Continuous production of pectic oligosaccharides from sugar beet pulp in a cross flow continuous enzyme membrane reactor.

Sugar beet pulp pectin is an attractive source for the production of pectic oligosaccharides, an emerging class of potential prebiotics. The main aim of the present work was to investigate a new process allowing to produce pectic oligosaccharides in a continuous way by means of a cross flow enzyme membrane reactor while using a low-cost crude enzyme mixture (viscozyme). Preliminary experiments in batch and semi-continuous setups allowed to identify suitable enzyme concentrations and assessing filtration suitability. Then, in continuous experiments in the enzyme membrane reactor, residence time and substrate loading were further optimized. The composition of the obtained oligosaccharide mixtures was assessed at the molecular level for the most promising conditions and was shown to be dominated by condition-specific arabinans, rhamnogalacturonans, and galacturonans. A continuous and stable production was performed for 28.5 h at the optimized conditions, obtaining an average pectic oligosaccharide yield of 82.9 ± 9.9% (w/w), a volumetric productivity of 17.5 ± 2.1 g/L/h, and a specific productivity of 8.0 ± 1.0 g/g E/h. This work demonstrated for the first time the continuous and stable production of oligosaccharide mixtures from sugar beet pulp using enzyme membrane reactor technology in a setup suitable for upscaling.

Geochemical and microbial community determinants of reductive dechlorination at a site biostimulated with glycerol.

Biostimulation is widely used to enhance reductive dechlorination of chlorinated ethenes in contaminated aquifers. However, the knowledge on corresponding biogeochemical responses is limited. In this study, glycerol was injected in an aquifer contaminated with cis-dichloroethene (cDCE), and geochemical and microbial shifts were followed for 265 days. Consistent with anoxic conditions and sulfate reduction after biostimulation, MiSeq 16S rRNA gene sequencing revealed temporarily increased relative abundance of Firmicutes, Bacteriodetes and sulfate reducing Deltaproteobacteria. In line with 13 C cDCE enrichment and increased Dehalococcoides mccartyi (Dcm) numbers, dechlorination was observed toward the end of the field experiment, albeit being incomplete with accumulation of vinyl chloride. This was concurrent with (i) decreased concentrations of dissolved organic carbon (DOC), reduced relative abundances of fermenting and sulfate reducing bacteria that have been suggested to promote Dcm growth by providing electron donor (H2 ) and essential corrinoid cofactors, (ii) increased sulfate concentration and increased relative abundance of Epsilonproteobacteria and Deferribacteres as putative oxidizers of reduced sulfur compounds. Strong correlations of DOC, relative abundance of fermenters and sulfate reducers, and dechlorination imply the importance of syntrophic interactions to sustain robust dechlorination. Tracking microbial and environmental parameters that promote/preclude enhanced reductive dechlorination should aid development of sustainable bioremediation strategies.

Geochemical Parameters and Reductive Dechlorination Determine Aerobic Cometabolic vs Aerobic Metabolic Vinyl Chloride Biodegradation at Oxic/Anoxic Interface of Hyporheic Zones.

Hyporheic zones mediate vinyl chloride (VC) biodegradation in groundwater discharging into surface waters. At the oxic/anoxic interface (OAI) of hyporheic zones subjected to redox oscillations, VC is degraded via coexisting aerobic ethenotrophic and anaerobic reductive dechlorination pathways. However, the identity of aerobic VC degradation pathways (cometabolic vs metabolic) and their interactions with reductive dechlorination in relation to riverbed sediment geochemistry remain ill-defined. We addressed this using microcosms containing OAI sediments incubated under fluctuating oxic/anoxic atmosphere. Under oxic atmosphere, aerobic metabolic VC oxidation was absent in sediments with high total organic carbon (TOC) and VC was reductively dechlorinated to ethene. Ethene was oxidized by ethenotrophs that can degrade VC cometabolically. Contrastingly, VC was metabolically oxidized by ethenotrophs in low-TOC sediments with low reductive dechlorination potential. Accordingly, enrichment and isolation of metabolic VC-oxidizing ethenotrophs was successful only from the low-TOC sediment. Sequence analysis of etnE genes from the microcosms as well phylogenetic typing of the isolates showed that ethenotrophs in the sediments were facultative anaerobic Proteobacteria capable of coping with OAI-associated redox fluctuations. Our results suggest that local sediment heterogeneity supports/selects divergent VC degradation processes at the OAI and that high reductive dechlorination potential suppresses development of aerobic metabolic VC oxidation potential.

