Microbial potential for carbon and nutrient cycling in a geogenic supercritical carbon dioxide reservoir.

Microorganisms catalyze carbon cycling and biogeochemical reactions in the deep subsurface and thus may be expected to influence the fate of injected supercritical (sc) CO2 following geological carbon sequestration (GCS). We hypothesized that natural subsurface scCO2 reservoirs, which serve as analogs for the long-term fate of sequestered scCO2 , harbor a 'deep carbonated biosphere' with carbon cycling potential. We sampled subsurface fluids from scCO2 -water separators at a natural scCO2 reservoir at McElmo Dome, Colorado for analysis of 16S rRNA gene diversity and metagenome content. Sequence annotations indicated dominance of Sulfurospirillum, Rhizobium, Desulfovibrio and four members of the Clostridiales family. Genomes extracted from metagenomes using homology and compositional approaches revealed diverse mechanisms for growth and nutrient cycling, including pathways for CO2 and N2 fixation, anaerobic respiration, sulfur oxidation, fermentation and potential for metabolic syntrophy. Differences in biogeochemical potential between two production well communities were consistent with differences in fluid chemical profiles, suggesting a potential link between microbial activity and geochemistry. The existence of a microbial ecosystem associated with the McElmo Dome scCO2 reservoir indicates that potential impacts of the deep biosphere on CO2 fate and transport should be taken into consideration as a component of GCS planning and modelling.

Relationship of Microbiota and Cyanobacterial Secondary Metabolites in Planktothricoides-Dominated Bloom.

The identification of phytoplankton species and microbial biodiversity is necessary to assess water ecosystem health and the quality of water resources. We investigated the short-term (2 days) vertical and diel variations in bacterial community structure and microbially derived secondary metabolites during a cyanobacterial bloom that emerged in a highly urbanized tropical reservoir. The waterbody was largely dominated by the cyanobacteria Planktothricoides spp., together with the Synechococcus, Pseudanabaena, Prochlorothrix, and Limnothrix. Spatial differences (i.e., water depth) rather than temporal differences (i.e., day versus night) better-explained the short-term variability in water quality parameters and bacterial community composition. Difference in bacterial structure suggested a resource-driven distribution pattern for the community. We found that the freshwater bacterial community associated with cyanobacterial blooms is largely conserved at the phylum level, with Proteobacteria (ß-proteobateria), Bacteroidetes, and Actinobacteria as the main taxa despite the cyanobacterial species present and geographical (Asia, Europe, Australia, and North America) or climatic distinctions. Through multivariate statistical analyses of the bacterial community, environmental parameters, and secondary metabolite concentrations, we observed positive relationships between the occurrences of cyanobacterial groups and off-flavor compounds (2-methyisoborneol and ß-ionone), suggesting a cyanobacterial origin. This study demonstrates the potential of 16S rRNA gene amplicon sequencing as a supporting tool in algal bloom monitoring or water-resource management.

Variation of Bacterial Communities with Water Quality in an Urban Tropical Catchment.

A major challenge for assessment of water quality in tropical environments is the natural occurrence and potential growth of Fecal Indicator Bacteria (FIB). To gain a better understanding of the relationship between measured levels of FIB and the distribution of sewage-associated bacteria, including potential pathogens, in the tropics this study compared the abundance of FIB (Total coliforms and E. coli) and the Bacteroidales (HF183 marker) with bacterial community structure determined by next-generation amplicon sequencing. Water was sampled twice over 6 months from 18 sites within a tropical urban catchment and reservoir, followed by extraction of DNA from microorganisms, and sequencing targeting the V3-V4 region of the 16S rRNA gene. Multivariate statistical analyses indicated that bacterial community composition (BCC) varied between reservoir and catchment, within catchment land-uses, and with E. coli concentration. Beta-regression indicated that the proportion of sequences from sewage-associated taxa (SAT) or pathogen-like sequences (PLS) were predicted most significantly by measured levels of E. coli(log MPN/100 mL) (χ2 > 8.7; p < 0.003). In addition, SAT were significantly predicted by log HF183 levels (χ2=13.1; p = 0.0003) while PLS were not. Our study suggests that measurements of E. coli concentration could be useful in predicting samples enriched in sewage-associated and pathogen-like bacteria in tropical environments despite the potential for nonconservative behavior.

Microbial growth under supercritical CO2.

