Continuous succinic acid production by Actinobacillus succinogenes on xylose‑enriched hydrolysate

BACKGROUND : Bio-manufacturing of high-value chemicals in parallel to renewable biofuels has the potential to dramatically improve the overall economic landscape of integrated lignocellulosic biorefineries. However, this will require the generation of carbohydrate streams from lignocellulose in a form suitable for efficient microbial conversion and downstream processing appropriate to the desired end use, making overall process development, along with selection of appropriate target molecules, crucial to the integrated biorefinery. Succinic acid (SA), a high-value target molecule, can be biologically produced from sugars and has the potential to serve as a platform chemical for various chemical and polymer applications. However, the feasibility of microbial SA production at industrially relevant productivities and yields from lignocellulosic biorefinery streams has not yet been reported. RESULTS : Actinobacillus succinogenes 130Z was immobilised in a custom continuous fermentation setup to produce SA on the xylose-enriched fraction of a non-detoxified, xylose-rich corn stover hydrolysate stream produced from deacetylation and dilute acid pretreatment. Effective biofilm attachment, which serves as a natural cell retention strategy to increase cell densities, productivities and resistance to toxicity, was accomplished by means of a novel agitator fitting. A maximum SA titre, yield and productivity of 39.6 g L−1, 0.78 g g−1 and 1.77 g L−1 h−1 were achieved, respectively. Steady states were obtained at dilution rates of 0.02, 0.03, 0.04, and 0.05 h−1 and the stirred biofilm reactor was stable over prolonged periods of operation with a combined fermentation time of 1550 h. Furthermore, it was found that a gradual increase in the dilution rate was required to facilitate adaptation of the culture to the hydrolysate, suggesting a strong evolutionary response to the toxic compounds in the hydrolysate. Moreover, the two primary suspected fermentation inhibitors, furfural and HMF, were metabolised during fermentation with the concentration of each remaining at zero across all steady states. CONCLUSIONS : The results demonstrate that immobilised A. succinogenes has the potential for effective conversion of an industrially relevant, biomass-derived feed stream to succinic acid. Furthermore, due to the attractive yields, productivities and titres achieved in this study, the process has the potential to serve as a means for value-added chemical manufacturing in the integrated biorefinery.The National Research Foundation (NRF) and the US Department of Energy Bioenergy Technologies Office.http://biotechnologyforbiofuels.biomedcentral.comam201

Succinic acid production on xylose‑enriched biorefinery streams by Actinobacillus succinogenes in batch fermentation

BACKGROUND : Co-production of chemicals from lignocellulosic biomass alongside fuels holds promise for improving the economic outlook of integrated biorefineries. In current biochemical conversion processes that use thermochemical pretreatment and enzymatic hydrolysis, fractionation of hemicellulose-derived and cellulose-derived sugar streams is possible using hydrothermal or dilute acid pretreatment (DAP), which then offers a route to parallel trains for fuel and chemical production from xylose- and glucose-enriched streams. Succinic acid (SA) is a co-product of particular interest in biorefineries because it could potentially displace petroleum-derived chemicals and polymer precursors for myriad applications. However, SA production from biomass-derived hydrolysates has not yet been fully explored or developed. RESULTS : Here, we employ Actinobacillus succinogenes 130Z to produce succinate in batch fermentations from various substrates including (1) pure sugars to quantify substrate inhibition, (2) from mock hydrolysates similar to those from DAP containing single putative inhibitors, and (3) using the hydrolysate derived from two pilot-scale pretreatments: first, a mild alkaline wash (deacetylation) followed by DAP, and secondly a single DAP step, both with corn stover. These latter streams are both rich in xylose and contain different levels of inhibitors such as acetate, sugar dehydration products (furfural, 5-hydroxymethylfurfural), and lignin-derived products (ferulate, p-coumarate). In batch fermentations, we quantify succinate and co-product (acetate and formate) titers as well as succinate yields and productivities. We demonstrate yields of 0.74 g succinate/g sugars and 42.8 g/L succinate from deacetylated DAP hydrolysate, achieving maximum productivities of up to 1.27 g/L-h. Moreover, A. succinogenes is shown to detoxify furfural via reduction to furfuryl alcohol, although an initial lag in succinate production is observed when furans are present. Acetate seems to be the main inhibitor for this bacterium present in biomass hydrolysates. CONCLUSION : Overall, these results demonstrate that biomass-derived, xylose-enriched hydrolysates result in similar yields and titers but lower productivities compared to clean sugar streams, which can likely be improved via fermentation process developments and metabolic engineering. Overall, this study comprehensively examines the behavior of A. succinogenes on xylose-enriched hydrolysates on an industrially relevant, lignocellulosic feedstock, which will pave the way for future work toward eventual SA production in an integrated biorefinery.Additional file 1. Supporting information.http://biotechnologyforbiofuels.biomedcentral.com/am2016Chemical Engineerin

