Roya R.R. Sardari

Biotransformation of glycerol to 3-hydroxypropionaldehyde: Improved production by in situ complexation with bisulfite in a fed-batch mode and separation on anion exchanger

3-Hydroxypropionaldehyde (3HPA) is an important C3 chemical that can be produced from renewable glycerol by resting whole cells of Lactobacillus reuteri. However the process efficiency is limited due to substrate inhibition, product-mediated loss of enzyme activity and cell viability, and also formation of by-products. Complex formation of 3HPA with sodium bisulfite and subsequent binding to Amberlite IRA-400 was investigated as a means of in situ product recovery and for overcoming inhibition. The adsorption capacity and -isotherm of the resin were evaluated using the Langmuir model. The resin exhibited maximum capacity of 2.92 mmol complex/g when equilibrated with 45 mL solution containing an equilibrium mixture of 2.74 mmol 3HPA-bisulfite complex and 2.01 mmol free 3HPA. The dynamic binding capacity based on the breakthrough curve of 3HPA and its complex on passing a solution with 2.49 mmol complex and 1.65 mmol free 3HPA was 2.01 mmol/g resin. The bound 3HPA was desorbed from the resin using 0.20 M NaCl with a high purity as a mixture of complexed- and free 3HPA at a ratio of 0.77 mol/mol. Fed-batch biotransformation of glycerol (818.85 mmol) with in situ 3HPA complexation and separation on the bisulfite-functionalized resin resulted in an improved process with consumption of 481.36 mmol glycerol yielding 325.54 mmol 3HPA at a rate of 17.13 mmol/h and a yield of 68 mol%. Also, the cell activity was maintained for at least 28 h.

Improved production of 3-hydroxypropionadehyde by complex formation with bisulfite during biotransformation of glycerol.

3-Hydroxypropionaldehyde (3HPA) is an important specialty chemical which can be produced from glycerol using resting cells of Lactobacillus reuteri. This biocatalytic route, however, suffers from substrate- and product-mediated loss of enzyme activity within 2 h of biotransformation. In order to overcome the inhibitory effects of 3HPA, complex formation with sodium bisulfite was investigated, optimized and applied for in situ capture of the aldehyde during biotransformation of glycerol in a fed-batch process. As a result, the activity of the cells was maintained for at least 18 h. The 3HPA produced per gram cell dry weight was increased 5.7 times compared to the batch production process, and 2.2 times compared to fed-batch process without in situ complex formation. This approach may have potential for production and in situ removal of 3HPA after further process development.

Coenzyme A-acylating propionaldehyde dehydrogenase (PduP) from Lactobacillus reuteri: Kinetic characterization and molecular modeling

3-Hydroxypropionic acid (3-HP), an important C3 chemical for a bio-based industry, is natively produced by Lactobacillus reuteri from glycerol. Conversion of glycerol occurs via the intermediate 3-hydroxypropionaldehyde (3-HPA), followed by an ATP-producing pathway initiated by the CoA-acylating propionaldehyde dehydrogenase (PduP). The pduP gene of L. reuteri was cloned and expressed in Escherichia coli and the recombinant enzyme was purified to homogeneity for characterization of its activity and properties. Kinetic studies with propionaldehyde as substrate showed a maximum specific activity of 28.9 U/mg, which is 80-fold higher than that reported previously. Maximum activity of 18 U/mg was obtained at 3-HPA concentration of 7 mM, above which substrate inhibition was observed. Substrate inhibition was also seen with coenzyme A at a concentration above 0.5mM and with NADP(+) above 9 mM. A structure of PduP is proposed based on homology modeling. In silico docking of the co-factors coenzyme A and NAD(+), respectively, showed a common binding site consisting of amino acids Thr145, Ile275, Cys277 and Ser417, which through site-directed mutagenesis to alanine and kinetic studies, were confirmed as essential for the catalytic activity of PduP.

Economic and environmental assessment of propionic acid production by fermentation using different renewable raw materials.

Production of propionic acid by fermentation of glycerol as a renewable resource has been suggested as a means for developing an environmentally-friendly route for this commodity chemical. However, in order to quantify the environmental benefits, life cycle assessment of the production, including raw materials, fermentation, upstream and downstream processing is required. The economic viability of the process also needs to be analysed to make sure that any environmental savings can be realised. In this study an environmental and economic assessment from cradle-to-gate has been conducted. The study highlights the need for a highly efficient bioprocess in terms of product titre (more than 100g/L and productivity more than 2g/(L · h)) in order to be sustainable. The importance of the raw materials and energy production for operating the process to minimize emissions of greenhouse gases is also shown.

Production of 3-hydroxypropionic acid from 3-hydroxypropionaldehyde by recombinant Escherichia coli co-expressing Lactobacillus reuteri propanediol utilization enzymes

3-Hydroxypropionic acid (3-HP) is an important platform chemical for the biobased chemical industry. Lactobacillus reuteri produces 3-HP from glycerol via 3-hydroxypropionaldehyde (3-HPA) through a CoA-dependent propanediol utilization (Pdu) pathway. This study was performed to verify and evaluate the pathway comprising propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL), and propionate kinase (PduW) for formation of 3-HP from 3-HPA. The pathway was confirmed using recombinant Escherichia coli co-expressing PduP, PduL and PduW of L. reuteri DSM 20016 and mutants lacking expression of either enzyme. Growing and resting cells of the recombinant strain produced 3-HP with a yield of 0.3mol/mol and 1mol/mol, respectively, from 3-HPA. 3-HP was the sole product with resting cells, while growing cells produced 1,3-propanediol as co-product. 3-HP production from glycerol was achieved with a yield of 0.68mol/mol by feeding recombinant E. coli with 3-HPA produced by L. reuteri and recovered using bisulfite-functionalized resin.