Growth of microorganisms in environments containing CO2 above its critical point is unexpected due to a combination of deleterious effects, including cytoplasmic acidification and membrane destabilization. Thus, supercritical CO2 (scCO2) is generally regarded as a sterilizing agent. We report isolation of bacteria from three sites targeted for geologic carbon dioxide sequestration (GCS) that are capable of growth in pressurized bioreactors containing scCO2. Analysis of 16S rRNA genes from scCO2 enrichment cultures revealed microbial assemblages of varied complexity, including representatives of the genus Bacillus. Propagation of enrichment cultures under scCO2 headspace led to isolation of six strains corresponding to Bacillus cereus, Bacillus subterraneus, Bacillus amyloliquefaciens, Bacillus safensis, and Bacillus megaterium. Isolates are spore-forming, facultative anaerobes and capable of germination and growth under an scCO2 headspace. In addition to these isolates, several Bacillus type strains grew under scCO2, suggesting that this may be a shared feature of spore-forming Bacillus spp. Our results provide direct evidence of microbial activity at the interface between scCO2 and an aqueous phase. Since microbial activity can influence the key mechanisms for permanent storage of sequestered CO2 (i.e., structural, residual, solubility, and mineral trapping), our work suggests that during GCS microorganisms may grow and catalyze biological reactions that influence the fate and transport of CO2 in the deep subsurface.

Microbiota of the major South Atlantic reef building coral Mussismilia.

The Brazilian endemic scleractinian corals, genus Mussismilia, are among the main reef builders of the South Atlantic and are threatened by accelerating rates of disease. To better understand how holobiont microbial populations interact with corals during health and disease and to evaluate whether selective pressures in the holobiont or neutral assembly shape microbial composition, we have examined the microbiota structure of Mussismilia corals according to coral lineage, environment, and disease/health status. Microbiota of three Mussismilia species (Mussismilia harttii, Mussismilia hispida, and Mussismilia braziliensis) was compared using 16S rRNA pyrosequencing and clone library analysis of coral fragments. Analysis of biological triplicates per Mussismilia species and reef site allowed assessment of variability among Mussismilia species and between sites for M. braziliensis. From 173,487 V6 sequences, 6,733 coral- and 1,052 water-associated operational taxonomic units (OTUs) were observed. M. braziliensis microbiota was more similar across reefs than to other Mussismilia species microbiota from the same reef. Highly prevalent OTUs were more significantly structured by coral lineage and were enriched in Alpha- and Gammaproteobacteria. Bacterial OTUs from healthy corals were recovered from a M. braziliensis skeleton sample at twice the frequency of recovery from water or a diseased coral suggesting the skeleton is a significant habitat for microbial populations in the holobiont. Diseased corals were enriched with pathogens and opportunists (Vibrios, Bacteroidetes, Thalassomonas, and SRB). Our study examines for the first time intra- and inter-specific variability of microbiota across the genus Mussismilia. Changes in microbiota may be useful indicators of coral health and thus be a valuable tool for coral reef management and conservation.

Balancing public health and group privacy: Ethics, rights, and obligations for wastewater surveillance systems.

As the threat of COVID-19 recedes, wastewater surveillance - unlike other pandemic-era public health surveillance methods - seems here to stay. Concerns have been raised, however, about the potential risks that wastewater surveillance might pose towards group privacy. Existing scholarship has focused upon using ethics- or human rights-based frameworks as a means of balancing the public health objectives of wastewater surveillance and the potential risks it might pose to group privacy. However, such frameworks greatly lack enforceability. In order to further the strong foundation laid by such frameworks - while addressing their lack of enforceability - this paper proposes the idea of the 'obligation' as an alternative way to regulate wastewater surveillance systems. The legal codification of said obligations provides a method of ensuring that wastewater surveillance systems can be deployed effectively and equitably. Our paper proposes that legal obligations for wastewater surveillance can be created and enforced through transparent and purposeful legislation (which would include limits on power and grant institutions substantial oversight) as well as paying heed to non-legislative legal means of enforcement, such as through courts or contracts. Introducing legal obligations for wastewater surveillance could therefore be highly useful to researchers, policymakers, corporate technologists, and government agencies working in this field.

Co-incidence of BA.1 and BA.2 at the start of Singapore's Omicron wave revealed by Community and University Campus wastewater surveillance.

