Michael Guarnieri

Lignin valorization through integrated biological funneling and chemical catalysis.

Significance For nearly a century, processes have been used to convert biomass-derived carbohydrates, such as glucose, into fuels and chemicals. However, plant cell walls also contain an aromatic polymer, lignin, that has not been cost-effectively converted into fuels or commodity chemicals. With the intensive development of lignocellulosic biorefineries around the world to produce fuels and chemicals from biomass-derived carbohydrates, the amount of waste lignin will dramatically increase, warranting new lignin upgrading strategies. In nature, some microorganisms have evolved pathways to catabolize lignin-derived aromatics. Our work demonstrates that the utilization of these natural aromatic catabolic pathways may enable new routes to overcome the lignin utilization barrier that, in turn, may enable a broader slate of molecules derived from lignocellulosic biomass. Lignin is an energy-dense, heterogeneous polymer comprised of phenylpropanoid monomers used by plants for structure, water transport, and defense, and it is the second most abundant biopolymer on Earth after cellulose. In production of fuels and chemicals from biomass, lignin is typically underused as a feedstock and burned for process heat because its inherent heterogeneity and recalcitrance make it difficult to selectively valorize. In nature, however, some organisms have evolved metabolic pathways that enable the utilization of lignin-derived aromatic molecules as carbon sources. Aromatic catabolism typically occurs via upper pathways that act as a “biological funnel” to convert heterogeneous substrates to central intermediates, such as protocatechuate or catechol. These intermediates undergo ring cleavage and are further converted via the β-ketoadipate pathway to central carbon metabolism. Here, we use a natural aromatic-catabolizing organism, Pseudomonas putida KT2440, to demonstrate that these aromatic metabolic pathways can be used to convert both aromatic model compounds and heterogeneous, lignin-enriched streams derived from pilot-scale biomass pretreatment into medium chain-length polyhydroxyalkanoates (mcl-PHAs). mcl-PHAs were then isolated from the cells and demonstrated to be similar in physicochemical properties to conventional carbohydrate-derived mcl-PHAs, which have applications as bioplastics. In a further demonstration of their utility, mcl-PHAs were catalytically converted to both chemical precursors and fuel-range hydrocarbons. Overall, this work demonstrates that the use of aromatic catabolic pathways enables an approach to valorize lignin by overcoming its inherent heterogeneity to produce fuels, chemicals, and materials.

Adipic acid production from lignin.

Lignin is an alkyl-aromatic polymer present in plant cell walls for defense, structure, and water transport. Despite exhibiting a high-energy content, lignin is typically slated for combustion in modern biorefineries due to its inherent heterogeneity and recalcitrance, whereas cellulose and hemicellulose are converted to renewable fuels and chemicals. However, it is critical for the viability of third-generation biorefineries to valorize lignin alongside polysaccharides. To that end, we employ metabolic engineering, separations, and catalysis to convert lignin-derived species into cis,cis-muconic acid, for subsequent hydrogenation to adipic acid, the latter being the most widely produced dicarboxylic acid. First, Pseudomonas putida KT2440 was metabolically engineered to funnel lignin-derived aromatics to cis,cis-muconate, which is an atom-efficient biochemical transformation. This engineered strain was employed in fed-batch biological cultivation to demonstrate a cis,cis-muconate titer of 13.5 g L−1 in 78.5 h from a model lignin-derived compound. cis,cis-Muconic acid was recovered in high purity (>97%) and yield (74%) by activated carbon treatment and crystallization (5 °C, pH 2). Pd/C was identified as a highly active catalyst for cis,cis-muconic acid hydrogenation to adipic acid with high conversion (>97%) and selectivity (>97%). Under surface reaction controlling conditions (24 °C, 24 bar, ethanol solvent), purified cis,cis-muconic acid exhibits a turnover frequency of 23–30 s−1 over Pd/C, with an apparent activation energy of 70 kJ mol−1. Lastly, cis,cis-muconate was produced with engineered P. putida grown on a biomass-derived, lignin-enriched stream, demonstrating an integrated strategy towards lignin valorization to an important commodity chemical.

Examination of triacylglycerol biosynthetic pathways via de novo transcriptomic and proteomic analyses in an unsequenced microalga.

