Starvation- and xenobiotic-related transcriptomic responses of the sulfanilic acid-degrading bacterium, Novosphingobium resinovorum SA1.

Novosphingobium resinovorum SA1 was the first single isolate capable of degrading sulfanilic acid, a widely used representative of sulfonated aromatic compounds. The genome of the strain was recently sequenced, and here, we present whole-cell transcriptome analyses of cells exposed to sulfanilic acid as compared to cells grown on glucose. The comparison of the transcript profiles suggested that the primary impact of sulfanilic acid on the cell transcriptome was a starvation-like effect. The genes of the peripheral, central, and common pathways of sulfanilic acid biodegradation had distinct transcript profiles. The peripheral genes located on a plasmid had very high basal expressions which were hardly upregulated by sulfanilic acid. The genomic context and the codon usage preference of these genes suggested that they were acquired by horizontal gene transfer. The genes of the central pathways were remarkably inducible by sulfanilic acid indicating the presence of a substrate-specific regulatory system in the cells. Surprisingly, the genes of the common part of the metabolic pathway had low and sulfanilic acid-independent transcript levels. The approach applied resulted in the identification of the genes of proteins involved in auxiliary processes such as electron transfer, substrate and iron transports, sulfite oxidases, and sulfite transporters. The whole transcriptome analysis revealed that the cells exposed to xenobiotics had multiple responses including general starvation-like, substrate-specific, and substrate-related effects. From the results, we propose that the genes of the peripheral, central, and common parts of the pathway have been evolved independently.

Exploitation of extracellular organic matter from Micrococcus luteus to enhance ex situ bioremediation of soils polluted with used lubricants.

Chronic pollution by used lubricant oils (ULOs) poses a serious challenge to the environment. Under stress conditions, microorganisms, including potential degraders, can enter a viable but non-culturable (VBNC) state, complicating the bioremediation of ULO-polluted areas. Resuscitation-promoting factors (Rpfs) can reverse this transition and/or enhance the biodegradation performance of both native and augmented strains. Here, Rpf-containing extracellular organic matter (EOM) from Micrococcus luteus was used to enhance the ex situ ULO removal in biostimulated and bioaugmented (with Rhodococcus qingshengii KAG C, R. erythropolis PR4) soils. ULO bioconversion, microbial activity, and CFUs were significantly higher in EOM-treated soils compared to corresponding control soils. After 60 days, the initial ULO concentration (52,500 mg kg-1) was reduced by 37% and 45% with EOM-supplemented biostimulation and bioaugmentation, respectively. Based on high-throughput 16S rRNA analysis, the enhancement was attributable both to the reactivation of EOM-responsive hydrocarbonoclastic bacterial genera (e.g., Pseudomonas, Comamonas, Stenotrophomonas, Gordonia) and to the long-term positive effect of EOM on the degradative efficacy of the introduced rhodococci. Ecotoxicological responses revealed that reduced ULO concentration did not correlate with decreased soil toxicity. Our findings provide an insight into the applicability of EOM in bioremediation and its effects on the soil microbial activity and community composition.

A comparative analysis of biogas production from tomato bio-waste in mesophilic batch and continuous anaerobic digestion systems.

Annually, agricultural activity produces an enormous amount of plant biomass by-product. Many studies have reported the biomethane potential of agro-industrial wastes, but only a few studies have investigated applying the substrates in both batch and continuous mode. Tomato is one of the most popular vegetables globally; its processing releases a substantial amount of by-product, such as stems and leaves. This study examined the BMP of tomato plant (Solanum lycopersicum Mill. L. cv. Alfred) waste. A comparative test revealed that the BMPs of corn stover, tomato waste,and their combination were approximately the same, around 280 mL methane/g Volatile Solid. In contrast, the relative biogas production decreased in the presence of tomato waste in a continuous mesophilic anaerobic digestion system; the daily biogas productions were 860 ± 80, 290 ± 50, and 570 ± 70 mL biogas/gVolatile Solid/day in the case of corn stover, tomato waste, and their mixture, respectively. The methane content of biogas was around 46-48%. The fermentation parameters of the continuous AD experiments were optimal in all cases; thus, TW might have an inhibitory effect on the microbial community. Tomato plant materials contain e.g. flavonoids, glycoalkaloids (such as tomatine and tomatidine), etc. known as antimicrobial and antifungal agents. The negative effect of tomatine on the biogas yield was confirmed in batch fermentation experiments. Metagenomic analysis revealed that the tomato plant waste caused significant rearrangements in the microbial communities in the continuously operated reactors. The results demonstrated that tomato waste could be a good mono-substrate in batch fermentations or a co-substrate with corn stover in a proper ratio in continuous anaerobic fermentations for biogas production. These results also point to the importance of running long-term continuous fermentations to test the suitability of a novel biomass substrate for industrial biogas production.

