BACTERIAL BIOTRANSFORMATION OF LIGNIN IN ANOXIC ENVIRONMENTS

There is a growing need to reduce reliance on non-renewable fuels, especially fossil fuels that contribute to the climate crisis. Plant lignocellulose is an abundant and undervalued source of energy, but its use is hindered due to the recalcitrance of the lignin-component. Current methods to remove lignin have sustainability concerns and are costly for industrial applications such as paper mill pulping. An alternative and greener approach is biopulping, which uses microbes and their enzymes to break down lignin. However, there are limitations to biopulping that prevent it from outcompeting other pulping processes, such as requiring constant aeration and mixing. The work presented in this dissertation investigates anaerobic bacteria as a promising alternative source for consolidated depolymerization of lignin and its conversion to valuable byproducts. We first ask if anaerobic aromatic metabolism is vertically inherited or horizontally transferred across bacteria. We analyzed seven out of the nine known central intermediate pathways. Of the seven, benzoyl-CoA metabolism had the strongest phylogenetic signal, suggesting vertical inheritance is the driver of its phylogenetic distribution. This information can be used in future studies to test if predictions can be made for uncharacterized taxa and anaerobic benzoyl-CoA related metabolism. We also investigated the mechanisms of two uncharacterized isolates, Sodalis sp. strain 159R and Tolumonas lignolytica BRL6-1. Strain 159R contains many genes related to both aerobic and anaerobic aromatic metabolism but lacks extracellular enzymes for anaerobic lignin depolymerization. Conversely, strain BRL6-1 did not demonstrate lignin metabolism but instead relies on iron redox and organic radicals to potentially modify lignin structure under anoxic conditions. The electron exchange between iron, lignin, and BRL6-1 suggests a protein that acts as a chelator and redox molecule is the intermediate between the bacteria and substrate. The two isolates demonstrate the importance that lignin depolymerization and metabolism may be found separately in organisms and should be considered in future designs for anaerobic biopulping and lignin valorization to be a competitive process on the market

A Membrane-Bound Cytochrome Enables Methanosarcina acetivorans To Conserve Energy from Extracellular Electron Transfer

The discovery of a methanogen that can conserve energy to support growth solely from the oxidation of organic carbon coupled to the reduction of an extracellular electron acceptor expands the possible environments in which methanogens might thrive. The potential importance of c-type cytochromes for extracellular electron transfer to syntrophic bacterial partners and/or Fe(III) minerals in some Archaea was previously proposed, but these studies with Methanosarcina acetivorans provide the first genetic evidence for cytochrome-based extracellular electron transfer in Archaea. The results suggest parallels with Gram-negative bacteria, such as Shewanella and Geobacter species, in which multiheme outer-surface c-type cytochromes are an essential component for electrical communication with the extracellular environment. M. acetivorans offers an unprecedented opportunity to study mechanisms for energy conservation from the anaerobic oxidation of one-carbon organic compounds coupled to extracellular electron transfer in Archaea with implications not only for methanogens but possibly also for Archaea that anaerobically oxidize methane.Extracellular electron exchange in Methanosarcina species and closely related Archaea plays an important role in the global carbon cycle and enhances the speed and stability of anaerobic digestion by facilitating efficient syntrophic interactions. Here, we grew Methanosarcina acetivorans with methanol provided as the electron donor and the humic analogue, anthraquione-2,6-disulfonate (AQDS), provided as the electron acceptor when methane production was inhibited with bromoethanesulfonate. AQDS was reduced with simultaneous methane production in the absence of bromoethanesulfonate. Transcriptomics revealed that expression of the gene for the transmembrane, multiheme, c-type cytochrome MmcA was higher in AQDS-respiring cells than in cells performing methylotrophic methanogenesis. A strain in which the gene for MmcA was deleted failed to grow via AQDS reduction but grew with the conversion of methanol or acetate to methane, suggesting that MmcA has a specialized role as a conduit for extracellular electron transfer. Enhanced expression of genes for methanol conversion to methyl-coenzyme M and the Rnf complex suggested that methanol is oxidized to carbon dioxide in AQDS-respiring cells through a pathway that is similar to methyl-coenzyme M oxidation in methanogenic cells. However, during AQDS respiration the Rnf complex and reduced methanophenazine probably transfer electrons to MmcA, which functions as the terminal reductase for AQDS reduction. Extracellular electron transfer may enable the survival of methanogens in dynamic environments in which oxidized humic substances and Fe(III) oxides are intermittently available. The availability of tools for genetic manipulation of M. acetivorans makes it an excellent model microbe for evaluating c-type cytochrome-dependent extracellular electron transfer in Archaea

