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ABSTRACT Aeration is a major cost at biological nutrient removal (BNR) plants. We report on microbial communities in a pilot-scale BNR system before and after a dissolved oxygen transition from 2.5 to 0.2 mg/L implemented over 18 months. Four PacBio metagenomes and 316 metagenome-assembled genomes are announced.

The growing significance of lignin-first biorefineries, which focus on upgrading the aromatics resulting from lignin depolymerization, presents opportunities for bioproduct synthesis using microbial strains capable of funneling a diverse array of phenolics into a single commodity chemical. In this study, we evaluated a biomass-to-bioproduct pipeline involving the reductive catalytic fractionation (RCF) of poplar biomass followed by biological funneling with a Novosphingobium aromaticivorans strain that produces 2-pyrone-4,6-dicarboxylic acid (PDC), a potential bioplastic precursor. Considering the impact of solvent on RCF reactor operating pressure, and the potential inhibitory effects of solvent on downstream microbial funneling, we performed an analysis of six puremore » solvents, namely methanol, ethanol, isopropanol, isobutanol, 1,4-dioxane and ethylene glycol, and different variations of their aqueous mixtures comprising 5 to 50 vol% water. For each pure solvent and solvent/water system, we measured phenolic monomer yields in the RCF process and PDC yields from the phenolic monomers. We then developed correlation models that relate phenolic monomer yields from RCF-derived samples to Hansen solubility parameters to determine solvent descriptors that contribute to high yields. Furthermore, we developed an integrated biorefinery system to estimate the minimum selling price (MSP) of PDC and the associated carbon footprint to identify solvent systems with better costs and sustainability metrics. These analyses resulted in the 50 vol% methanol/water system being identified as optimal because it reduces RCF reactor pressure and is compatible with microbial funneling with N. aromaticivorans. This solvent system produced 63 g PDC per kg biomass (264 g PDC per kg lignin) from 85 g phenolic monomers per kg biomass at a reduced reactor pressure of 48 bar (reduced by 26% compared to our previous poplar-to-PDC pipeline). The MSP for this system is $$\$$$$13.98 per kg of purified PDC (carbon footprint of 1.47 kg CO₂e per kg), which is about 24% lower than a previously described poplar-to-PDC pipeline and 46% lower than a lignin-to-PDC pipeline that used pure methanol as the solvent. The results from this study illustrate improvements that can be made in lignocellulosic biorefineries that are compatible with the hybrid chemical and biological processes needed to gain value from lignin.« less

Operating biological nutrient removal (BNR) wastewater treatment plants with low dissolved oxygen (DO) conditions can reduce energy costs. We report on five metagenomes and 492 metagenome-assembled genomes (MAGs) obtained from samples collected at the Pomona water reclamation plant before and after a DO reduction from 3.5 to 0.7 mg/L.

Achieving high biochemical production in biotransformations of renewable resources requires using concentrated cultures that not only generate the product of interest but also produce abundant microbial cell waste. We explored the concept of gaining value from microbial cells by producing intracellular products in tandem with a desired extracellular product. Specifically, we engineered a strain ofNovosphingobium aromaticivorans to extracellularly produce 2-pyrone-4,6-dicarboxylic acid (PDC) from aromatic substrates and to intracellularly accumulate astaxanthin along with coenzyme Q₁₀, all of which are products of industrial interest. Achieving the goal of concurrent production of intracellular and extracellular products required the creative application of bioreactor engineeringmore » principles. Although a continuously fed membrane bioreactor (MBR) maximized extracellular product biosynthesis, it had a negative effect on intracellular product accumulation. However, operating the MBR as a sequencing batch reactor (MBR-SBR) with a step-feed resulted in stable concurrent production of both extracellular and intracellular products. With aromatics extracted from poplar biomass, we achieved productivities of 1.14 g of PDC/L-h for the extracellular product and 0.04 mg of astaxanthin/L-h and 0.64 mg of CoQ₁₀/L-h for intracellular products, respectively. Our findings demonstrate that the mode of operation of a bioreactor impacts the simultaneous production of intracellular and extracellular products byN. aromaticivorans.« less

Aromatic compounds are a ubiquitous and diverse family of chemicals with functions as biomolecules, natural products, industrial chemicals, and pollutants. Novosphingobium aromaticivorans DSM 12444 uses multiple inducible pathways to catabolize H-, G-, and S-type aromatics that contain zero, one, or two methoxy groups, respectively. Here, we obtain a systems-level view of the transcriptional control of its aromatic metabolic pathways. Several in vitro analyses found that a N. aromaticivorans homolog of the Sphingobium lignivorans SYK-6 transcription factor LigR bound genomic DNA upstream of genes involved in metabolism of multiple aromatic types. We found that a ΔLigR mutant had growth defects onmore » all three types of aromatics as sole carbon sources. Transcriptomic analysis revealed that LigR was required to increase expression of gene products that function in metabolism of all three aromatic types. We also found that, in media containing both glucose and an aromatic carbon source, the ΔLigR mutant directed intermediates through alternative aromatic metabolic pathways. Protein-DNA binding assays showed that N. aromaticivorans LigR binds immediately upstream of promoters of genes involved in aromatic metabolism. We found that N. aromaticivorans LigR coordinates the expression of enzymes that function in the catabolism of H-, G-, and S-type aromatics, and that there are differences in the role of LigR in N. aromaticivorans and S. lignivorans. A comparative genomic analysis predicted that LigR homologs and the aromatic-metabolizing genes that it directly regulates are often co-localized in the genomes of Sphingomonadales, but often not found in this arrangement in many other known aromatic metabolizing bacteria.« less

