Biology subjects

Khunjar, W.

Publications and source records attributed to Khunjar, W..

4 recordsLinked to original sources

bioRxiv2025

Quantitative metagenomics using a portable protocol.

A field-deployable DNA sequencing approach for quantitative microbial community profiling can enable rapid responses for a range of applications in the water sector - from process control to wastewater surveillance. Current quantitative approaches have complex instrumentation requirements and long turnaround time for DNA recovery and absolute quantitation. In this study, we report a field-deployable rapid detection and absolute quantitation (rD+rQ) workflow that leverages the real-time sequencing capabilities of Nanopore sequencing for quantitative metagenomics. This workflow integrates a high-molecular-weight DNA recovery protocol for diverse environmental matrices of relevance to the water sector and multiplexed Nanopore sequencing with barcoded spike-in-based calibration (BSINC). BSINC using multi-species genomic spike-in controls exhibits significantly higher calibration accuracy compared to conventional approaches that utilize either single DNA fragment or single organism spike in controls. Dynamic detection and quantitation limits were established based on coverage fraction of sequenced genomes and the coefficient of variation of genome copy numbers across replicates to enhance the accuracy and precision of microbial quantitation. The rD+rQ workflow achieves species-level identification and absolute quantitative results comparable to digital PCR in environmental samples. This portable laboratory and easy-to-use rD+rQ workflow should facilitate rapid decision-making for the water industry.

microbiology↗

bioRxiv2022

Divergent microbial structure still results in convergent microbial function during arrested anaerobic digestion of food waste at different hydraulic retention times

In this study, two arrested anaerobic digestion bioreactors fed with food waste operated under different hydraulic retention times (HRTs) exhibited long-term stable volatile fatty acid (VFA) production performance including similar total yields (p = .085) with propionic acid (PA) being the most abundant VFA. Meta-omics analysis revealed distinct microbial structures (p = .02) at the two HRTs while there were no differences in potential and extant functionality as indicated by the whole-genome and whole-transcriptome sequencing, respectively. The highest potential (relative abundance of DNA sequence reads) and extant (relative abundance of mRNA sequence reads) functionality corresponded with PA production compared to other acids. The most abundant genus Prevotella produced PA mainly through the acryloyl-CoA pathway. Based on our results, the mechanistic basis for the similar VFA production performance observed under the HRTs tested lies in the community-level redundancy in convergent acidification functions and pathways, rather than trends in community structure.

microbiology↗

bioRxiv2022

Low diversity and microdiversity of comammox bacteria in wastewater systems suggests wastewater-specific adaptation within the Ca. Nitrospira nitrosa cluster.

Studies have found Ca. Nitrospira nitrosa-like bacteria to be the principal or sole comammox bacteria in nitrogen removal systems for wastewater treatment. In contrast, multiple populations of strict ammonia and nitrite oxidizers co-exist in similar systems. This apparent lack of diversity is surprising and could impact the feasibility of leveraging comammox bacteria for nitrogen removal. We used full-length 16S rRNA gene sequencing and genome-resolved metagenomics to compare population-level (i.e., species) diversity of comammox bacteria with that of strict nitrifiers in full-scale wastewater treatment systems and assess whether these observations were consistent or diverged at the strain-level. Full-length 16S rRNA gene sequencing indicated that while Nitrosomonas-like bacteria exhibited higher population-level diversity, the effective microdiversity of most Nitrospira-like bacteria were comparatively higher except for one Nitrospira Lineage II population. Comammox bacterial metagenome assembled genomes (MAGs) were associated with Ca. Nitrospira nitrosa. The average amino acid identity between comammox bacterial MAGs (93% {+/-} 3) across systems was significantly higher than that of the Nitrosomonas-like ammonia oxidizers (73%{+/-}8) and the Nitrospira-like nitrite oxidizer MAGs (75%{+/-}13), suggesting that the same comammox population was detected in all systems. Comammox bacteria and some ammonia oxidizers MAGs were significantly less microdiverse than most ammonia and nitrite oxidizers. Interestingly, strain-resolved analysis also indicates that different nitrogen removal systems harbor different comammox bacterial strains within the Ca. Nitrospira nitrosa cluster. These results suggest that comammox bacteria associated with Ca. Nitrospira nitrosa have low species- and strain-level diversity in nitrogen removal systems and may thus harbor specific adaptations to the wastewater ecosystem.

microbiology↗

bioRxiv2019

Long solids retention times and attached growth phase favor prevalence of comammox bacteria in nitrogen removal systems.

The discovery of the complete ammonia oxidizing (comammox) bacteria overturns the traditional two-organism nitrification paradigm which largely underpins the design and operation of nitrogen removal during wastewater treatment. Quantifying the abundance, diversity, and activity of comammox bacteria in wastewater treatment systems is important for ensuring a clear understanding of the nitrogen biotransformations responsible for ammonia removal. To this end, we conducted a yearlong survey of 14 full-scale nitrogen removal systems including mainstream conventional and simultaneous nitrification-denitrification and side-stream partial nitrification-anammox systems with varying process configurations. Metagenomics and genome-resolved metagenomics identified comammox bacteria in mainstream conventional and simultaneous nitrification-denitrification systems, with no evidence for their presence in side-stream partial nitrification-anammox systems. Further, comammox bacterial diversity was restricted to clade A and these clade A comammox bacteria were detected in systems with long solids retention times (>10 days) and/or in the attached growth phase. Using a newly designed qPCR assay targeting the amoB gene of clade A comammox bacteria in combination with quantitation of other canonical nitrifiers, we show that long solids retention time is the key process parameter associated with the prevalence and abundance of comammox bacteria. The increase in comammox bacterial abundance was not associated with concomitant decrease in the abundance of canonical nitrifiers; however, systems with comammox bacteria showed significantly better and temporally stable ammonia removal compared to systems where they were not detected. Finally, in contrast to recent studies, we do not find any significant association of comammox bacterial prevalence and abundance with dissolved oxygen concentrations in this study.\n\nHighlightsO_LIClade A comammox bacteria were detected in wastewater nitrogen removal systems.\nC_LIO_LINew qPCR assay targeting the amoB gene of clade A comammox bacteria was developed.\nC_LIO_LIComammox bacteria are prevalent in mainstream conventional and simultaneous nitrification-denitrification systems with long solids retention times (>10 days).\nC_LIO_LIComammox bacteria were not detected in sidestream partial nitrification-anammox systems included in this study.\nC_LI\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC=\"FIGDIR/small/696351v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (28K):\norg.highwire.dtl.DTLVardef@3228c9org.highwire.dtl.DTLVardef@113dfb8org.highwire.dtl.DTLVardef@1c4adaforg.highwire.dtl.DTLVardef@166f997_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT\n\nC_FIG

microbiology↗