Wastewater surveillance (WWS) has been globally recognised to be a useful tool in quantifying SARS-CoV-2 RNA at the community and residential levels without biases associated with case-reporting. The emergence of variants of concern (VOCs) have given rise to an unprecedented number of infections even though populations are increasingly vaccinated. This is because VOCs have been reported to possess higher transmissibility and can evade host immune responses. The B.1.1.529 lineage (Omicron) has severely disrupted global plans to return to normalcy. In this study, we developed an allele-specific (AS) RT-qPCR assay which simultaneously targets the stretch of deletions and mutations in the spike protein from position 24-27 for quantitative detection of Omicron BA.2. Together with previous assays that detect mutations associated with Omicron BA.1 (deletion at position 69 and 70) and all Omicron (mutation at position 493 and 498), we report the validation and time series of these assays from September 2021 to May 2022 using influent samples from two wastewater treatment plants and across four University campus sites in Singapore. Viral RNA concentrations at the treatment plants corroborate with locally reported clinical cases, AS RT-qPCR assays revealed co-incidence of Omicron BA.1 and BA.2 on 12 January 2022, almost two months after initial BA.1 detection in South Africa and Botswana. BA.2 became the dominant variant by the end of January 2022 and completely displaced BA.1 by mid-March 2022. University campus sites were similarly positive for BA.1 and/or BA.2 in the same week as first detection at the treatment plants, where BA.2 became rapidly established as the dominant lineage within three weeks. These results corroborate clinical incidence of the Omicron lineages in Singapore and indicate minimal silent circulation prior to January 2022. The subsequent simultaneous spread of both variant lineages followed strategic relaxation of safe management measures upon meeting nationwide vaccination goals.

Palau's warmest reefs harbor thermally tolerant corals that thrive across different habitats.

Ocean warming is killing corals, but heat-tolerant populations exist; if protected, they could replenish affected reefs naturally or through restoration. Palau's Rock Islands experience consistently higher temperatures and extreme heatwaves, yet their diverse coral communities bleach less than those on Palau's cooler outer reefs. Here, we combined genetic analyses, bleaching histories and growth rates of Porites cf. lobata colonies to identify thermally tolerant genotypes, map their distribution, and investigate potential growth trade-offs. We identified four genetic lineages of P. cf. lobata. On Palau's outer reefs, a thermally sensitive lineage dominates. The Rock Islands harbor two lineages with enhanced thermal tolerance; one of which shows no consistent growth trade-off and also occurs on several outer reefs. This suggests that the Rock Islands provide naturally tolerant larvae to neighboring areas. Finding and protecting such sources of thermally-tolerant corals is key to reef survival under 21st century climate change.

Gut Ruminococcaceae levels at baseline correlate with risk of antibiotic-associated diarrhea.

Antibiotic-associated diarrhea (AAD) affects a significant proportion of patients receiving antibiotics. We sought to understand if differences in the gut microbiome would influence the development of AAD. We administered a 3-day course of amoxicillin-clavulanate to 30 healthy adult volunteers, and analyzed their stool microbiome, using 16S rRNA gene sequencing, at baseline and up to 4 weeks post antibiotic administration. Lower levels of gut Ruminococcaceae were significantly and consistently observed from baseline until day 7 in participants who developed AAD. Overall, participants who developed AAD experienced a greater decrease in microbial diversity. The probability of AAD could be predicted based on qPCR-derived levels of Faecalibacterium prausnitzii at baseline. Our findings suggest that a lack of gut Ruminococcaceae influences development of AAD. Quantification of F. prausnitzii in stool prior to antibiotic administration may help identify patients at risk of AAD, and aid clinicians in devising individualized treatment regimens to minimize such adverse effects.

Coral mucus rapidly induces chemokinesis and genome-wide transcriptional shifts toward early pathogenesis in a bacterial coral pathogen.