The in-planta accumulation of coproducts in crops can enhance the value of lignocellulosic biomass and facilitate a sustainable bioeconomy. Corn stover represents a major renewable source of lignocellulose for the production of advanced biofuels and bioproducts. In this study, we engineered corn with a bacterial gene encoding a dehydroshikimate dehydratase (QsuB) to overproduce protocatechuate (DHBA). Transgenic corn lines accumulate up to 2.9% DHBA on a dry weight basis in leaf and stem biomass. DHBA occurs in the form of glucosides that are extractable from biomass using aqueous methanol as the solvent. The analysis of lignin did not show any evidencemore » for the incorporation of DHBA; however, an increase in the lignin syringyl to guaiacyl ratio and a higher relative abundance of p-coumarate groups compared with total lignin units were observed in QsuB-modified corn. Alkaline hydrolysates prepared from QsuB corn were enriched in DHBA compared to the hydrolysates obtained from wild-type biomass, which contained mostly p-coumarate and ferulate. Using engineered Novosphingobium aromaticivorans as a production host, a 375% improvement in 2-pyrone-4,6-dicarboxylate titers was achieved through biological upgrading of alkaline hydrolysates derived from QsuB corn compared to unmodified biomass. Our data demonstrate an engineering strategy to overproduce DHBA in corn that can facilitate sustainable manufacturing of other valuable bioproducts using stover as a feedstock.« less

Replacing biorefinery designs that use stepwise fractionation, depolymerization, and conversion processes with designed tandem process steps can greatly reduce a biorefinery's operating costs and environmental impact. Reductive Catalytic Fractionation (RCF) is a highly efficient lignin-first approach that combines biomass fractionation and lignin depolymerization to generate a hydrogenolysis oil and pulp. The oil is composed of a complex mixture of phenolic monomers, dimers, and oligomers. Intergrating this chemical deconstruction process with microbial funneling of phenolics can simplify the product mixture and make high-value products. We applied RCF to poplar biomass in a biomass-to-bioproduct processing chain in which the phenolics were funneledmore » to 2-pyrone-4,6-dicarboxylic acid (PDC) by an engineered strain of Novosphingobium aromaticivorans DSM12444. The pulp was enzymatically digested and the glucose and xylose was funneled to ethanol by an engineered strain of Saccharomyces cerevisiae GLBRCY945. By combining biomass fractionation and lignin depolymerization we removed a costly processing step that directly translated into a 29% reduction in the minimum selling price of PDC. This work combines experimentation with process modeling of an integrated biorefinery design to show how utilizing tandem process steps can significantly reduce operating expenses and environmental impact of upgrading lignocellulosic biomass to a portfolio of high value products.« less

Spent liquors of biomass pretreatment provide a source for renewable chemical production. These liquors require treatment before being discharged; otherwise, they negatively impact the environment. Herein, spent liquors from aqueous ammonia pretreatment of poplar wood are characterized for phenolic content via liquid chromatography–mass spectrometry and nuclear magnetic resonance spectroscopy. The main phenolics are phenol, p-hydroxybenzamide (pHBAm), and p-hydroxybenzoic acid (pHBA), of which pHBAm and pHBA are produced from the ester-linked p-hydroxybenzoates in poplar wood. Phenol is produced from pHBA via decarboxylation. The potential biotransformation of the extracted phenolics into 2-pyrone-4,6-dicarboxylic acid (PDC) is assessed using an engineered strain of Novosphingobiummore » aromaticivorans DSM12444 (PDC strain). Biotransformation of pHBAm to PDC is shown to be possible in the presence of pHBA, but not when pHBAm is the sole phenolic substrate, this is the first reported observation of N. aromaticivorans producing PDC from an aromatic amide. The phenol present is not transformed to PDC and does not inhibit PDC production. This study demonstrates that the phenolic amide in spent liquor from ammonia pretreatment can be valorized via biotransformation using N. aromaticivorans, which adds to the growing versatility of N. aromaticivorans as a microbial chassis for converting plant-derived compounds to useful products.« less

The aromatic compounds used in a variety of industrial products are currently obtained from nonrenewable petroleum sources. Alternatively, the plant polymer lignin is an abundant renewable source of aromatics, and its depolymerization generates a variety of products that can include acetovanillone, a vanillin derivative containing an acetyl side chain. The Alphaproteobacterium Novosphingobium aromaticivorans DSM12444 can metabolize several chemically modified aromatics in deconstructed lignin, but not acetovanillone. In this work, adaptive laboratory evolution identified a single amino acid change in the previously uncharacterized gene product Saro_1862 that is necessary and sufficient for N. aromaticivorans growth with acetovanillone as a sole growthmore » substrate, as well as other aromatic monomers not metabolized by wild-type cells. We show that a glutamate (E) to lysine (K) substitution at amino acid residue 16 of Saro_1862 results in a ~1600-fold increase in the rate of ATP-dependent acetovanillone phosphorylation. We also find that recombinant Saro_1862 E16K phosphorylates several other aromatic compounds in vitro, defining the first reported catalytic activity for the widespread UPF0261 protein domain contained in Saro_1862. Thus, we propose naming Saro_1862 MarK, for multiple aromatic kinase. A 1.57 Å crystal structure of MarK E16K predicts that the E16K substitution lies in a potential ATP binding site, suggesting how this amino acid change increased catalytic activity. A search for homologs of MarK and other proteins required for acetovanillone degradation predicts that this pathway for aromatic metabolism exists throughout the bacterial phylogeny.« less

The SARO_RS14285 gene, encoding a transcription factor, was deleted in Novosphingobium aromaticivorans DSM12444. The transcriptomes of the parent and ΔSARO_RS14285 strains were determined when grown in medium containing glucose with or without protocatechuic, vanillic, syringic, or 4-coumaric acid. We present the raw RNA sequencing data obtained from these cultures.