Biofuels derived from algal lipids represent an opportunity to dramatically impact the global energy demand for transportation fuels. Systems biology analyses of oleaginous algae could greatly accelerate the commercialization of algal-derived biofuels by elucidating the key components involved in lipid productivity and leading to the initiation of hypothesis-driven strain-improvement strategies. However, higher-level systems biology analyses, such as transcriptomics and proteomics, are highly dependent upon available genomic sequence data, and the lack of these data has hindered the pursuit of such analyses for many oleaginous microalgae. In order to examine the triacylglycerol biosynthetic pathway in the unsequenced oleaginous microalga, Chlorella vulgaris, we have established a strategy with which to bypass the necessity for genomic sequence information by using the transcriptome as a guide. Our results indicate an upregulation of both fatty acid and triacylglycerol biosynthetic machinery under oil-accumulating conditions, and demonstrate the utility of a de novo assembled transcriptome as a search model for proteomic analysis of an unsequenced microalga.

Bioconversion of natural gas to liquid fuel: Opportunities and challenges

Natural gas is a mixture of low molecular weight hydrocarbon gases that can be generated from either fossil or anthropogenic resources. Although natural gas is used as a transportation fuel, constraints in storage, relatively low energy content (MJ/L), and delivery have limited widespread adoption. Advanced utilization of natural gas has been explored for biofuel production by microorganisms. In recent years, the aerobic bioconversion of natural gas (or primarily the methane content of natural gas) into liquid fuels (Bio-GTL) by biocatalysts (methanotrophs) has gained increasing attention as a promising alternative for drop-in biofuel production. Methanotrophic bacteria are capable of converting methane into microbial lipids, which can in turn be converted into renewable diesel via a hydrotreating process. In this paper, biodiversity, catalytic properties and key enzymes and pathways of these microbes are summarized. Bioprocess technologies are discussed based upon existing literature, including cultivation conditions, fermentation modes, bioreactor design, and lipid extraction and upgrading. This review also outlines the potential of Bio-GTL using methane as an alternative carbon source as well as the major challenges and future research needs of microbial lipid accumulation derived from methane, key performance index, and techno-economic analysis. An analysis of raw material costs suggests that methane-derived diesel fuel has the potential to be competitive with petroleum-derived diesel.

Immunochemical measurement of myelin basic protein in developing rat brain: An index of myelin synthesis

Abstract The initial time and rate of myelin basic protein synthesis in neural tissues of the rat have been measured from birth to 120 days. The protein was quantitated by a radioimmunoassay directly applied to unfractionated cerebrum, cerebellum, olfactory bulb, midbrain, brain stem, optic and trigeminal nerve, and areas of the spinal cord. Because the protein is a specific myelin constituent and its appearance correlates precisely with the synthesis of myelin lipids, the data in this report can be interpreted in terms of myelin synthesis and oligodendrocyte activity. The results show striking heterogeneity in the initial time and rate of myelin synthesis in neural tissue.

Proteomic analysis of Chlorella vulgaris: potential targets for enhanced lipid accumulation.

UNLABELLED Oleaginous microalgae are capable of producing large quantities of fatty acids and triacylglycerides. As such, they are promising feedstocks for the production of biofuels and bioproducts. Genetic strain-engineering strategies offer a means to accelerate the commercialization of algal biofuels by improving the rate and total accumulation of microalgal lipids. However, the industrial potential of these organisms remains to be met, largely due to the incomplete knowledgebase surrounding the mechanisms governing the induction of algal lipid biosynthesis. Such strategies require further elucidation of genes and gene products controlling algal lipid accumulation. In this study, we have set out to examine these mechanisms and identify novel strain-engineering targets in the oleaginous microalga, Chlorella vulgaris. Comparative shotgun proteomic analyses have identified a number of novel targets, including previously unidentified transcription factors and proteins involved in cell signaling and cell cycle regulation. These results lay the foundation for strain-improvement strategies and demonstrate the power of translational proteomic analysis. BIOLOGICAL SIGNIFICANCE We have applied label-free, comparative shotgun proteomic analyses, via a transcriptome-to-proteome pipeline, in order to examine the nitrogen deprivation response in the oleaginous microalga, C. vulgaris. Herein, we identify potential targets for strain-engineering strategies targeting enhanced lipid accumulation for algal biofuels applications. Among the identified targets are proteins involved in transcriptional regulation, lipid biosynthesis, cell signaling and cell cycle progression. This article is part of a Special Issue entitled: Translational Plant Proteomics.

