Search

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.

We combine metabolic engineering and polymer chemistry to convert cresols, a main component of biomass-derived catalytic fast pyrolysis wastewater, to methyl muconic acids that have performance-advantaged properties in nylons and plasticizers.

Lignin is the largest source of bio-based aromatic compounds in nature, and its valorization is essential to the sustainability of lignocellulosic biorefining. Characterizing lignin-derived compounds remains challenging due to the heterogeneity of this biopolymer. Tandem mass spectrometry is a promising tool for lignin structural analytics, as fragmentation patterns of model compounds can be extrapolated to identify characteristic moieties in complex samples. This work extended previous resonance excitation-type collision-induced dissociation (CID) methods that identified lignin oligomers containing β-O-4, β-5, and β-β bonds, to also identify characteristics of 5-5, β-1, and 4-O-5 dimers, enabled by quadrupole time-of-flight (QTOF) CID with energy-resolved mass spectrometry (ERMS). Overall, QTOF-ERMS offers in-depth structural information and could ultimately contribute to tools for high-throughput lignin dimer identification.

Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic–catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.

While biomass-derived carbohydrates have been predominant substrates for biological production of renewable fuels, chemicals, and materials, organic waste streams are growing in prominence as potential alternative feedstocks to improve the sustainability of manufacturing processes. Catalytic fast pyrolysis (CFP) is a promising approach to generate biofuels from lignocellulosic biomass, but it generates a complex, carbon-rich, and toxic wastewater stream that is challenging to process catalytically but could be biologically upgraded to valuable co-products. In this work, we implemented modular, heterologous catabolic pathways in the Pseudomonas putida KT2440-derived EM42 strain along with the overexpression of native toxicity tolerance machinery to enable utilization of 89% (w/w) of carbon in CFP wastewater. The dmp monooxygenase and meta-cleavage pathway from Pseudomonas putida CF600 were constitutively expressed to enable utilization of phenol, cresols, 2- and 3-ethyl phenol, and methyl catechols, and the native chaperones clpB, groES, and groEL were overexpressed to improve toxicity tolerance to diverse aromatic substrates. Next, heterologous furfural and acetone utilization pathways were incorporated, and a native alcohol dehydrogenase was overexpressed to improve methanol utilization, generating reducing equivalents. All pathways (encoded by genes totaling ~30 kilobases of DNA) were combined into a single strain that can catabolize a mock CFP wastewater stream as a sole carbon source. Further engineering enabled conversion of all aromatic compounds in the mock wastewater stream to (methyl)muconates with a ~90% (mol/mol) yield. Biological upgrading of CFP wastewater as outlined in this work provides a roadmap for future applications in valorizing other heterogeneous waste streams.

Summary To drive innovation in chemical and material applications beyond what has been afforded by the mature petrochemical industry, new molecules that possess diverse chemical functionality are needed. One source of such molecules lies in the varied metabolic pathways that soil microbes utilize to catabolize aromatic compounds generated during plant decomposition. Here, we have engineered Pseudomonas putida KT2440 to convert these aromatic compounds to 15 catabolic intermediates that exhibit substantial chemical diversity. Bioreactor cultivations, analytical methods, and bench-scale separations were developed to enable production (up to 58 g/L), detection, and purification of each target molecule. We further engineered strains for production of a subset of these molecules from glucose, achieving a 41% molar yield of muconic acid. Finally, we produce materials from three compounds to illustrate the potential for realizing performance-advantaged properties relative to petroleum-derived analogs.