Enzymatic pectic oligosaccharides (POS) production from sugar beet pulp using response surface methodology.

Pectic oligosaccharides (POS) have been indicated as novel candidate prebiotics. Traditionally, POS are produced from pectin-rich by-products using a two-step process involving extraction of the pectin, followed by its hydrolysis into POS. A one-step approach, in which the POS is directly produced from the raw material, might provide a more efficient alternative. Thus, the main aim of this paper was to investigate a one-step enzymatic hydrolysis approach to directly produce POS from sugar beet pulp (SBP). The POS yield was investigated as a function of the process parameters, as well as raw material characteristics. A statistically-based response surface methodology, using a central composite design was applied, to investigate the individual as well as the combined influences of the diverse parameters. The model was confirmed by a validation experiment, carried out at 135 g/l substrate concentration, 0.75 FPU/g SBP enzyme concentration, 0.8 mm particle size and 3 h hydrolysis time. Under these conditions, a POS-rich hydrolysate was obtained, containing rhamnose, arabinose, galactose, xylose and galacturonic acid, at 0.9, 15.2, 5.1, 1.4, and 13.2 g/l, respectively, enzymes were added each at 20 FPU/g dry matter (DM).

Pectic oligosaccharides from agricultural by-products: production, characterization and health benefits.

Pectin containing agricultural by-products are potential sources of a new class of prebiotics known as pectic oligosaccharides (POS). In general, pectin is made up of homogalacturonan (HG, α-1,4-linked galacturonic acid monomers) and rhamnogalacturonan (RG, alternate galacturonic acid and rhamnose backbone with neutral side chains). Controlled hydrolysis of pectin containing agricultural by-products like sugar beet, apple, olive and citrus by chemical, enzymatic and hydrothermal can be used to produce oligo-galacturonides (GalpOS), galacto-oligosaccharides (GalOS), rhamnogalacturonan-oligosaccharides (RGOS), etc. However, extensive research is needed to establish the role of POS, both as a prebiotic as well as therapeutic agent. This review comprehensively covers different facets of POS, including the nature and chemistry of pectin and POS, potential agricultural residual sources of pectin, pre-treatment methods for facilitating selective extraction of pectin, identification and characterization of POS, health benefits and important applications of POS in food and feed. This review has been compiled to establish a platform for future research in the purification and characterization of POS and for in vivo and in vitro studies of important POS, so that they could be commercially exploited.

Biodegradation: Updating the concepts of control for microbial cleanup in contaminated aquifers.

Biodegradation is one of the most favored and sustainable means of removing organic pollutants from contaminated aquifers but the major steering factors are still surprisingly poorly understood. Growing evidence questions some of the established concepts for control of biodegradation. Here, we critically discuss classical concepts such as the thermodynamic redox zonation, or the use of steady state transport scenarios for assessing biodegradation rates. Furthermore, we discuss if the absence of specific degrader populations can explain poor biodegradation. We propose updated perspectives on the controls of biodegradation in contaminant plumes. These include the plume fringe concept, transport limitations, and transient conditions as currently underestimated processes affecting biodegradation.

Evaluation of solid polymeric organic materials for use in bioreactive sediment capping to stimulate the degradation of chlorinated aliphatic hydrocarbons.