Mixed plastics waste valorization through tandem chemical oxidation and biological funneling

115 p.-4 fig.-45 fig. supl.-14 tab supl.Mixed plastics waste represents an abundant and largely untapped feedstock for the production of valuable products. The chemical diversity and complexity of thesematerials, however, present major barriers to realizing this opportunity. In this work, we show that metal-catalyzed autoxidation depolymerizes comingled polymers into a mixture of oxygenated small molecules that are advantaged substrates for biological conversion. We engineer a robust soil bacterium, Pseudomonas putida, to funnel these oxygenated compounds into a single exemplary chemical product, either b-ketoadipate or polyhydroxyalkanoates. This hybrid process establishes a strategy for the selective conversion of mixed plastics waste into useful chemical products.Funding was provided by the US Department of Energy, Office of Energy Efficiency and Renewable Energy, Advanced Manufacturing Office (AMO), and Bioenergy Technologies Office (BETO). This work was performed as part of the BOTTLE Consortium and was supported by AMO and BETO under contract no. DE-AC36- 08GO28308 with the National Renewable Energy Laboratory (NREL),operated by the Alliance for Sustainable Energy, LLC. The BOTTLE Consortium includes members from MIT, funded under contract no. DE-AC36-08GO28308 with NREL. Contributions by S.S.S. were supported by the US Department of Energy, Office of Basic Energy Sciences, under award no. DEFG02-05ER15690.Peer reviewe

Smoke, curtains and mirrors: the production of race through time and title registration

This article analyses the temporal effects of title registration and their relationship to race. It traces the move away from the retrospection of pre-registry common law conveyancing and toward the dynamic, future-oriented Torrens title registration system. The Torrens system, developed in early colonial Australia, enabled the production of ‘clean’, fresh titles that were independent of their predecessors. Through a process praised by legal commentators for ‘curing’ titles of their pasts, this system produces indefeasible titles behind its distinctive ‘curtain’ and ‘mirror’, which function similarly to magicians’ smoke and mirrors by blocking particular realities from view. In the case of title registries, those realities are particular histories of and relationships with land, which will not be protected by property law and are thus made precarious. Building on interdisciplinary work which theorises time as a social tool, I argue that Torrens title registration produces a temporal order which enables land market coordination by rendering some relationships with land temporary and making others indefeasible. This ordering of relationships with land in turn has consequences for the human subjects who have those relationships, cutting futures short for some and guaranteeing permanence to others. Engaging with Renisa Mawani and other critical race theorists, I argue that the categories produced by Torrens title registration systems materialise as race

EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies), 2013. Scientific Opinion on nutrient requirements and dietary intakes of infants and young children in the European Union.

Following a request from the European Commission, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver a Scientific Opinion on the nutrient requirements and dietary intakes of infants and young children in the European Union. This Opinion describes the dietary requirements of infants and young children, compares dietary intakes and requirements in infants and young children in Europe and, based on these findings, concludes on the potential role of young-child formulae in the diets of infants and young children, including whether they have any nutritional benefits when compared with other foods that may be included in the normal diet of infants and young children. The Panel concluded on the levels of nutrient and energy intakes that are considered adequate for the majority of infants and young children, and evaluated the risk of inadequate nutrient intakes in infants and young children in living Europe. Dietary intakes of alpha-linolenic acid (ALA), docosahexaenoic acid (DHA), iron, vitamin D and iodine (in some European countries) are low in infants and young children living in Europe, and particular attention should be paid to ensuring an appropriate supply of ALA, DHA, iron, vitamin D and iodine in infants and young children with inadequate or at risk of inadequate status of these nutrients. No unique role of young -child formulae with respect to the provision of critical nutrients in the diet of infants and young children living in Europe can be identified, so that they cannot be considered as a necessity to satisfy the nutritional requirements of young children when compared with other foods that may be included in the normal diet of young children (such as breast milk, infant formulae, follow-on formulae and cow\u2018s milk)

Identification of Conjugated Linoleic Acid (CLA) Isomers by Silver Ion-Liquid Chromatography/In-line Ozonolysis/Mass Spectrometry (Ag⁺‑LC/O₃‑MS)

A novel method for the identification of conjugated linoleic acid (CLA) isomers has been developed in which silver ion liquid chromatography is coupled to in-line ozonolysis/mass spectrometry (Ag⁺-LC/O₃-MS). The mobile phase containing CLA isomers eluting from the Ag⁺-LC column flows through a length of gas-permeable tubing within an ozone rich environment. Ozone penetrating the tubing wall reacts with the conjugated double bonds forming ozonolysis product aldehydes. These, and their corresponding methanol loss fragment ions formed within the atmospheric pressure photoionization (APPI) source, were detected by in-line MS and used for the direct assignment of double bond positions. Assignment of positional isomers is based entirely on the two pairs of diagnostic ions seen in the in-line O₃-MS mass spectra. In this way, de novo identification of CLA positional isomers, i.e. without requiring comparison to CLA standards, was achieved. The Ag⁺-LC/O₃-MS method was applied to the analysis of CLA isomers in a commercial CLA supplement, milk fat, and the lipid extract from a <i>Lactobacillus plantarum</i> TMW1460 culture. The results demonstrate how Ag⁺-LC/O₃-MS can be used for the direct and fast determination of CLA isomers at low concentrations and in complex lipid mixtures