Thermochemical conversion of lignocellulosic biomass is a promising route to produce fuels and oxygenated chemicals and could enable circular carbon utilization. In most thermochemical conversion processes, however, some chemical co-products are lost in aqueous waste streams that are both dilute and heterogeneous. Cost-competitive isolation of these chemical co-products is challenging due to the high-purity requirements typically necessary for bulk chemical production. Here, we demonstrate the production and isolation of two biomass-derived monomers, phenol and catechol, from a comprehensively characterized aqueous waste stream generated via catalytic fast pyrolysis. Specifically, we separate phenol and catechol to 97 wt% purity using the industrially relevant processes of liquid–liquid extraction, distillation, and recrystallization. Techno-economic analysis predicts that a mixed phenolics stream can be produced from the waste stream at a minimum selling price of $1.06 kg−1. Overall, this work demonstrates an approach to high-purity oxygenated aromatic compounds that is potentially economically feasible and technically achievable which increases the atom efficiency of thermochemical conversion through waste stream valorization.

Carboxylic acids are common products produced from the bioconversion of renewable feedstocks. In these processes the separation of the acid product from fermentation broth is the most energy and cost intensive unit operation. Thus, the development of robust, scalable separation approaches that can be applied to a variety of carboxylates is of critical importance to the development of processes that utilize carboxylic acids as platform chemicals. Here we report a batch separation method that includes cell and particulate removal, cation exchange, activated carbon treatment, dewatering with a polymer resin, and product recovery. This method is demonstrated on two unique fermentation broths both derived from corn stover hydrolysate to separate neat succinic and propionic acid. For succinic acid, a crystallization yield of 91% with a product purity of 99.93% was achieved. To our knowledge this is the highest reported crystallization yield and purity for the recovery of succinic acid. Additionally, the method ...

Oleaginous microbes are promising platform strains for the production of renewable diesel and fatty-acid derived chemicals given their innate capacity to produce high lipid yields from lignocellulose-derived sugars. Substantial efforts have been conducted to engineer model oleaginous yeasts primarily on model feedstocks, but to enable lipid production from biomass, judicious strain selection based on phenotypes beneficial for processing, performance on realistic feedstocks, and process integration aspects from sugars to fuels should be examined holistically. To that end, here we report the bench-scale production of diesel blendstock using a biological-catalytic hybrid process based on oleaginous yeast. This work includes flask screening of 31 oleaginous yeast strains, evaluated based on baseline lipid profiles and sugar consumption with corn stover hydrolysate. Three strains were down-selected for bioreactor performance evaluation. The cultivation results reveal that Cryptococcus curvatus ATCC 20509 and Rhodosporodium toruloides DSM-4444 exhibit equivalent fatty acid methyl ester (FAME) yield (0.24 g g−1), whereas the highest overall FAME productivity (0.22 g L−1 h−1) was obtained with C. curvatus, and R. toruloides displayed the highest final FAME titer (23.3 g L−1). Time-resolved lipid profiling (including neutral and polar lipid classing) demonstrated triacylglycerol accumulation as the predominant lipid class in all strains. When evaluating tolerance mechanisms to inhibitory compounds, all strains could reduce and oxidize 5-(hydroxymethyl)furfural, illustrating parallel detoxification mechanisms. The R. toruloides strain was also capable of growth on four aromatic compounds as a sole carbon source, suggesting its use as a strain for simultaneous sugar and lignin conversion. Lipids from R. toruloides were recovered using a mild acid treatment and extraction, hydrogenated, and isomerized to produce a renewable diesel blendstock. The blendstock exhibited a cloud point of −14.5 °C and simulated distillation showed that approximately 75% of the product was in the diesel range with a T90 consistent with no. 2 diesel fuel. Taken together, these results demonstrate an integrated process for renewable diesel production, identify oleaginous strains for further development, and highlight opportunities for improvements to an oleaginous microbial platform for the production of renewable diesel blendstock.