In situ bioreactive capping is a promising technology for mitigation of surface water contamination by discharging polluted groundwater. Organohalide respiration (OHR) of chlorinated ethenes in bioreactive caps can be stimulated through incorporation of solid polymeric organic materials (SPOMs) that provide a sustainable electron source for organohalide respiring bacteria. In this study, wood chips, hay, straw, tree bark and shrimp waste, were assessed for their long term applicability as an electron donor for OHR of cis-dichloroethene (cDCE) and vinyl chloride (VC) in sediment microcosms. The initial release of fermentation products, such as acetate, propionate and butyrate led to the onset of extensive methane production especially in microcosms amended with shrimp waste, straw and hay, while no considerable stimulation of VC dechlorination was obtained in any of the SPOM amended microcosms. However, in the longer term, short chain fatty acids accumulation decreased as well as methanogenesis, whereas high dechlorination rates of VC and cDCE were established with concomitant increase of Dehalococcoides mccartyi and vcrA and bvcA gene numbers both in the sediment and on the SPOMs. A numeric simulation indicated that a capping layer of 40 cm with hay, straw, tree bark or shrimp waste is suffice to reduce the groundwater VC concentration below the threshold level of 5 µg/l before discharging into the Zenne River, Belgium. Of all SPOMs, the persistent colonization of tree bark by D. mccartyi combined with the lowest stimulation of methanogenesis singled out tree bark as a long-term electron donor for OHR of cDCE/VC in bioreactive caps.

Quantitative and functional dynamics of Dehalococcoides spp. and its tceA and vcrA genes under TCE exposure.

This study aimed at monitoring the dynamics of phylogenetic and catabolic genes of a dechlorinating enrichment culture before, during, and after complete dechlorination of chlorinated compounds. More specifically, the effect of 40 µM trichloroethene (TCE) and 5.6 mM lactate on the gene abundance and activity of an enrichment culture was investigated for 40 days. Although tceA and vcrA gene copy numbers were relatively stable in DNA extracts over time, tceA and vcrA mRNA abundances were upregulated from undetectable levels to 2.96 × and 6.33 × 104 transcripts/mL, respectively, only after exposure to TCE and lactate. While tceA gene transcripts decreased over time with TCE dechlorination, the vcrA gene was expressed steadily even when the concentration of vinyl chloride was at undetectable levels. In addition, ratios between catabolic and phylogenetic genes indicated that tceA and vcrA gene carrying organisms dechlorinated TCE and its produced daughter products, while vcrA gene was mainly responsible for the dechlorination of the lower VC concentrations in a later stage of degradation.

Methyl oleate for plant protection products formulations: Enzymatic synthesis, reaction kinetics and application testing.

This study presents a solvent-free enzymatic approach for the synthesis of fatty acid methyl esters (FAMEs), such as methyl oleate, for their application as adjuvant in plant protection products (PPP) formulations. The direct esterification between free fatty acid and methanol was optimized to achieve 98% acid conversion. The kinetics of this conversion was accurately described by a simple second order mechanism and non-linear regression was applied to calculate the rate constants of the forward and backward reactions based on full progress curves data. The rate constant of the forward reaction (synthesis) was one order of magnitude higher than the backward reaction (hydrolysis) and favored formation of the target methyl ester product, rendering the removal of water unnecessary. Enzymatically synthesized methyl oleate was benchmarked against the chemically synthesized compound, showing matching results in terms of stability, spreadability and emulsifying capacity in plant care formulations. The enzymatic synthesis of FAMEs under solvent free conditions allows to achieve a safer and more sustainable character for carrier solvents in PPP formulations.

Selective Enzymatic Esterification of Lignin-Derived Phenolics for the Synthesis of Lipophilic Antioxidants.

Lignin is an abundant and renewable source of phenolic compounds that can be used as natural antioxidants to substitute synthetic, petroleum-based alternatives. The development of lignin depolymerization techniques has improved the accessibility of low-molecular-weight phenolic fractions with enhanced antioxidant activity compared to native lignin. The selective esterification of the aliphatic OH groups in these compounds is necessary in order to increase their compatibility with hydrophobic product matrixes, while preserving their antioxidant capacity. In the present work, lipase was chosen as a selective catalyst for the esterification of the monolignol dihydroconiferyl alcohol (DCA), in order to target the esterification of aliphatic OHs without modifying the aromatic groups. The reaction was studied under solvent-assisted and solvent-free conditions, using different fatty acids and substrate ratios. A product yield of 97% could be obtained after 24 h in a solvent-assisted reaction with 2 molar equivalents of fatty acid, or after 3 h in a solvent-free reaction with 10 molar equivalents of the fatty acid. The esterified monolignol showed relevant long-term radical scavenging activity, comparable to other commercial, petroleum-based antioxidants. Different lignin fractions were also used as substrates for enzymatic esterification with different fatty acids, resulting in esterification degrees of 20-58% (of the total aliphatic OH), depending on the specific combination of fatty acid-lignin fractions.

