Revealing oxidative pentose metabolism in new Pseudomonas putida isolates.

The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.

Complete Genome Sequences of Five Isolated Pseudomonas Strains that Catabolize Pentose Sugars and Aromatic Compounds Obtained from Lignocellulosic Biomass.

We report on complete genome sequences of five Pseudomonas soil isolates that are capable of metabolizing pentose sugars and aromatic monomers. These complete genome sequence data provide insight into possible alternative hosts for the production of biofuels and bio-based chemicals from lignocellulosic feedstock.

Response of Pseudomonas putida to Complex, Aromatic-Rich Fractions from Biomass.

There is strong interest in the valorization of lignin to produce valuable products; however, its structural complexity has been a conversion bottleneck. Chemical pretreatment liberates lignin-derived soluble fractions that may be upgraded by bioconversion. Cholinium ionic liquid pretreatment of sorghum produced soluble, aromatic-rich fractions that were converted by Pseudomonas putida (P. putida), a promising host for aromatic bioconversion. Growth studies and mutational analysis demonstrated that P. putida growth on these fractions was dependent on aromatic monomers but unknown factors also contributed. Proteomic and metabolomic analyses indicated that these unknown factors were amino acids and residual ionic liquid; the oligomeric aromatic fraction derived from lignin was not converted. A cholinium catabolic pathway was identified, and the deletion of the pathway stopped the ability of P. putida to grow on cholinium ionic liquid. This work demonstrates that aromatic-rich fractions obtained through pretreatment contain multiple substrates; conversion strategies should account for this complexity.

Aquaponic Systems for Sustainable Resource Recovery: Linking Nitrogen Transformations to Microbial Communities.

Aquaponics is a technology for food production (fish and vegetables/fruits) with concomitant remediation of nitrogen-rich aquaculture effluent. There is, however, a critical need to improve the nitrogen use efficiency (NUE) in aquaponics. Here, we employed quantitative polymerase chain reactions and next-generation sequencing to evaluate the bacterial communities and their links to nitrogen transformations for improving NUEs in four bench-scale plant-based floating-raft aquaponics (pak choi, lettuce, chive, and tomato) and three pH levels (7.0, 6.0, and 5.2). Low relative abundance of nitrifiers in plant roots and biofilters suggested nitrogen loss, which decreased NUE in aquaponics. Low pH level was a major factor that shifted the microbial communities and reduced the relative abundance of nitrifiers in aquaponic systems, leading to total ammonia nitrogen accumulation in recirculating water. In plant roots, the abundance of nitrite-oxidizing bacteria (e.g., Nitrospira spp.) did not decrease at low pH levels, suggesting the benefit of growing plants in aquaponics for efficient nitrification and improving NUE. These findings on microbial communities and nitrogen transformations provided complementary strategies to improve the performance of the aquaponics regarding water quality and extent of nutrient recovery from aquaculture effluent.

Fate of antibiotic resistance genes and metal resistance genes during thermophilic aerobic digestion of sewage sludge.

This study examines the fate of twenty-three representative antibiotic resistance genes (ARGs) encoding tetracyclines, sulfonamides, quinolones, ß-lactam antibiotics, macrolides, florfenicol and multidrug resistance during thermophilic aerobic digestion (TAD) of sewage sludge. The bacterial community, class 1 integrons (intI1) and four metal resistance genes (MRGs) were also quantified to determine the key drivers of changes in ARGs during TAD. At the end of digestion, significant decreases in the quantities of ARGs, MRGs and intI1 as well as 16S rRNA genes were observed. Partial redundancy analysis (RDA) showed that shifts in temperature were the key factors affecting a decrease in ARGs. Shifts in temperature led to decreased amounts of ARGs by reducing resistome and bacterial diversity, rather than by lowering horizontal transfer potential via intI1 or co-resistance via MRGs.

Molecular and Kinetic Characterization of Planktonic Nitrospira spp. Selectively Enriched from Activated Sludge.

Nitrospira spp. are chemolithoautotrophic nitrite-oxidizing bacteria (NOB), which are ubiquitous in natural and engineered environments. However, there exist few independent biokinetic studies on Nitrospira spp., likely because their isolation and selective enrichment from environmental consortia such as activated sludge can be challenging. Herein, planktonic Nitrospira spp. cultures closely related to Candidatus Nitrospira defluvii (Nitrospira lineage I) were successfully enriched from activated sludge in a sequencing batch reactor by maintaining sustained limiting extant nitrite and dissolved oxygen concentrations. Morphologically, the enrichment consisted largely of planktonic cells with an average characteristic diameter of 1.3 ± 0.6 µm. On the basis of respirometric assays, estimated maximum specific growth rate (µmax), nitrite half saturation coefficient (KS), oxygen half saturation coefficient (KO), and biomass yield coefficient (Y) of the enriched cultures were 0.69 ± 0.10 d-1, 0.52 ± 0.14 mg-N/L, 0.33 ± 0.14 mg-O2/L, and 0.14 ± 0.02 mg-COD/mg-N, respectively. These parameters collectively reflect not just higher affinities of this enrichment for nitrite and oxygen, respectively, but also a higher biomass yield and energy transfer efficiency relative to Nitrobacter spp. Used in combination, these kinetic and thermodynamic parameters can help toward the development and application of energy-efficient biological nutrient removal processes through effective Nitrospira out-selection.

Impact of nitrate dose on toluene degradation under denitrifying condition.

In this study, we investigated the impact of nitrate dose on toluene degradation by Pseudomonas putida to elucidate the upper limit of nitrate concentration and whether an optimum ratio of nitrate to toluene concentration exists. Batch microcosm studies were conducted in order to monitor toluene degradation for various ratios (2-20) of nitrate to toluene with nitrate concentrations ranging from 0 to 700 mg L(-1) for a given toluene concentration of 50 and 25 mg L(-1) during 4-day (short term) and 14-day (long term) incubation time, respectively. The short-term study revealed that nitrate concentration of 500 mg L(-1) was toxic to bacteria and the optimum concentration was 300 mg L(-1) yielding the highest toluene degradation rate (0.083 mg L(-1) h(-1)). In the batch study of long term, toluene degradation was limited to 6 days after which the nitrate at 50 mg L(-1) was depleted, indicating that nitrate was a necessary electron acceptor. For both batch studies, an optimum ratio of 6 was found yielding the highest toluene degradation rate. This indicates that an appropriate nitrate dose is essential for efficient degradation of toluene when bioremediation of groundwater contaminated with toluene is under consideration.