Thermochemical (TC) biomass conversion processes such as pyrolysis and liquefaction generate considerable amounts of wastewater, which often contains highly toxic compounds that are incredibly challenging to convert via standard wastewater treatment approaches such as anaerobic digestion. These streams represent a cost for TC biorefineries, and a potential valorization opportunity, if effective conversion methods are developed. The primary challenge hindering microbial conversion of TC wastewater is toxicity. In this study, we employ a robust bacterium, Pseudomonas putida, with TC wastewater streams to demonstrate that aldehydes are the most inhibitory compounds in these streams. Proteomics, transcriptomics, and fluorescence-based immunoassays of P. putida grown in a representative wastewater stream indicate that stress results from protein damage, which we hypothesize is a primary toxicity mechanism. Constitutive overexpression of the chaperone genes, groEL, groES, and clpB, in a genome-reduced P. putida strain improves the tolerance towards multiple TC wastewater samples up to 200-fold. Moreover, the concentration ranges of TC wastewater are industrially relevant for further bioprocess development for all wastewater streams examined here, representing different TC process configurations. Furthermore, we demonstrate proof-of-concept polyhydroxyalkanoate production from the usable carbon in an exemplary TC wastewater stream. Overall, this study demonstrates that protein quality control machinery and repair mechanisms can enable substantial gains in microbial tolerance to highly toxic substrates, including heterogeneous waste streams. When coupled to other metabolic engineering advances such as expanded substrate utilization and enhanced product accumulation, this study generally enables new strategies for biological conversion of highly-toxic, organic-rich wastewater via engineered aerobic monocultures or designer consortia.

Muconic acid (MA) is a bio-based platform chemical that can be converted into the commodity petrochemical building blocks adipic acid or terephthalic acid, or used in emerging, performance-advantaged materials. MA is a metabolic intermediate in the β-ketoadipate pathway, and can be produced from carbohydrates or other traditional carbon sources via the shikimate pathway. MA can also be produced from lignin-derived aromatic compounds with high atom efficiency through aromatic-catabolic pathways. Metabolic engineering efforts to date have developed efficient muconic acid-producing strains of the aromatic-catabolic microbe Pseudomonas putida KT2440, but the titers, productivities, and yields from aromatic compounds in most cases remain below the thresholds needed for industrially-relevant bioreactor cultivations. To that end, this work presents further process and host development towards improving MA titers, yields, and productivities, using the hydroxycinnamic acids, p-coumaric acid and ferulic acid, as model aromatic compounds. Coupling strain engineering and bioprocess development enabled the discovery of new bottlenecks in P. putida that hinder MA production from these compounds. A combination of gene overexpression and removal of a global catabolic regulator resulted in high-yielding strains (100% molar yield). Maximum MA titers of 50 g L−1, which is near the lethal toxicity limit in this bacterium, and productivities over 0.5 g L−1 h−1 were achieved in separate process configurations. Additionally, a high-pH feeding strategy, which could potentially reduce the salt load and enable higher titers by decreasing product dilution, was tested with model compounds and lignin-rich streams from corn stover and a complete conversion of the primary monomeric aromatic compounds to MA was demonstrated, obtaining a titer of 4 g L−1. Overall, this study presents a step forward for the production of value-added chemicals from lignin and highlights critical needs for further strain improvement and bioprocess development that can be applied in the biological valorization of lignin.

Catalytic fast pyrolysis (CFP) of biomass produces a liquid product consisting of organic and aqueous streams. The organic stream is typically slated for hydrotreating to produce hydrocarbon biofuels, while the aqueous stream is considered a waste stream, resulting in the loss of residual biogenic carbon. Here, we report the detailed characterization and catalytic conversion of a CFP wastewater stream with the ultimate aim to improve overall biomass utilization within a thermochemical biorefinery. An aqueous stream derived from CFP of beech wood was comprehensively characterized, quantifying 53 organic compounds to a total of 17% organics. The most abundant classes of compounds are acids, aldehydes, and alcohols. The most abundant components identified in the aqueous stream were C1-C2 organics, comprising 6.40% acetic acid, 2.16% methanol, and 1.84% formaldehyde on wet basis. The CFP aqueous stream was catalytically upgraded to olefins and aromatic hydrocarbons using a Ga/HZSM-5 catalyst at 500°C. When th...

