Methods for Culturing Anaerobic Microorganisms.

Anaerobic microorganisms (anaerobes) proliferate in diverse oxygen-free environments. They inhabit Earth's soils and aquatic sediments, the rumen and gut of mammals, and the gut of insects among many other oxygen-free environments. Anaerobes impact biotechnological, biomedical, ecological, and astrobiological fields. Sensitivity to oxygen is of prime consideration for successful culturing which is essential to understand function. Although cultivated for many years, the protocols and media components have been modified and adapted to the special needs of species, as well as conditions and variables for experimental evaluations. Here we describe a revised method used in our laboratories for the growth of methane-producing anaerobes (methanogenic archaea) which are among the most oxygen sensitive. The method is an example for the preparation of more specific media to cultivate a wide diversity of anaerobes.

Gut microbes contribute to nitrogen provisioning in a wood-feeding cerambycid.

Xylophagous insects often thrive on nutritionally suboptimal diets through symbiotic associations with microbes that supplement their nutritional requirements, particularly nitrogen. The wood-feeding cerambycid Anoplophora glabripennis (Motschulsky) feeds on living, healthy host trees and harbors a diverse gut microbial community. We investigated gut microbial contributions to larval nitrogen requirements through nitrogen fixing and recycling (urea hydrolysis) processes, using a combination of molecular, biochemical, and stable isotope approaches. Genes and transcripts of conserved regions of the urease operon (ureC) and nitrogen fixing (nif) regulon (nifH) were detected in A. glabripennis eggs and larvae from naturally infested logs and from larvae reared on artificial diet. Significant nitrogen fixation and recycling were documented in larvae using (15)N2 gas and (15)N-urea, respectively. Subsequent (15)N-routing of incorporated recycled nitrogen into larval essential and nonessential amino acids was shown for (15)N-urea diet-fed larvae. Results from this study show significant gut microbial contributions to this insect's metabolic nitrogen utilization through nitrogenous waste product recycling and nitrogen fixation.