New approach to tumor therapy for inoperable areas of the brain: chronic intraparenchymal drug delivery

Because the brainstem has little functional redundancy, diffuse lesions have been regarded as inoperable. To determine whether local drug therapy can prolong survival in a rodent model of a tumor in such eloquent tissue, lethal doses of F98 and 9L tumor cells were injected into the brainstems of Fischer 344 rats. Five days after inoculations, 0.5 mg/ml solutions of carboplatin were infused at 1 μl/h for 7 days. Compared to control groups that survived 13–17 days with F98 tumors and 22–23 days with 9L tumors, animals locally infused with 0.1 mg of carboplatin survived 27–30 days (Prob > Chi Sq = 0.0003), and 32 days (Prob > Chi Sq = 0.01), respectively. Measurements of tissue platinum levels at autopsy suggested that infusions distributed pharmacologically relevant levels of carbo-platin through a volume of tissue at least 0.5 cm in diameter. The results suggest that chronic low-flow infusions provide a promising approach to therapy for CNS lesions in tissues considered to be inoperable.

Bioconversion of methane to lactate by an obligate methanotrophic bacterium

Methane is the second most abundant greenhouse gas (GHG), with nearly 60% of emissions derived from anthropogenic sources. Microbial conversion of methane to fuels and value-added chemicals offers a means to reduce GHG emissions, while also valorizing this otherwise squandered high-volume, high-energy gas. However, to date, advances in methane biocatalysis have been constrained by the low-productivity and limited genetic tractability of natural methane-consuming microbes. Here, leveraging recent identification of a novel, tractable methanotrophic bacterium, Methylomicrobium buryatense, we demonstrate microbial biocatalysis of methane to lactate, an industrial platform chemical. Heterologous overexpression of a Lactobacillus helveticus L-lactate dehydrogenase in M. buryatense resulted in an initial titer of 0.06 g lactate/L from methane. Cultivation in a 5 L continuously stirred tank bioreactor enabled production of 0.8 g lactate/L, representing a 13-fold improvement compared to the initial titer. The yields (0.05 g lactate/g methane) and productivity (0.008 g lactate/L/h) indicate the need and opportunity for future strain improvement. Additionally, real-time analysis of methane utilization implicated gas-to-liquid transfer and/or microbial methane consumption as process limitations. This work opens the door to develop an array of methanotrophic bacterial strain-engineering strategies currently employed for biocatalytic sugar upgrading to “green” chemicals and fuels.

Algal omics: unlocking bioproduct diversity in algae cell factories

Rapid advances in “omic” technologies are helping to unlock the full potential of microalgae as multi-use feedstocks, with utility in an array of industrial biotechnology, biofuel, and biomedical applications. In turn, algae are emerging as highly attractive candidates for development as microbial cell factories. In this review, we examine the wide array of potential algal bioproducts, with a focus upon the role of omic technologies in driving bioproduct discovery and optimization in microalgal systems.

De novo transcriptomic analysis of hydrogen production in the green alga Chlamydomonas moewusii through RNA-Seq

BackgroundMicroalgae can make a significant contribution towards meeting global renewable energy needs in both carbon-based and hydrogen (H2) biofuel. The development of energy-related products from algae could be accelerated with improvements in systems biology tools, and recent advances in sequencing technology provide a platform for enhanced transcriptomic analyses. However, these techniques are still heavily reliant upon available genomic sequence data. Chlamydomonas moewusii is a unicellular green alga capable of evolving molecular H2 under both dark and light anaerobic conditions, and has high hydrogenase activity that can be rapidly induced. However, to date, there is no systematic investigation of transcriptomic profiling during induction of H2 photoproduction in this organism.ResultsIn this work, RNA-Seq was applied to investigate transcriptomic profiles during the dark anaerobic induction of H2 photoproduction. 156 million reads generated from 7 samples were then used for de novo assembly after data trimming. BlastX results against NCBI database and Blast2GO results were used to interpret the functions of the assembled 34,136 contigs, which were then used as the reference contigs for RNA-Seq analysis. Our results indicated that more contigs were differentially expressed during the period of early and higher H2 photoproduction, and fewer contigs were differentially expressed when H2-photoproduction rates decreased. In addition, C. moewusii and C. reinhardtii share core functional pathways, and transcripts for H2 photoproduction and anaerobic metabolite production were identified in both organisms. C. moewusii also possesses similar metabolic flexibility as C. reinhardtii, and the difference between C. moewusii and C. reinhardtii on hydrogenase expression and anaerobic fermentative pathways involved in redox balancing may explain their different profiles of hydrogenase activity and secreted anaerobic metabolites.ConclusionsHerein, we have described a workflow using commercial software to analyze RNA-Seq data without reference genome sequence information, which can be applied to other unsequenced microorganisms. This study provided biological insights into the anaerobic fermentation and H2 photoproduction of C. moewusii, and the first transcriptomic RNA-Seq dataset of C. moewusii generated in this study also offer baseline data for further investigation (e.g. regulatory proteins related to fermentative pathway discussed in this study) of this organism as a H2-photoproduction strain.