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...

Summary Microbial conversion offers a promising strategy for overcoming the intrinsic heterogeneity of the plant biopolymer, lignin. Soil microbes that natively harbour aromatic‐catabolic pathways are natural choices for chassis strains, and Pseudomonas putida KT2440 has emerged as a viable whole‐cell biocatalyst for funnelling lignin‐derived compounds to value‐added products, including its native carbon storage product, medium‐chain‐length polyhydroxyalkanoates (mcl‐PHA). In this work, a series of metabolic engineering targets to improve mcl‐PHA production are combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p‐coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene phaZ was knocked out, and the genes involved in β‐oxidation (fadBA1 and fadBA2) were deleted. Additionally, to increase carbon flux into mcl‐PHA biosynthesis, phaG, alkK, phaC1 and phaC2 were overexpressed. The best performing strain – which contains all the genetic modifications detailed above – demonstrated a 53% and 200% increase in mcl‐PHA titre (g l−1) and a 20% and 100% increase in yield (g mcl‐PHA per g cell dry weight) from p‐coumaric acid and lignin, respectively, compared with the wild type strain. Overall, these results present a promising strain to be employed in further process development for enhancing mcl‐PHA production from aromatic compounds and lignin., In this work, a series of metabolic engineering targets to improve mcl‐PHA production were combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p‐coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene (phaZ) and genes involved in β‐oxidation (fadBA1 and fadBA2) were deleted, while phaG, alkK, phaC1, and phaC2 were overexpressed to increase carbon flux from fatty acid biosynthesis to mcl‐PHA production. This strain demonstrated an increase in mcl‐PHA titer (g l−1) and yield (g mcl‐PHA per g cell dry weight) from p‐coumaric acid and from lignin compared to the wild‐type strain, resulting in a promising strain to be employed in further process development for enhancing mcl‐PHA production from aromatic compounds and lignin.

Correction for ‘Thermochemical wastewater valorization via enhanced microbial toxicity tolerance’ by Lahiru N. Jayakody et al., Energy Environ. Sci., 2018, 11, 1625–1638, DOI: 10.1039/C8EE00460A.

Biomass pyrolysis offers a promising means to rapidly depolymerize lignocellulosic biomass for subsequent catalytic upgrading to renewable fuels. Substantial efforts are currently ongoing to optimize pyrolysis processes including various fast pyrolysis and catalytic fast pyrolysis schemes. In all cases, complex aqueous streams are generated containing solubilized organic compounds that are not converted to target fuels or chemicals and are often slated for wastewater treatment, in turn creating an economic burden on the biorefinery. Valorization of the species in these aqueous streams, however, offers significant potential for substantially improving the economics and sustainability of thermochemical biorefineries. To that end, here we provide a thorough characterization of the aqueous streams from four pilot-scale pyrolysis processes: namely, from fast pyrolysis, fast pyrolysis with downstream fractionation, in situ catalytic fast pyrolysis, and ex situ catalytic fast pyrolysis. These configurations and ...

The production of chemicals alongside fuels will be essential to enhance the feasibility of lignocellulosic biorefineries. Succinic acid (SA), a naturally occurring C4-diacid, is a primary intermediate of the tricarboxylic acid cycle and a promising building block chemical that has received significant industrial attention. Basfia succiniciproducens is a relatively unexplored SA-producing bacterium with advantageous features such as broad substrate utilization, genetic tractability, and facultative anaerobic metabolism. Here B. succiniciproducens is evaluated in high xylose-content hydrolysates from corn stover and different synthetic media in batch fermentation. SA titers in hydrolysate at an initial sugar concentration of 60 g/L reached up to 30 g/L, with metabolic yields of 0.69 g/g, and an overall productivity of 0.43 g/L/h. These results demonstrate that B. succiniciproducens may be an attractive platform organism for bio-SA production from biomass hydrolysates.