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<p>Venn diagrams of differentially expressed genes, where <b>(A)</b> green circles indicate number of total genes are up-regulated each time point, and <b>(B)</b> red circles indicate number of total genes were down-regulated each time point. EE indicates cells analyzed in early exponential phase, ME, mid-exponential phase and ES, early stationary phase, where actual times of sampling each cell population is indicated in the methods as well as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186440#pone.0186440.g001" target="_blank">Fig 1A</a>. <b>(C)</b> Changes in the gene profile as a result of lignin exposure in early exponential phase, mid-exponential and early stationary phase of growth. In the right side of each chart (blue), number of genes with increased relative abundance, and in the left side (red), number of genes with lower relative abundance. The genes are grouped according to functional class as defined by COG annotation.</p

Lignin induced iron reduction by novel sp., Tolumonas lignolytic BRL6-1

Lignin is the second most abundant carbon polymer on earth and despite having more fuel value than cellulose, it currently is considered a waste byproduct in many industrial lignocellulose applications. Valorization of lignin relies on effective and green methods of de-lignification, with a growing interest in the use of microbes. Here we investigate the physiology and molecular response of the novel facultative anaerobic bacterium, Tolumonas lignolytica BRL6-1, to lignin under anoxic conditions. Physiological and biochemical changes were compared between cells grown anaerobically in either lignin-amended or unamended conditions. In the presence of lignin, BRL6-1 accumulates higher biomass and has a shorter lag phase compared to unamended conditions, and 14% of the proteins determined to be significantly higher in abundance by log fold-change of 2 or greater were related to Fe(II) transport in late logarithmic phase. Ferrozine assays of the supernatant confirmed that Fe(III) was bound to lignin and reduced to Fe(II) only in the presence of BRL6-1, suggesting redox activity by the cells. LC-MS/MS analysis of the secretome showed an extra band at 20 kDa in lignin-amended conditions. Protein sequencing of this band identified a protein of unknown function with homology to enzymes in the radical SAM superfamily. Expression of this protein in lignin-amended conditions suggests its role in radical formation. From our findings, we suggest that BRL6-1 is using a protein in the radical SAM superfamily to interact with the Fe(III) bound to lignin and reducing it to Fe(II) for cellular use, increasing BRL6-1 yield under lignin-amended conditions. This interaction potentially generates organic free radicals and causes a radical cascade which could modify and depolymerize lignin. Further research should clarify the extent to which this mechanism is similar to previously described aerobic chelator-mediated Fenton chemistry or radical producing lignolytic enzymes, such as lignin peroxidases, but under anoxic conditions

Lignin induced iron reduction by novel sp., Tolumonas lignolytic BRL6-1.

Lignin is the second most abundant carbon polymer on earth and despite having more fuel value than cellulose, it currently is considered a waste byproduct in many industrial lignocellulose applications. Valorization of lignin relies on effective and green methods of de-lignification, with a growing interest in the use of microbes. Here we investigate the physiology and molecular response of the novel facultative anaerobic bacterium, Tolumonas lignolytica BRL6-1, to lignin under anoxic conditions. Physiological and biochemical changes were compared between cells grown anaerobically in either lignin-amended or unamended conditions. In the presence of lignin, BRL6-1 accumulates higher biomass and has a shorter lag phase compared to unamended conditions, and 14% of the proteins determined to be significantly higher in abundance by log2 fold-change of 2 or greater were related to Fe(II) transport in late logarithmic phase. Ferrozine assays of the supernatant confirmed that Fe(III) was bound to lignin and reduced to Fe(II) only in the presence of BRL6-1, suggesting redox activity by the cells. LC-MS/MS analysis of the secretome showed an extra band at 20 kDa in lignin-amended conditions. Protein sequencing of this band identified a protein of unknown function with homology to enzymes in the radical SAM superfamily. Expression of this protein in lignin-amended conditions suggests its role in radical formation. From our findings, we suggest that BRL6-1 is using a protein in the radical SAM superfamily to interact with the Fe(III) bound to lignin and reducing it to Fe(II) for cellular use, increasing BRL6-1 yield under lignin-amended conditions. This interaction potentially generates organic free radicals and causes a radical cascade which could modify and depolymerize lignin. Further research should clarify the extent to which this mechanism is similar to previously described aerobic chelator-mediated Fenton chemistry or radical producing lignolytic enzymes, such as lignin peroxidases, but under anoxic conditions

Total genes up and down regulated in each of the three points monitored during growth of SCF1 in lignin-amended cultures.

<p>The number in parenthesis indicates the percentage of the genes that were differentially expressed under each time.</p