Elevated seawater temperatures have contributed to the rise of coral disease mediated by bacterial pathogens, such as the globally distributed Vibrio coralliilyticus, which utilizes coral mucus as a chemical cue to locate stressed corals. However, the physiological events in the pathogens that follow their entry into the coral host environment remain unknown. Here, we present simultaneous measurements of the behavioral and transcriptional responses of V. coralliilyticus BAA-450 incubated in coral mucus. Video microscopy revealed a strong and rapid chemokinetic behavioral response by the pathogen, characterized by a two-fold increase in average swimming speed within 6 min of coral mucus exposure. RNA sequencing showed that this bacterial behavior was accompanied by an equally rapid differential expression of 53% of the genes in the V. coralliilyticus genome. Specifically, transcript abundance 10 min after mucus exposure showed upregulation of genes involved in quorum sensing, biofilm formation, and nutrient metabolism, and downregulation of flagella synthesis and chemotaxis genes. After 60 min, we observed upregulation of genes associated with virulence, including zinc metalloproteases responsible for causing coral tissue damage and algal symbiont photoinactivation, and secretion systems that may export toxins. Together, our results suggest that V. coralliilyticus employs a suite of behavioral and transcriptional responses to rapidly shift into a distinct infection mode within minutes of exposure to the coral microenvironment.

Making waves: Wastewater surveillance of SARS-CoV-2 for population-based health management.

Worldwide, clinical data remain the gold standard for disease surveillance and tracking. However, such data are limited due to factors such as reporting bias and inability to track asymptomatic disease carriers. Disease agents are excreted in the urine and feces of infected individuals regardless of disease symptom severity. Wastewater surveillance - that is, monitoring disease via human effluent - represents a valuable complement to clinical approaches. Because wastewater is relatively inexpensive and easy to collect and can be monitored at different levels of population aggregation as needed, wastewater surveillance can offer a real-time, cost-effective view of a community's health that is independent of biases associated with case-reporting. For SARS-CoV-2 and other disease-causing agents we envision an aggregate wastewater-monitoring system at the level of a wastewater treatment plant and exploratory or confirmatory monitoring of the sewerage system at the neighborhood scale to identify or confirm clusters of infection or assess impact of control measures where transmission has been established. Implementation will require constructing a framework with collaborating government agencies, public or private utilities, and civil society organizations for appropriate use of data collected from wastewater, identification of an appropriate scale of sample collection and aggregation to balance privacy concerns and risk of stigmatization with public health preservation, and consideration of the social implications of wastewater surveillance.

A molecular and physiological survey of a diverse collection of hydrothermal vent Thermococcus and Pyrococcus isolates.

Strains of hyperthermophilic anaerobic hydrothermal vent archaea maintained in the culture collection assembled by Holger Jannasch at the Woods Hole Oceanographic Institution between 1984 and 1998 were identified and partially characterized by Denaturing Gradient Gel Electrophoresis, 16S rRNA gene sequencing, and by growth tests at different temperatures and on different organic carbon and nitrogen sources. All strains were members of the genera Thermococcus and Pyrococcus. The greatest phylogenetic diversity was found in strains from a single Guaymas Basin core isolated by serial dilution from four different depth horizons of heated sediment incubated at the corresponding in situ temperatures. In contrast, geographically distinct vent locations and sample materials yielded a lower diversity of isolates when enriched under uniform temperature regimes and without prior dilution of the source material.

Shrimp pathogenicity, hemolysis, and the presence of hemolysin and TTSS genes in Vibrio harveyi isolated from Thailand.

The virulence factors of Vibrio harveyi, the causative agent of luminous vibriosis, are not completely understood. We investigated the correlations between shrimp mortality, hemolysis, the presence of a hemolysin gene (vhh), and a gene involved in the type III secretion system (the Vibrio calcium response gene vcrD). V harveyi HY01 was isolated from a shrimp that died from vibriosis, and 36 other V. harveyi isolates were obtained from fish and shellfish in Hat Yai city, Thailand. An ocean isolate of V. harveyi BAA-1116 was also included. Thirteen isolates including V harveyi HYO1 caused shrimp death 12 h after injection. Most V harveyi isolates in this group (designated as Group A) caused hemolysis on prawn blood agar. None of the shrimp died after injection with V harveyi BAA-1116. Molecular analysis of all V harveyi isolates revealed the presence of vcrD in both pathogenic and non-pathogenic strains. Although vhh was detected in all V harveyi isolates, some isolates did not cause hemolysis, indicating that vhh gene expression might be regulated. Analysis of the V harveyi HYO1 genome revealed a V cholerae like-hemolysin gene, hlyA (designated as hhl). Specific primers designed for hhl detected this gene in 3 additional V harveyi isolates but the presence of this gene was not correlated with pathogenicity. Random amplified polymorphic DNA (RAPD) analysis revealed a high degree of genetic diversity in all V harveyi isolates, and there were no correlations among the hhl-positive isolates or the pathogenic strains.