New Frontiers of Anaerobic Hydrocarbon Biodegradation in the Multi-Omics Era.

The accumulation of petroleum hydrocarbons in the environment substantially endangers terrestrial and aquatic ecosystems. Many microbial strains have been recognized to utilize aliphatic and aromatic hydrocarbons under aerobic conditions. Nevertheless, most of these pollutants are transferred by natural processes, including rain, into the underground anaerobic zones where their degradation is much more problematic. In oxic zones, anaerobic microenvironments can be formed as a consequence of the intensive respiratory activities of (facultative) aerobic microbes. Even though aerobic bioremediation has been well-characterized over the past few decades, ample research is yet to be done in the field of anaerobic hydrocarbon biodegradation. With the emergence of high-throughput techniques, known as omics (e.g., genomics and metagenomics), the individual biodegraders, hydrocarbon-degrading microbial communities and metabolic pathways, interactions can be described at a contaminated site. Omics approaches provide the opportunity to examine single microorganisms or microbial communities at the system level and elucidate the metabolic networks, interspecies interactions during hydrocarbon mineralization. Metatranscriptomics and metaproteomics, for example, can shed light on the active genes and proteins and functional importance of the less abundant species. Moreover, novel unculturable hydrocarbon-degrading strains and enzymes can be discovered and fit into the metabolic networks of the community. Our objective is to review the anaerobic hydrocarbon biodegradation processes, the most important hydrocarbon degraders and their diverse metabolic pathways, including the use of various terminal electron acceptors and various electron transfer processes. The review primarily focuses on the achievements obtained by the current high-throughput (multi-omics) techniques which opened new perspectives in understanding the processes at the system level including the metabolic routes of individual strains, metabolic/electric interaction of the members of microbial communities. Based on the multi-omics techniques, novel metabolic blocks can be designed and used for the construction of microbial strains/consortia for efficient removal of hydrocarbons in anaerobic zones.

Intensification of Ex Situ Bioremediation of Soils Polluted with Used Lubricant Oils: A Comparison of Biostimulation and Bioaugmentation with a Special Focus on the Type and Size of the Inoculum.

Used lubricant oils (ULOs) strongly bind to soil particles and cause persistent pollution. In this study, soil microcosm experiments were conducted to model the ex situ bioremediation of a long term ULO-polluted area. Biostimulation and various inoculation levels of bioaugmentation were applied to determine the efficacy of total petrol hydrocarbon (TPH) removal. ULO-contaminated soil microcosms were monitored for microbial respiration, colony-forming units (CFUs) and TPH bioconversion. Biostimulation with inorganic nutrients was responsible for 22% of ULO removal after 40 days. Bioaugmentation using two hydrocarbon-degrader strains: Rhodococcus quingshengii KAG C and Rhodococcus erythropolis PR4 at a small inoculum size (107 CFUs g-1 soil), reduced initial TPH concentration by 24% and 29%, respectively; the application of a higher inoculum size (109 CFUs g-1 soil) led to 41% and 32% bioconversion, respectively. After 20 days, all augmented CFUs decreased to the same level as measured in the biostimulated cases, substantiating the challenge for the newly introduced hydrocarbon-degrading strains to cope with environmental stressors. Our results not only highlight that an increased number of degrader cells does not always correlate with enhanced TPH bioconversion, but they also indicate that biostimulation might be an economical solution to promote ULO biodegradation in long term contaminated soils.

Characterization of the Rhodococcus sp. MK1 strain and its pilot application for bioremediation of diesel oil-contaminated soil.

Petroleum hydrocarbons and derivatives are widespread contaminants in both aquifers and soil, their elimination is in the primary focus of environmental studies. Microorganisms are key components in biological removal of pollutants. Strains capable to utilize hydrocarbons usually appear at the contaminated sites, but their metabolic activities are often restricted by the lack of nutrients and/or they can only utilize one or two components of a mixture. We isolated a novel Rhodococcus sp. MK1 strain capable to degrade the components of diesel oil simultaneously. The draft genome of the strain was determined and besides the chromosome, the presence of one plasmid could be revealed. Numerous routes for oxidation of aliphatic and aromatic compounds were identified. The strain was tested in ex situ applications aiming to compare alternative solutions for microbial degradation of hydrocarbons. The results of bioaugmentation and biostimulation experiments clearly demonstrated that - in certain cases - the indigenous microbial community could be exploited for bioremediation of oil-contaminated soils. Biostimulation seems to be efficient for removal of aged contaminations at lower concentration range, whereas bioaugmentation is necessary for the treatment of freshly and highly polluted sites.