Antifungal Lipids Produced by Lactobacilli and Their Structural Identification by Normal Phase LC/Atmospheric Pressure Photoionization–MS/MS

Lactobacillus hammesii converts linoleic acid into an antifungal hydroxy fatty acid. High speed counter-current chromatography (HSCCC) using a hexane/ethyl acetate/methanol/water solvent system [3.5:1.5:3:2 (v/v/v/v)] allowed isolation of the antifungal hydroxy fatty acid. A method was developed for characterization of antifungal hydroxy fatty acids using normal phase liquid chromatography combined with atmospheric pressure photoionization–tandem mass spectrometry (LC/APPI-MS/MS). The position of unsaturations and hydroxyl groups was determined directly from crude lipid extracts and their hydroxylated derivatives. The antifungal compounds were identified as a racemic mixture of 10-hydroxy-<i>cis</i>-12-octadecenoic and 10-hydroxy-<i>trans</i>-12-octadecenoic acid. Additionally, HSCCC and LC/APPI-MS/MS methods were used to elucidate the pathway of conversion of linoleic acid by Lactobacillus sanfranciscensis, Lactobacillus plantarum, and L. hammesii to hydroxy fatty acids and conjugated linoleic acid. This study links previously reported 10-hydroxy-12-octadecenoic acid producing Lactobacillus strains to antifungal activities

Alkaline Peroxide Delignification of Corn Stover

Selective biomass fractionation into carbohydrates and lignin is a key challenge in the conversion of lignocellulosic biomass to fuels and chemicals. In the present study, alkaline hydrogen peroxide (AHP) pretreatment was investigated to fractionate lignin from polysaccharides in corn stover (CS), with a particular emphasis on the fate of the lignin for subsequent valorization. The influence of peroxide loading on delignification during AHP pretreatment was examined over the range of 30–500 mg H₂O₂/g dry CS at 50 °C for 3 h. Mass balances were conducted on the solid and liquid fractions generated after pretreatment for each of the three primary components, lignin, hemicellulose, and cellulose. AHP pretreatment at 250 mg H₂O₂/g dry CS resulted in the pretreated solids with more than 80% delignification consequently enriching the carbohydrate fraction to >90%. Two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy of the AHP pretreated residue shows that, under high peroxide loadings (>250 mg H₂O₂/g dry CS), most of the side chain structures were oxidized and the aryl-ether bonds in lignin were partially cleaved, resulting in significant delignification of the pretreated residues. Gel permeation chromatography (GPC) analysis shows that AHP pretreatment effectively depolymerizes CS lignin into low molecular weight (LMW) lignin fragments in the aqueous fraction. Imaging of AHP pretreated residues shows a more granular texture and a clear lamellar pattern in secondary walls, indicative of layers of varying lignin removal or relocalization. Enzymatic hydrolysis of this pretreated residue at 20 mg/g of glucan resulted in 90% and 80% yields of glucose and xylose, respectively, after 120 h. Overall, AHP pretreatment is able to selectively remove more than 80% of the lignin from biomass in a form that has potential for downstream valorization processes and enriches the solid pulp into a highly digestible material

Structural Identification of Novel Oligosaccharides Produced by Lactobacillus bulgaricus and Lactobacillus plantarum

β-Galactosidases (β-Gal) of lactic acid bacteria produce oligosaccharides from lactose when suitable acceptor carbohydrates are present. This study aimed to elucidate the structure of oligosaccharides formed by galactosylation of <i>N</i>-acetylglucosamine (GlcNAc) and fucose. Crude cellular extract of Lactobacillus bulgaricus and LacLM of Lactobacillus plantarum were used as sources of β-Gal activity. Disaccharides obtained by galactosylation of GlcNAc were identified as Gal-β-(1→4)-GlcNAc or Gal-β-(1→6)-GlcNAc by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and comparison with external standards. Trisaccharides were identified as Gal-β-(1→6)-Gal-β-(1→[4 or 6])-GlcNAc by LC-MS, analysis of the MS/MS spectra of selected in-source fragment ions, and their relative retention times. LC-MS analysis revealed the presence of five galactosylated fucosides, but their linkage type could not be identified, partly due to the lack of reference compounds. β-Gal of lactic acid bacteria may serve as suitable tools for the chemoenzymatic synthesis of therapeutic oligosaccharides