In Nature, powerful oxidative enzymes secreted by white rot fungi and some bacteria catalyze lignin depolymerization and some microbes are able to catabolize the resulting aromatic compounds as carbon and energy sources. Taken together, these two processes offer a potential route for microbial valorization of lignin. However, many challenges remain in realizing this concept, including that oxidative enzymes responsible for lignin depolymerization also catalyze polymerization of low molecular weight (LMW) lignin. Here, multiple basidiomycete secretomes were screened for ligninolytic enzyme activities in the presence of a residual lignin solid stream from a corn stover biorefinery, dubbed DMR-EH (Deacetylation, Mechanical Refining, and Enzymatic Hydrolysis) lignin. Two selected fungal secretomes, with high levels of laccases and peroxidases, were utilized for DMR-EH lignin depolymerization assays. The secretome from Pleurotus eryngii, which exhibited the highest laccase activity, reduced the lignin average molecular weight (Mw) by 63% and 75% at pH 7 compared to the Mw of the control treated at the same conditions and the initial DMR-EH lignin, respectively, and was applied in further depolymerization assays as a function of time. As repolymerization was observed after 3 days of incubation, an aromatic-catabolic microbe (Pseudomonas putida KT2440) was incubated with the fungal secretome and DMR-EH lignin. These experiments demonstrated that the presence of the bacterium enhances lignin depolymerization, likely due to bacterial catabolism of LMW lignin, which may partially prevent repolymerization. In addition, proteomics was also applied to the P. eryngii secretome to identify the enzymes present in the fungal cocktail utilized for the depolymerization assays, which highlighted a significant number of glucose/methanol/choline (GMC) oxidoreductases and laccases. Overall, this study demonstrates that ligninolytic enzymes can be used to partially depolymerize a solid, high lignin content biorefinery stream and that the presence of an aromatic-catabolic bacterium as a “microbial sink” improves the extent of enzymatic lignin depolymerization.

This study determined whether chitin-oligosaccharides and chitosan-oligosaccharides (COS) are suitable acceptor carbohydrates for the LacLM-type β-galactosidase of Lactobacillus plantarum . Enzymatic reactions were carried out with lactose as galactosyl-donor and chitinbiose (N,N′-diacetyl chitobiose), chitintriose (N,N′N″-triacetyl chitotriose) or a highly deacetylated COS preparation with a degree of polymerization ranging from 2 to 4 as galactosyl acceptors. Transgalactosylated oligosaccharides were identified by liquid chromatography-mass spectrometry (LC-MS/MS). LC-MS/MS analysis demonstrated formation of mono- and digalactosylated chitinbiose, mono-galactosylated chitintriose, and mono-, di-, and trigalactosylated COS. β-(1→4)-Linkages were formed during the galactosylation of chitinbiose and chitintriose. In conclusion, the conversion of lactose with β-Gal from Lb. plantarum and chitin-oligosaccharides or COS as glycosyl acceptors allows the production of novel oligosaccharides.

© 2017 American Chemical Society. Lignin valorization offers significant potential to enhance the economic viability of lignocellulosic biorefineries. However, because of its heterogeneous and recalcitrant nature, conversion of lignin to value-added coproducts remains a considerable technical challenge. In this study, we employ base-catalyzed depolymerization (BCD) using a process-relevant solid lignin stream produced via deacetylation, mechanical refining, and enzymatic hydrolysis to enable biological lignin conversion. BCD was conducted with the solid lignin substrate over a range of temperatures at two NaOH concentrations, and the results demonstrate that the lignin can be partially extracted and saponified at temperatures as low as 60 °C. At 120 °C and 2% NaOH, the high extent of lignin solubility was accompanied by a considerable decrease in the lignin average molecular weight and the release of lignin-derived monomers including hydroxycinnamic acids. BCD liquors were tested for microbial growth using seven aromatic-catabolizing bacteria and two yeasts. Three organisms (Pseudomonas putida KT2440, Rhodotorula mucilaginosa, and Corynebacterium glutamicum) tolerate high BCD liquor concentrations (up to 90% v/v) and rapidly consume the main lignin-derived monomers, resulting in lignin conversion of up to 15%. Furthermore, as a proof of concept, muconic acid production from a representative lignin BCD liquor was demonstrated with an engineered P. putida KT2440 strain. These results highlight the potential for a mild lignin depolymerization process to enhance the microbial conversion of solid lignin-rich biorefinery streams.

