Genetic redundancy in the naphthalene-degradation pathway of Cycloclasticus pugetii strain PS-1 enables response to varying substrate concentrations.

Polycyclic aromatic hydrocarbon (PAH) contamination in marine environments range from low-diffusive inputs to high loads. The influence of PAH concentration on the expression of functional genes [e.g. those encoding ring-hydroxylating dioxygenases (RHDs)] has been overlooked in PAH biodegradation studies. However, understanding marker-gene expression under different PAH loads can help to monitor and predict bioremediation efficiency. Here, we followed the expression (via RNA sequencing) of Cycloclasticus pugetii strain PS-1 in cell suspension experiments under different naphthalene (100 and 30 mg L-1) concentrations. We identified genes encoding previously uncharacterized RHD subunits, termed rhdPS1α and rhdPS1ß, that were highly transcribed in response to naphthalene-degradation activity. Additionally, we identified six RHD subunit-encoding genes that responded to naphthalene exposure. By contrast, four RHD subunit genes were PAH-independently expressed and three other RHD subunit genes responded to naphthalene starvation. Cycloclasticus spp. could, therefore, use genetic redundancy in key PAH-degradation genes to react to varying PAH loads. This genetic redundancy may restrict the monitoring of environmental hydrocarbon-degradation activity using single-gene expression. For Cycloclasticus pugetii strain PS-1, however, the newly identified rhdPS1α and rhdPS1ß genes might be potential target genes to monitor its environmental naphthalene-degradation activity.

Substrate-independent expression of key functional genes in Cycloclasticus pugetii strain PS-1 limits their use as markers for PAH biodegradation.

Microbial degradation of petroleum hydrocarbons is a crucial process for the clean-up of oil-contaminated environments. Cycloclasticus spp. are well-known polycyclic aromatic hydrocarbon (PAH) degraders that possess PAH-degradation marker genes including rhd3α, rhd2α, and pahE. However, it remains unknown if the expression of these genes can serve as an indicator for active PAH degradation. Here, we determined transcript-to-gene (TtG) ratios with (reverse transcription) qPCR in cultures of Cycloclasticus pugetii strain PS-1 grown with naphthalene, phenanthrene, a mixture of these PAHs, or alternate substrates (i.e., no PAHs). Mean TtG ratios of 1.99 × 10-2, 1.80 × 10-3, and 3.20 × 10-3 for rhd3α, rhd2α, and pahE, respectively, were measured in the presence or absence of PAHs. The TtG values suggested that marker-gene expression is independent of PAH degradation. Measurement of TtG ratios in Arctic seawater microcosms amended with water-accommodated crude oil fractions, and incubated under in situ temperature conditions (i.e., 1.5°C), only detected Cycloclasticus spp. rhd2α genes and transcripts (mean TtG ratio of 4.15 × 10-1). The other marker genes-rhd3α and pahE-were not detected, suggesting that not all Cycloclasticus spp. carry these genes and a broader yet-to-be-identified repertoire of PAH-degradation genes exists. The results indicate that the expression of PAH marker genes may not correlate with PAH-degradation activity, and transcription data should be interpreted cautiously.

Dosage concentration and pulsing frequency affect the degradation efficiency in simulated bacterial polycyclic aromatic hydrocarbon-degrading cultures.

A major source of anthropogenic polycyclic aromatic hydrocarbon (PAH) inputs into marine environments are diffuse emissions which result in low PAH concentrations in the ocean water, posing a potential threat for the affected ecosystems. However, the remediation of low-dosage PAH contaminations through microbial processes remains largely unknown. Here, we developed a process-based numerical model to simulate batch cultures receiving repeated low-dosage naphthalene pulses compared to the conventionally used one-time high-dosage. Pulsing frequency as well as dosage concentration had a large impact on the degradation efficiency. After 10 days, 99.7%, 97.2%, 86.6%, or 83.5% of the 145 mg L-1 naphthalene was degraded when given as a one-time high-dosage or in 2, 5, or 10 repeated low-concentration dosages equally spaced throughout the experiment, respectively. If the simulation was altered, giving the system that received 10 pulses time to recover to 99.7%, pulsing patterns affected the degradation of naphthalene. When pulsing 10 days at once per day, naphthalene accumulated following each pulse and if the degradation was allowed to continue until the recovered state was reached, the incubation time was prolonged to 17 days with a generation time of 3.81 days. If a full recovery was conditional before the next pulse was added, the scenario elongated to 55 days and generation time increased to 14.15 days. This indicates that dissolution kinetics dominate biodegradation kinetics, and the biomass concentration of PAH-degrading bacteria alone is not a sufficient indicator for quantifying active biodegradation. Applying those findings to the environment, a one-time input of a high dosage is potentially degraded faster than repeated low-dosage PAH pollution and repeated low-dosage input could lead to PAH accumulation in vulnerable pristine environments. Further research on the overlooked field of chronic low-dosage PAH contamination is necessary.