Variably improved microbial source tracking with digital droplet PCR.

This study addressed whether digital droplet PCR (ddPCR) could improve sensitivity and specificity of human-associated Bacteroidales genetic markers, BacHum and B. theta, and their quantification in environmental and fecal composite samples. Human markers were quantified by qPCR and ddPCR platforms obtained from the same manufacturer. A total of 180 samples were evaluated by each platform including human and animal feces, sewage, and environmental water. The sensitivity of ddPCR and qPCR marker assays in sewage and human stool was 0.85-1.00 with marginal reduction in human stool by ddPCR relative to qPCR (<10%). The prevalence and distribution of markers across complex sample types was similar (74-100% agreement) by both platforms with qPCR showing higher sensitivity for markers in environmental and composite samples and ddPCR showing greater reproducibility for marker detection in fecal composites. Determination of BacHum prevalence in fecal samples by ddPCR increased specificity relative to qPCR (from 0.58 to 0.88) and accuracy (from 0.77 to 0.94), while the B. theta assay performed similarly on both platforms (specificity = 0.98). In silico analysis indicated higher specificity of ddPCR for BacHum was not solely attributed to reduced sensitivity relative to qPCR. Marker concentrations measured by ddPCR for all sample types were consistently lower than those measured by qPCR, by a factor of 2.6 ±â€¯2.8 for B. theta and 18.7 ±â€¯10.0 for BacHum. We suggest that differences in assay performance on ddPCR and qPCR platforms may be linked to the characteristics of the assay targets (that is, genes with multiple versus single copies and encoding proteins versus ribosomal RNA) however further work is needed to validate these ideas. We conclude that ddPCR is a suitable tool for microbial source tracking, however, other factors such as cost-effectiveness and assay-specific performance should be considered.

Engineered microbial biofuel production and recovery under supercritical carbon dioxide.

Culture contamination, end-product toxicity, and energy efficient product recovery are long-standing bioprocess challenges. To solve these problems, we propose a high-pressure fermentation strategy, coupled with in situ extraction using the abundant and renewable solvent supercritical carbon dioxide (scCO2), which is also known for its broad microbial lethality. Towards this goal, we report the domestication and engineering of a scCO2-tolerant strain of Bacillus megaterium, previously isolated from formation waters from the McElmo Dome CO2 field, to produce branched alcohols that have potential use as biofuels. After establishing induced-expression under scCO2, isobutanol production from 2-ketoisovalerate is observed with greater than 40% yield with co-produced isopentanol. Finally, we present a process model to compare the energy required for our process to other in situ extraction methods, such as gas stripping, finding scCO2 extraction to be potentially competitive, if not superior.

Isolation, Development, and Genomic Analysis of Bacillus megaterium SR7 for Growth and Metabolite Production Under Supercritical Carbon Dioxide.

Supercritical carbon dioxide (scCO2) is an attractive substitute for conventional organic solvents due to its unique transport and thermodynamic properties, its renewability and labile nature, and its high solubility for compounds such as alcohols, ketones, and aldehydes. However, biological systems that use scCO2 are mainly limited to in vitro processes due to its strong inhibition of cell viability and growth. To solve this problem, we used a bioprospecting approach to isolate a microbial strain with the natural ability to grow while exposed to scCO2. Enrichment culture and serial passaging of deep subsurface fluids from the McElmo Dome scCO2 reservoir in aqueous media under scCO2 headspace enabled the isolation of spore-forming strain Bacillus megaterium SR7. Sequencing and analysis of the complete 5.51 Mbp genome and physiological characterization revealed the capacity for facultative anaerobic metabolism, including fermentative growth on a diverse range of organic substrates. Supplementation of growth medium with L-alanine for chemical induction of spore germination significantly improved growth frequencies and biomass accumulation under scCO2 headspace. Detection of endogenous fermentative compounds in cultures grown under scCO2 represents the first observation of bioproduct generation and accumulation under this condition. Culturing development and metabolic characterization of B. megaterium SR7 represent initial advancements in the effort toward enabling exploitation of scCO2 as a sustainable solvent for in vivo bioprocessing.

