Organic matter sources and distribution along land-use gradient in a Himalayan Foothills River: Insights from molecular markers.

The analysis of hydrocarbon biomarkers in surface sediments along the Markanda River in the foothills of the Indian Himalayas was conducted to gain insights into the distribution and composition of organic matter (OM) within the sediments. This investigation is essential for comprehending how anthropogenic changes are influencing the OM dynamics in river systems. The study involved identification and quantification of various compound groups such as n-alkanes, hopanes, steranes, polycyclic aromatic hydrocarbons (PAHs), linear alkyl benzenes (LABs) and phthalate esters along with their respective parametric ratios. The variation in distribution of n-alkanes and associated indices (odd-even carbon number predominance (OEP), average chain length (ACL), terrigenous to aquatic ratio (TAR), carbon preference index (CPI), and natural n-alkanes ratio (NAR)) were used to distinguish the natural source of organic content from those influenced by anthropogenic contamination. The detection of petroleum contamination was indicated by the presence of prominent unresolved complex mixtures (UCM) as well as specific petroleum biomarkers such as hopanes, diasteranes, and steranes. The study revealed varying concentrations of the analyzed organic pollutants, with the average of PAHs at 24.6 ng/g dw, LABs at 18.1 ng/g dw, and phthalates at 8.3 µg/g dw. The variability in concentration of the investigated compound groups across different locations indicated spatial heterogeneity, and the land use patterns appears to modulate the sources of OM in surface sediments. The source contribution of PAHs and phthalates were determined by positive matrix factorization (PMF) shows the predominant sources of the anthropogenic hydrocarbons were linked primarily to petroleum/ petroleum-derived products emissions, industrial discharges, cultural practices and common household waste/ sewage disposal. This analysis provides insights for developing mitigation strategies and informing relevant policy changes globally, thereby contributing to the broader understanding of anthropogenic impacts on water ecosystems.

The potential of R. toruloides mevalonate pathway genes in increasing isoprenoid yields in S. cerevisiae: Evaluation of GGPPS and HMG-CoA reductase.

The enzymes of the mevalonate pathway need to be improved to achieve high yields of isoprenoids in the yeast Saccharomyces cerevisiae. The red yeast Rhodosporidium toruloides produces high levels of carotenoids and may have evolved to carry a naturally high flux of isoprenoids. Enzymes from such yeasts are likely to be promising candidates for improvement. Towards this end, we have systematically investigated the various enzymes of the mevalonate pathway of R. toruloides and custom synthesized, expressed, and evaluated six key enzymes in S. cerevisiae. The two nodal enzymes geranyl pyrophosphate synthase (RtGGPPS) and truncated HMG-CoA reductase (RttHMG) of R. toruloides showed a significant advantage to the cells for isoprenoid production as seen by a visual carotenoid screen. These two were analyzed further, and attempts were also made at further improvement. RtGGPPS was confirmed to be superior to the S. cerevisiae enzyme, as seen from in vitro activity determinations and in vivo production of the heterologous diterpenoid sclareol. Four mutants were created through rational mutagenesis but were unable to improve the activity further. In the case of RttHMG, functional evaluation of the enzyme revealed that it was very unstable despite functioning very well in S. cerevisiae. We succeeded in stabilizing the enzyme through mutation of a conserved serine in the catalytic region, which did not alter the enzyme activity per se. In vivo evaluation of the mutant revealed that it could enable better sclareol yields. Therefore, these two enzymes from the red yeast are excellent candidates for heterologous isoprenoid production.

Glucose 6-phosphate dehydrogenase variants increase NADPH pools for yeast isoprenoid production.

Isoprenoid biosynthesis has a significant requirement for the co-factor NADPH. Thus, increasing NADPH levels for enhancing isoprenoid yields in synthetic biology is critical. Previous efforts have focused on diverting flux into the pentose phosphate pathway or overproducing enzymes that generate NADPH. In this study, we instead focused on increasing the efficiency of enzymes that generate NADPH. We first established a robust genetic screen that allowed us to screen improved variants. The pentose phosphate pathway enzyme, glucose 6-phosphate dehydrogenase (G6PD), was chosen for further improvement. Different gene fusions of G6PD with the downstream enzyme in the pentose phosphate pathway, 6-phosphogluconolactonase (6PGL), were created. The linker-less G6PD-6PGL fusion displayed the highest activity, and although it had slightly lower activity than the WT enzyme, the affinity for G6P was higher and showed higher yields of the diterpenoid sclareol in vivo. A second gene fusion approach was to fuse G6PD to truncated HMG-CoA reductase, the rate-limiting step and also the major NADPH consumer in the pathway. Both domains were functional, and the fusion also yielded higher sclareol levels. We simultaneously carried out a rational mutagenesis approach with G6PD, which led to the identification of two mutants of G6PD, N403D and S238QI239F, that showed 15-25% higher activity in vitro. The diterpene sclareol yields were also increased in the strains overexpressing these mutants relative to WT G6PD, and these will be very beneficial in synthetic biology applications.

Occurrence, distribution and sources of petroleum contamination in reef-associated sediments of the Gulf of Mannar, India.

The distribution of saturated hydrocarbons and polycyclic aromatic hydrocarbons (PAHs) was assessed in superficial sediment samples collected from Mandapam island groups, Gulf of Mannar, India. The hydrocarbon distribution pattern and the n-alkane indices (e.g., carbon preference index (CPI) and natural n-alkanes ratio (NAR)) were deployed to differentiate between the biogenic and anthropogenic sources. Petroleum pollution was indicated by the pristane/phytane ratio close to 1. Presence of a prominent unresolved complex mixture (UCM) as well as hopane concentrations further supported this assertion. The evaluation of petrogenic sources of contamination were also comprehended by various diagnostic ratios of PAHs. The sites associated with shipping activities, tourism, and located near the mainland and accessible portions of the islands exhibited high petroleum contamination. Correlation analysis underlines the significance of combining petroleum-specific marker compounds and diagnostic ratios to improve the assessment of human influence on marine ecosystems.

Distribution and characteristics of microplastics and phthalate esters from a freshwater lake system in Lesser Himalayas.

The occurrence, distribution, characterization and quantification of microplastics (MPs) and phthalic acid esters (PAEs) from the freshwater aquatic environment are not thoroughly explored in the Indian Himalayas despite concern over their adverse effects on human health and ecosystem. In this study, we have investigated the presence of MPs and PAEs in an aquatic system from Indian subcontinent. The MPs were detected in all water and sediment samples with abundances ranging from 02-64 particles/L and 15-632 particles/kg dw, respectively. The abundance of MPs, dominated by polyethylene and polystyrene, with the majority being fibres and fragments indicated that they were derived from plastic paints, boats or synthetic products. The concentrations of PAEs in the surface sediment samples varied from 06-357 ng/g dw. The most abundant PAEs in the sediments were dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP), since they were present in all the samples collected from the lake basin. The relatively higher abundances of MPs and higher concentrations of PAEs were generally found in the vicinity of areas impacted by anthropogenic activities. A clear correlation between the abundance of microplastics and PAEs concentration was observed suggesting that they are closely attributed to a single source. This study also provides an alternative approach to utilize the chemical additives in plastics as markers to trace the presence and distribution of MPs in the aquatic environment.