Toward Renewable-Based Prebiotics from Woody Biomass: Potential of Tailored Xylo-Oligosaccharides Obtained by Enzymatic Hydrolysis of Beechwood Xylan as a Prebiotic Feed Supplement for Young Broilers.

Although antibiotic resistance emerges naturally, this process has been accelerated by the worldwide overuse and misuse of antibiotics. It is essential to find effective alternatives in the broiler industry to improve poultry health while maintaining production efficiency and product safety. In this study, we aimed to evaluate a potential alternative: wood-derived xylo-oligosaccharides (XOS). The objective of this research was to investigate the potential of XOS prepared using enzymatic hydrolysis of beechwood xylan as a prebiotic feed supplement for broilers. A pilot study was conducted to explore the optimal XOS fraction profile by in vitro fermentation. Subsequently, a semi-continuous enzyme membrane reactor was used, allowing for the production of tailored XOS in large quantities. Given the strong bidirectional relationship between intestinal health, nutrition, and intestinal microbiota composition in broilers, an in vivo experiment was performed to explore the potential of XOS as a prebiotic feed supplement by investigating growth performance, feed conversion ratio, caecal short and medium chain fatty acid (SCFA and MCFA) concentration, and microbiological composition of the caecal content. Results from the pilot study indicated that higher enzyme concentrations in the hydrolysis process yield a product that leads to a higher total SCFA and MCFA- and butyric acid production during in vitro fermentation by caecal bacteria. Supplementation of the tailored XOS to the broiler diet (day 1 (d1)-d8 0.13% wt/wt XOS, d9-d15 0.32% XOS) resulted in higher Bifidobacterium counts, beneficial to the health of birds, on d11 and d15.

Enzymatic Production of Xylo-oligosaccharides from Destarched Wheat Bran and the Impact of Their Degree of Polymerization and Substituents on Their Utilization as a Carbon Source by Probiotic Bacteria.

The enzymatic production of xylo-oligosaccharides (XOs) from destarched wheat bran with a GH11 xylanase was studied. Xylo-oligosaccharides (XOs) produced were separated into different fractions according to their degree of polymerization (DP) and the nature of their substituents: arabinoxylo-oligosaccharides (AXOs) with a DP from 2 to 3 and DP from 2 to 6 and feruloylated arabinoxylo-oligosaccharides (FAXOs) esterified by ferulic and p-coumaric acids with a DP from 3 to 6. Both AXOs (short and long DP) and FAXOs stimulated the growth of Bifidobacterium adolescentis, Faecalibacterium prausnitzii, and Prevotella copri similarly but not Lactobacillus rhamnosus. The utilization of AXOs and FAXOs as a carbon source resulted in the increase in turbidity, decrease in pH, and production of short-chain fatty acids (SCFAs) in the culture broth. The highest amount of SCFAs was produced by F. prausnitzii using FAXOs. Results suggest that FAXOs and AXOs have the potential to be considered as prebiotics.

Investigating Pervaporation for In Situ Acetone Removal as Process Intensification Tool in ω-Transaminase Catalyzed Chiral Amine Synthesis.

Hydrophobic pervaporation (PV), allowing for the separation of an organic component from an aqueous stream, was investigated for in situ acetone removal from a transamination reaction. A poly(dimethylsiloxane) membrane was applied in a coupled enzymatic process at 5 L scale. Among the four components, there was no loss of donor and product amines through PV which was highly desirable. However, in addition to removal of acetone, there was also an unwanted loss of acetophenone (substrate ketone) because of PV. The coupled enzyme-PV process resulted in 13% more product formation compared to the control process (where no PV was applied) after 9 h. Results from a qualitative simulation study (based on partial vapor pressures and a vapor-liquid equilibrium of the feed solution) indicated that PV might have an advantage over direct distillation strategy for selective removal of acetone from the reaction medium. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2731, 2019.