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 Lactobacillus plantarum 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.

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-cis-12-octadecenoic and 10-hydroxy-trans-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.

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 N-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.

α-Galacto-oligosaccharides (α-GOS) are produced by transgalactosylation reactions of α-galactosidase (α-Gal) or by conversion of raffinose family oligosaccharides by levansucrase. Similarly to β-GOS, α-GOS have the potential to mimic glycan receptors on eukaryotic cells and act as molecular decoys to prevent bacterial infection; however, data on transgalactosylation reactions of α-Gal remain scarce. The α-Gal gene sequence from Lactobacillus reuteri was cloned into an α-Gal negative strain of Lactococcus lactis. Transgalactosylation reactions were achieved using crude cell extracts with melibiose or raffinose as galactosyl donor and fucose, N-acetylglucosamine or lactose as galactosyl acceptor. The composition, sequence and most linkage types of α-GOS formed with acceptors saccharides were determined by liquid chromatography-tandem mass spectrometry. α-Gal of Lactobacillus reuteri formed (1 → 3)-, (1 → 4)- or (1 → 6)-linked α-GOS but exhibited a preference for formation of (1 → 6)-linkages. Fucose, N-acetylglucosamine and lactose were suitable galactosyl acceptors for α-Gal of L. reuteri, resulting in formation of (1 → 3)-, (1 → 4)- or (1 → 6)-linked hetero-oligosaccharides. By determining the structural specificity of α-Gal and increasing the variation of oligosaccharides produced by introducing alternative acceptor sugars, this work supports further studies to assess α-GOS pathogen adhesion prevention in mammalian hosts.

Lactobacilli convert linoleic acid to hydroxy fatty acids; however, this conversion has not been demonstrated in food fermentations and it remains unknown whether hydroxy fatty acids produced by lactobacilli have antifungal activity. This study aimed to determine whether lactobacilli convert linoleic acid to metabolites with antifungal activity and to assess whether this conversion can be employed to delay fungal growth on bread. Aqueous and organic extracts from seven strains of lactobacilli grown in modified De Man Rogosa Sharpe medium or sourdough were assayed for antifungal activity. Lactobacillus hammesii exhibited increased antifungal activity upon the addition of linoleic acid as a substrate. Bioassay-guided fractionation attributed the antifungal activity of L. hammesii to a monohydroxy C 18:1 fatty acid. Comparison of its antifungal activity to those of other hydroxy fatty acids revealed that the monohydroxy fraction from L. hammesii and coriolic (13-hydroxy-9,11-octadecadienoic) acid were the most active, with MICs of 0.1 to 0.7 g liter −1 . Ricinoleic (12-hydroxy-9-octadecenoic) acid was active at a MIC of 2.4 g liter −1 . L. hammesii accumulated the monohydroxy C 18:1 fatty acid in sourdough to a concentration of 0.73 � 0.03 g liter −1 (mean � standard deviation). Generation of hydroxy fatty acids in sourdough also occurred through enzymatic oxidation of linoleic acid to coriolic acid. The use of 20% sourdough fermented with L. hammesii or the use of 0.15% coriolic acid in bread making increased the mold-free shelf life by 2 to 3 days or from 2 to more than 6 days, respectively. In conclusion, L. hammesii converts linoleic acid in sourdough and the resulting monohydroxy octadecenoic acid exerts antifungal activity in bread.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0425-1) contains supplementary material, which is available to authorized users.

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.