Efficient poly(3-hydroxypropionate) production from glycerol using Lactobacillus reuteri and recombinant Escherichia coli harboring L. reuteri propionaldehyde dehydrogenase and Chromobacterium sp. PHA synthase genes.

Poly(3-hydroxypropionate), P(3HP), is a polymer combining good biodegradability with favorable material properties. In the present study, a production system for P(3HP) was designed, comprising conversion of glycerol to 3-hydroxypropionaldehyde (3HPA) as equilibrium mixture with 3HPA-hydrate and -dimer in aqueous system (reuterin) using resting cells of native Lactobacillus reuteri in a first stage followed by transformation of the 3HPA to P(3HP) using recombinant Escherichia coli strain co-expressing highly active coenzyme A-acylating propionaldehyde dehydrogenase (PduP) from L. reuteri and polyhydroxyalkanoate synthase (PhaCcs) from Chromobacterium sp. P(3HP) content of up to 40% (w/w) cell dry weight was reached, and the yield with respect to the reuterin consumed by the cells was 78%. Short biotransformation period (4.5h), lack of additives or expensive cofactors, and use of a cheap medium for cultivation of the recombinant strain, provides a new efficient and potentially economical system for P(3HP) production.

Valorization of Brewer's spent grain to prebiotic oligosaccharide: Production, xylanase catalyzed hydrolysis, in-vitro evaluation with probiotic strains and in a batch human fecal fermentation model

Brewers spent grain (BSG) accounts for around 85% of the solid by-products from beer production. BSG was first extracted to obtain water-soluble arabinoxylan (AX). Using subsequent alkali extraction (0.5 M KOH) it was possible to dissolve additional AX. In total, about 57% of the AX in BSG was extracted with the purity of 45-55%. After comparison of nine xylanases, Pentopan mono BG, a GH11 enzyme, was selected for hydrolysis of the extracts to oligosaccharides with minimal formation of monosaccharides. Growth of Bifidobacterium adolescentis (ATCC 15703) was promoted by the enzymatic hydrolysis to arabinoxylooligosaccharides, while Lactobacillus brevis (DSMZ 1264) utilized only unsubstituted xylooligosaccharides. Furthermore, utilization of the hydrolysates by human gut microbiota was also assessed in a batch human fecal fermentation model. Results revealed that the rates of fermentation of the BSG hydrolysates by human gut microbiota were similar to that of commercial prebiotic fructooligosaccharides, while inulin was fermented at a slower rate. In summary, a sustainable process to valorize BSG to functional food ingredients has been proposed.

Characterization of carotenoids in Rhodothermus marinus

Rhodothermus marinus, a marine aerobic thermophile, was first isolated from an intertidal hot spring in Iceland. In recent years, the R. marinus strain PRI 493 has been genetically modified, which opens up possibilities for targeted metabolic engineering of the species, such as of the carotenoid biosynthetic pathway. In this study, the carotenoids of the R. marinus type‐strain DSM 4252T, strain DSM 4253, and strain PRI 493 were characterized. Bioreactor cultivations were used for pressurized liquid extraction and analyzed by ultra‐high performance supercritical fluid chromatography with diode array and quadropole time‐of‐flight mass spectrometry detection (UHPSFC‐DAD‐QTOF/MS). Salinixanthin, a carotenoid originally found in Salinibacter ruber and previously detected in strain DSM 4253, was identified in all three R. marinus strains, both in the hydroxylated and nonhydroxylated form. Furthermore, an additional and structurally distinct carotenoid was detected in the three strains. MS/MS fragmentation implied that the mass difference between salinixanthin and the novel carotenoid structure corresponded to the absence of a 4‐keto group on the ß‐ionone ring. The study confirmed the lack of carotenoids for the strain SB‐71 (ΔtrpBΔpurAcrtBI’::trpB) in which genes encoding two enzymes of the proposed pathway are partially deleted. Moreover, antioxidant capacity was detected in extracts of all the examined R. marinus strains and found to be 2–4 times lower for the knock‐out strain SB‐71. A gene cluster with 11 genes in two operons in the R. marinusDSM 4252T genome was identified and analyzed, in which several genes were matched with carotenoid biosynthetic pathway genes in other organisms.

Marine Poly- and Oligosaccharides as Prebiotics

The marine environment can increase the global production of biomass. Interest in marine macroalgae and microorganisms has increased tremendously as a result of international agendas and market trends promoting sustainability as well as healthy food. Macroalgae and marine microorganisms contain unique poly- and oligosaccharides with different substitutions, e.g., sulfation or carboxylation. There is great potential to find prebiotic compounds from these marine-derived saccharides. However, the exact composition and substituent distribution needed for the activity is to a large extent unexplored. In depth investigations of these compounds will provide us with novel insights on the specific structures required for the observed functions.