Architecture and spatial organization in a triple-species bacterial biofilm synergistically degrading the phenylurea herbicide linuron.

Members of a triple-species 3-(3,4-dichlorophenyl)-1-methoxy-1-methyl urea (linuron)-mineralizing consortium, i.e. the linuron- and 3,4-dichloroaniline-degrading Variovorax sp. WDL1, the 3,4-dichloroaniline-degrading Comamonas testosteroni WDL7 and the N,O-dimethylhydroxylamine-degrading Hyphomicrobium sulfonivorans WDL6, were cultivated as mono- or multi-species biofilms in flow cells irrigated with selective or nonselective media, and examined with confocal laser scanning microscopy. In contrast to mono-species biofilms of Variovorax sp. WDL1, the triple-species consortium biofilm degraded linuron completely through apparent synergistic interactions. The triple-species linuron-fed consortium biofilm displayed a heterogeneous structure with an irregular surface topography that most resembled the topography of linuron-fed mono-species WDL1 biofilms, indicating that WDL1 had a dominating influence on the triple-species biofilm architecture. This architecture was dependent on the carbon source supplied, as the biofilm architecture of WDL1 growing on alternative carbon sources was different from that observed under linuron-fed conditions. Linuron-fed triple-species consortium biofilms consisted of mounds composed of closely associated WDL1, WDL7 and WDL6 cells, while this association was lost when the consortium was grown on a nonselective carbon source. In addition, under linuron-fed conditions, microcolonies displaying associated growth developed rapidly after inoculation. These observations indicate that the spatial organization in the linuron-fed consortium biofilm reflected the metabolic interactions within the consortium.

Continuous production of pectic oligosaccharides from onion skins with an enzyme membrane reactor.

The aim of this research was to valorize onion skins, an under-utilized agricultural by-product, into pectic oligosaccharides (POS), compounds with potential health benefits. To achieve high hydrolysis performance with the multi-activity enzyme Viscozyme L, an innovative approach was investigated based on a cross-flow continuous membrane enzyme bioreactor (EMR). The influence of the various process conditions (residence time, enzyme concentration, substrate concentration) was investigated on productivity and yield. The composition of the POS mixtures in terms of mono- and oligosaccharides was assessed at the molecular level. At optimized conditions, a stable POS production with 22.0g/L/h volumetric productivity and 4.5g/g POS/monosaccharides was achieved. Compared to previous results obtained in batch for the enzyme Viscozyme L, EMR provided a 3-5× higher volumetric productivity for the smallest POS. Moreover, it gave competitive results even when compared to batch production with a pure endo-galacturonase enzyme, demonstrating its feasibility for efficient POS production.

Contrasting dual (C, Cl) isotope fractionation offers potential to distinguish reductive chloroethene transformation from breakdown by permanganate.

cis-1,2-Dichloroethene (cis-DCE) and trichloroethene (TCE) are persistent, toxic and mobile pollutants in groundwater systems. They are both conducive to reductive dehalogenation and to oxidation by permanganate. In this study, the potential of dual element (C, Cl) compound specific isotope analyses (CSIA) for distinguishing between chemical oxidation and anaerobic reductive dechlorination of cis-DCE and TCE was investigated. Well-controlled cis-DCE degradation batch tests gave similar carbon isotope enrichment factors εC (‰), but starkly contrasting dual element isotope slopes Δδ13C/Δδ37Cl for permanganate oxidation (εC=-26‰±6‰, Δδ13C/Δδ37Cl≈-125±47) compared to reductive dechlorination (εC=-18‰±4‰, Δδ13C/Δδ37Cl≈4.5±3.4). The difference can be tracked down to distinctly different chlorine isotope fractionation: an inverse isotope effect during chemical oxidation (εCl=+0.2‰±0.1‰) compared to a large normal isotope effect in reductive dechlorination (εCl=-3.3‰±0.9‰) (p≪0.05). A similar trend was observed for TCE. The dual isotope approach was evaluated in the field before and up to 443days after a pilot scale permanganate injection in the subsurface. Our study indicates, for the first time, the potential of the dual element isotope approach for distinguishing cis-DCE (and TCE) concentration drops caused by dilution, oxidation by permanganate and reductive dechlorination both at laboratory and field scale.

PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites.

tmoA and related genes encode the alpha-subunit of the hydroxylase component of the major group (subgroup 1 of subfamily 2) of bacterial multicomponent mono-oxygenase enzyme complexes involved in aerobic benzene, toluene, ethylbenzene and xylene (BTEX) degradation. A PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess the diversity of tmoA-like gene sequences in environmental samples using a newly designed moderately degenerate primer set suitable for that purpose. In 35 BTEX-degrading bacterial strains isolated from a hydrocarbon polluted aquifer, tmoA-like genes were only detected in two o-xylene degraders and were identical to the touA gene of Pseudomonas stutzeri OX1. The diversity of tmoA-like genes was examined in DNA extracts from contaminated and non-contaminated subsurface samples at a site containing a BTEX-contaminated groundwater plume. Differences in DGGE patterns were observed between strongly contaminated, less contaminated and non-contaminated samples and between different depths, suggesting that the diversity of tmoA-like genes was determined by environmental conditions including the contamination level. Phylogenetic analysis of the protein sequences deduced from the amplified amplicons showed that the diversity of TmoA-analogues in the environment is larger than suggested from described TmoA-analogues from cultured isolates, which was translated in the DGGE patterns. Although different positions on the DGGE gel can correspond to closely related TmoA-proteins, relationships could be noticed between the position of tmoA-like amplicons in the DGGE profile and the phylogenetic position of the deduced protein sequence.

Alternative primer sets for PCR detection of genotypes involved in bacterial aerobic BTEX degradation: distribution of the genes in BTEX degrading isolates and in subsurface soils of a BTEX contaminated industrial site.

Eight new primer sets were designed for PCR detection of (i) mono-oxygenase and dioxygenase gene sequences involved in initial attack of bacterial aerobic BTEX degradation and of (ii) catechol 2,3-dioxygenase gene sequences responsible for meta-cleavage of the aromatic ring. The new primer sets allowed detection of the corresponding genotypes in soil with a detection limit of 10(3)-10(4) or 10(5)-10(6) gene copies g(-1) soil, assuming one copy of the gene per cell. The primer sets were used in PCR to assess the distribution of the catabolic genes in BTEX degrading bacterial strains and DNA extracts isolated from soils sampled from different locations and depths (vadose, capillary fringe and saturated zone) within a BTEX contaminated site. In both soil DNA and the isolates, tmoA-, xylM- and xylE1-like genes were the most frequently recovered BTEX catabolic genes. xylM and xylE1 were only recovered from material from the contaminated samples while tmoA was detected in material from both the contaminated and non-contaminated samples. The isolates, mainly obtained from the contaminated locations, belonged to the Actinobacteria or Proteobacteria (mainly Pseudomonas). The ability to degrade benzene was the most common BTEX degradation phenotype among them and its distribution was largely congruent with the distribution of the tmoA-like genotype. The presence of tmoA and xylM genes in phylogenetically distant strains indicated the occurrence of horizontal transfer of BTEX catabolic genes in the aquifer. Overall, these results show spatial variation in the composition of the BTEX degradation genes and hence in the type of BTEX degradation activity and pathway, at the examined site. They indicate that bacteria carrying specific pathways and primarily carrying tmoA/xylM/xylE1 genotypes, are being selected upon BTEX contamination.

Enzymatic milk clotting activity in artichoke (Cynara scolymus) leaves and alpine thistle (Carduus defloratus) flowers. Immobilization of alpine thistle aspartic protease.

Two different milk clotting enzymes, belonging to the aspartic protease family, were extracted from both artichoke leaves and alpine thistle flowers, and the latter was covalently immobilized by using a polyacrylic support containing polar epoxy groups. Our findings showed that the alpine thistle aspartic protease was successfully immobilized at pH 7.0 on Immobeads IB-150P beads and that, under these experimental conditions, an immobilization yield of about 68% and a recovery of about 54% were obtained. Since the enzyme showed an optimal pH of 5.0, a value very similar to the one generally used for milk clotting during cheese making, and exhibited a satisfactory stability over time, the use of such immobilized vegetable rennet for the production of novel dairy products is suggested.