Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli.

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for more than 35 years and 77,000 bacterial generations. The setup and procedures used in the LTEE epitomize reliable and reproducible methods for studying microbial evolution. In this protocol, we first describe how the LTEE populations are transferred to fresh medium and cultured each day. Then, we describe how the LTEE populations are regularly checked for possible signs of contamination and archived to provide a permanent frozen "fossil record" for later study. Multiple safeguards included in these procedures are designed to prevent contamination, detect various problems when they occur, and recover from disruptions without appreciably setting back the progress of the experiment. One way that the overall tempo and character of evolutionary changes are monitored in the LTEE is by measuring the competitive fitness of populations and strains from the experiment. We describe how co-culture competition assays are conducted and provide both a spreadsheet and an R package (fitnessR) for calculating relative fitness from the results. Over the course of the LTEE, the behaviors of some populations have changed in interesting ways, and new technologies like whole-genome sequencing have provided additional avenues for investigating how the populations have evolved. We end by discussing how the original LTEE procedures have been updated to accommodate or take advantage of these changes. This protocol will be useful for researchers who use the LTEE as a model system for studying connections between evolution and genetics, molecular biology, systems biology, and ecology. More broadly, the LTEE provides a tried-and-true template for those who are beginning their own evolution experiments with new microbes, environments, and questions.

Stress induced by crude glycerol in a thermophilic digester: microbial community divergence and resilience, but slow process recovery.

Recovery from stress is an important property for anaerobic digestion (AD). Although AD is quite adaptable with regard to waste composition, new substrates added to stable systems may cause process decline. We tested whether crude glycerol would cause stress to a thermophilic AD microbiome previously stabilized long-term on a low C/N ratio feedstock. Three-percent (v/v) crude glycerol was added to the basal substrate (poultry litter) for two hydraulic retention time (HRT) periods. This caused stress where biogas volume and methane percentage dramatically decreased and VFA levels increased. When the basal substrate was resumed, secondary inhibition occurred, resulting in even greater stress (biogas production ceased, methane 3.6%). Unassisted recovery of system processes required eight HRT periods. In contrast, crude glycerol applied at a lower organic loading rate did not cause inhibition. Crude glycerol caused changes in dominance in the microbial community (16S rRNA pyrotags). Although process resilience was slow, the recovery of digester functions occurred in conjunction with the recovery of community structure, particularly putative syntrophic acetate-oxidizing bacteria. KEY POINTS: • Crude glycerol caused stress in thermophilic co-digestion with poultry litter. • Unassisted resilience of digester functions (methane) required 8 HRT. • Syntrophic acetate-oxidizing bacteria implicated for keystone resilience functions. Graphical abstract.

Metagenome Sequences of a Thermophilic Anaerobic Digester Adapted to a Low C/N Ratio, High-Ammonia Feedstock (Poultry Litter).

We sequenced the metagenome of a pilot-scale thermophilic digester with long-term, stable performance on poultry litter feedstock which has a very low C/N ratio, a high ammonia level, and high lignocellulose content. Firmicutes were the dominant phylum (68.9%). Other abundant phyla included Bacteroidetes, Euryarchaeota, and Thermotogae This microbiome represents a hydrogenotrophic methanogenic community with high diversity.

Metagenome Sequences of Sediment from a Recovering Industrialized Appalachian River in West Virginia.

We sequenced two metagenomes from upper sediment layers (0 to 5 and 6 to 10 cm) from the Kanawha River, West Virginia. The watershed includes inputs from the forested Appalachian Mountains, surface coal mining, municipal residues, and extensive chemical manufacturing. The dominant bacterial phyla were Proteobacteria, Bacteriodetes, Firmicutes, Actinobacteria, and Chloroflexi Xenobiotic degradation pathways were present.