergillus niger produces an arsenal of extracellular enzymes that allow synergistic degradation of plant biomass found in its environment. Pectin is a heteropolymer abundantly present in the primary cell wall of plants. The complex structure of pectin requires multiple enzymes to act together. Production of pectinolytic enzymes in A. niger is highly regulated, which allows flexible and efficient capture of nutrients. So far, three transcriptional activators have been linked to regulation of pectin degradation in A. niger. The L-rhamnose-responsive regulator RhaR controls the production of enzymes that degrade rhamnogalacturonan-I. The L-arabinose-responsive regulator AraR controls the production of enzymes that decompose the arabinan and arabinogalactan side chains of rhamnogalacturonan-II. The D-galacturonic acid-responsive regulator GaaR controls the production of enzymes that act on the polygalacturonic acid backbone of pectin. This project aims to better understand how RhaR, AraR and GaaR co-regulate pectin degradation. For that reason, we constructed single, double and triple disruptant strains of these regulators and analyzed their growth phenotype and pectinolytic gene expression in A. niger grown on sugar beet pectin.

A distinct class of vesicles derived from the trans-Golgi mediates secretion of xylogalacturonan in the root border cell.

Root border cells lie on the surface of the root cap and secrete massive amounts of mucilage that contains polysaccharides and proteoglycans. Golgi stacks in the border cells have hypertrophied margins, reflecting elevated biosynthetic activity to produce the polysaccharide components of the mucilage. To investigate the three-dimensional structures and macromolecular compositions of these Golgi stacks, we examined high-pressure frozen/freeze-substituted alfalfa root cap cells with electron microscopy/tomography. Golgi stacks in border cells and peripheral cells, precursor cells of border cells, displayed similar morphological features, such as proliferation of trans cisternae and swelling of the trans cisternae and trans-Golgi network (TGN) compartments. These swollen margins give rise to two types of vesicles larger than other Golgi-associated vesicles. Margins of trans-Golgi cisternae accumulate the LM8 xylogalacturonan (XGA) epitope, and they become darkly stained large vesicles (LVs) after release from the Golgi. Epitopes for xyloglucan (XG), polygalacturonic acid/rhamnogalacturonan-I (PGA/RG-I) are detected in the trans-most cisternae and TGN compartments. LVs produced from TGN compartments (TGN-LVs) stained lighter than LVs and contained the cell wall polysaccharide epitopes seen in the TGN. LVs carrying the XGA epitope fuse with the plasma membrane only in border cells, whereas TGN-LVs containing the XG and PGA/RG-I epitopes fuse with the plasma membrane of both peripheral cells and border cells. Taken together, these results indicate that XGA is secreted by a novel type of secretory vesicles derived from trans-Golgi cisternae. Furthermore, we simulated the collapse in the central domain of the trans-cisternae accompanying polysaccharide synthesis with a mathematical model.

Effects of Pectin Molecular Weight Changes on the Structure, Dynamics, and Polysaccharide Interactions of Primary Cell Walls of Arabidopsis thaliana: Insights from Solid-State NMR.

Significant cellulose-pectin interactions in plant cell walls have been reported recently based on 2D 13C solid-state NMR spectra of intact cell walls, but how these interactions affect cell growth has not been probed. Here, we characterize two Arabidopsis thaliana lines with altered expression of the POLYGALACTURONASE INVOLVED IN EXPANSION1 (PGX1) gene, which encodes a polygalacturonase that cleaves homogalacturonan (HG). PGX1AT plants overexpress PGX1, have HG with lower molecular weight, and grow larger, whereas pgx1-2 knockout plants have HG with higher molecular weight and grow smaller. Quantitative 13C solid-state NMR spectra show that PGX1AT cell walls have lower galacturonic acid and xylose contents and higher HG methyl esterification than controls, whereas high molecular weight pgx1-2 walls have similar galacturonic acid content and methyl esterification as controls. 1H-transferred 13C INEPT spectra indicate that the interfibrillar HG backbones are more aggregated whereas the RG-I side chains are more dispersed in PGX1AT cell walls than in pgx1-2 walls. In contrast, the pectins that are close to cellulose become more mobile and have weaker cross peaks with cellulose in PGX1AT walls than in pgx1-2 walls. Together, these results show that polygalacturonase-mediated plant growth is accompanied by increased esterification and decreased cross-linking of HG, increased aggregation of interfibrillar HG, and weaker HG-cellulose interactions. These structural and dynamical differences give molecular insights into how pectins influence wall dynamics during cell growth.

Scaling up and scaling down the production of galactaric acid from pectin using Trichoderma reesei.

BACKGROUND: Bioconversion of D-galacturonic acid to galactaric (mucic) acid has previously been carried out in small scale (50-1000 mL) cultures, which produce tens of grams of galactaric acid. To obtain larger amounts of biologically produced galactaric acid, the process needed to be scaled up using a readily available technical substrate. Food grade pectin was selected as a readily available source of D-galacturonic acid for conversion to galactaric acid. RESULTS: We demonstrated that the process using Trichoderma reesei QM6a Δgar1 udh can be scaled up from 1 L to 10 and 250 L, replacing pure D-galacturonic acid with commercially available pectin. T. reesei produced 18 g L-1 galactaric acid from food-grade pectin (yield 1.00 g [g D-galacturonate consumed]-1) when grown at 1 L scale, 21 g L-1 galactaric acid (yield 1.11 g [g D-galacturonate consumed]-1) when grown at 10 L scale and 14 g L-1 galactaric acid (yield 0.77 g [g D-galacturonate consumed]-1) when grown at 250 L scale. Initial production rates were similar to those observed in 500 mL cultures with pure D-galacturonate as substrate. Approximately 2.8 kg galactaric acid was precipitated from the 250 L culture, representing a recovery of 77% of the galactaric acid in the supernatant. In addition to scaling up, we also demonstrated that the process could be scaled down to 4 mL for screening of production strains in 24-well plate format. Production of galactaric acid from pectin was assessed for three strains expressing uronate dehydrogenase under alternative promoters and up to 11 g L-1 galactaric acid were produced in the batch process. CONCLUSIONS: The process of producing galactaric acid by bioconversion with T. reesei was demonstrated to be equally efficient using pectin as it was with D-galacturonic acid. The 24-well plate batch process will be useful screening new constructs, but cannot replace process optimisation in bioreactors. Scaling up to 250 L demonstrated good reproducibility with the smaller scale but there was a loss in yield at 250 L which indicated that total biomass extraction and more efficient DSP would both be needed for a large scale process.