Geospatial distribution of viromes in tropical freshwater ecosystems.

This study seeks to understand the general distribution of virome abundance and diversity in tropical freshwater ecosystems in Singapore and the geospatial distribution of the virome under different landuse patterns. Correlations between diversity, environmental parameters and land use patterns were analyzed and significant correlations were highlighted. Overall, the majority (65.5%) of the annotated virome belonged to bacteriophages. The percentage of Caudovirales was higher in reservoirs whereas the percentages of Dicistroviridae, Microviridae and Circoviridae were higher in tributaries. Reservoirs showed a higher Shannon-index virome diversity compared to upstream tributaries. Land use (urbanized, agriculture and parkland areas) influenced the characteristics of the virome distribution pattern. Dicistroviridae and Microviridae were enriched in urbanized tributaries while Mimiviridae, Phycodnaviridae, Siphoviridae and Podoviridae were enriched in parkland reservoirs. Several sequences closely related to the emerging zoonotic virus, cyclovirus, and the human-related virus (human picobirnavirus), were also detected. In addition, the relative abundance of PMMoV (pepper mild mottle virus) sequences was significantly correlated with RT-qPCR measurements (0.588 < r < 0.879, p < 0.05). This study shows that spatial factors (e.g., reservoirs/tributaries, land use) are the main drivers of the viral community structure in tropical freshwater ecosystems.

Corrigendum: Characterization of Metagenomes in Urban Aquatic Compartments Reveals High Prevalence of Clinically Relevant Antibiotic Resistance Genes in Wastewaters.

[This corrects the article on p. 2200 in vol. 8, PMID: 29201017.].

Quantitative Detection of Active Vibrios Associated with White Plague Disease in Mussismilia braziliensis Corals.

Over recent decades several coral diseases have been reported as a significant threat to coral reef ecosystems causing the decline of corals cover and diversity around the world. The development of techniques that improve the ability to detect and quantify microbial agents involved in coral disease will aid in the elucidation of disease cause, facilitating coral disease detection and diagnosis, identification and pathogen monitoring, pathogen sources, vectors, and reservoirs. The genus Vibrio is known to harbor pathogenic strains to marine organisms. One of the best-characterized coral pathogens is Vibrio coralliilyticus, an aetilogic agent of White Plague Disease (WPD). We used Mussismilia coral tissue (healthy and diseased specimens) to develop a rapid reproducible detection system for vibrios based on RT-QPCR and SYBR chemistry. We were able to detect total vibrios in expressed RNA targeting the 16S rRNA gene at 5.23 × 106 copies/µg RNA and V. coralliilyticus targeting the pyrH gene at 5.10 × 103 copies/µg RNA in coral tissue. Detection of V. coralliilyticus in diseased and in healthy samples suggests that WPD in the Abrolhos Bank may be caused by a consortium of microorganism and not only a single pathogen. We developed a more practical and economic system compared with probe uses for the real-time detection and quantification of vibrios from coral tissues by using the 16S rRNA and pyrH gene. This qPCR assay is a reliable tool for the monitoring of coral pathogens, and can be useful to prevent, control, or reduce impacts in this ecosystem.

Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'.

Although it has been recognized that PCR amplification of mixed templates may generate sequence artifacts, the mechanisms of their formation, frequency and potential elimination have not been fully elucidated. Here evidence is presented for heteroduplexes as a major source of artifacts in mixed-template PCR. Nearly equal proportions of homoduplexes and heteroduplexes were observed after co-amplifying 16S rDNA from three bacterial genomes and analyzing products by constant denaturing capillary electrophoresis (CDCE). Heteroduplexes became increasingly prevalent as primers became limiting and/or template diversity was increased. A model exploring the fate of cloned heteroduplexes during MutHLS-mediated mismatch repair in the Escherichia coli host demonstrates that the diversity of artifactual sequences increases exponentially with the number of both variable nucleotides and of original sequence variants. Our model illustrates how minimization of heteroduplex molecules before cloning may reduce artificial genetic diversity detected during sequence analysis by clone screening. Thus, we developed a method to eliminate heteroduplexes from mixed-template PCR products by subjecting them to 'reconditioning PCR', a low cycle number re-amplification of a 10-fold diluted mixed-template PCR product. This simple modification to the protocol may ensure that sequence richness encountered in clone libraries more closely reflects genetic diversity in the original sample.