Susan E. Lowe

Growth of Anaerobic Rumen Fungi on Defined and Semi-defined Media Lacking Rumen Fluid

Summary: Anaerobic fungi were isolated from rumen digesta of sheep and cattle and were purified using a plate culture technique. The isolates were successfully cultured on a semi-defined medium which lacked rumen fluid, and on a defined medium.

The Life Cycle and Growth Kinetics of an Anaerobic Rumen Fungus

SUMMARY: The life cycle of an anaerobic fungus isolated from the rumen of sheep was studied and was found to be similar to that of the chytrids. The organism was monocentric. At 39 °C the duration of the life cycle varied from about 26 to 32 h. The zoospores were spherical or oval in shape and were polyflagellate (8 to 17 flagella per zoospore). During the first 6·5 h of growth there was a rapid development of an extensive, non-septate, highly branched rhizoidal system; during this period the ‘main’ rhizoid increased in length exponentially with a doubling time of 2·49 h. Between 6·5 to 9·5 h after inoculation, the rate of extension of the main rhizoid declined, and no further extension occurred after 9·5 h. The main rhizoid increased in width at its base from 2·2 to 15·0 μm during the first 13 h after inoculation, indicating that intercalary wall growth occurred. Nuclei were occasionally observed in the rhizomycelium using DAPI (4′,6-diamidino-2-phenylindole) staining. Zoosporangia varied in shape from spherical to columnar, and some columnar zoosporangia were observed to become spherical. The zoosporangium initially increased in volume at an exponential rate with a doubling time of 1·56 h. Between 14 to 20 h after inoculation, growth of the zoosporangium decelerated and little growth occurred after 20 h: the zoosporangium had a final volume of 2·5 × 105μm3. At about 21 h after inoculation, a septum was formed at the base of the zoosporangium, delimiting it from the rhizoidal system. The formation of this septum was correlated with the cessation of zoosporangial growth and the onset of zoosporogenesis. After zoosporogenesis, zoospores (about 88 zoospores per zoosporangium) were liberated through a pore formed in the zoosporangial wall opposite the main rhizoid. About 3 h after zoospore release the rhizoidal system became less refractile, suggesting that autolysis had occurred. Growth of the isolate was inhibited by nikkomycin (an inhibitor of chitin synthase), but not by amphotericin B or nystatin (antibiotics which bind to sterols).

Isolation of Anaerobic Fungi from Saliva and Faeces of Sheep

SUMMARY: Anaerobic fungi were isolated from the faeces (isolates F1 and F2) and saliva (isolates S1 and S2) of a sheep. Isolate S1 and F1 were morphologically similar and both resembled Neocallimastix spp. They produced polyflagellate zoospores and were monocentric; the zoosporangium was supported by an extensive, highly branched, non-septate rhizomycelium. Isolate F1 utilized a variety of polysaccharides (including cellulose) and disaccharides, but of the eight monosaccharides tested, only fructose, glucose and xylose supported growth. Isolates S2 and F2 were morphologically similar to each other and resembled Sphaeromonas communis. These organisms were isolated on glucose but not on cellulose and they formed large spherical bodies which were closely associated with small ‘zoosporangia’; no rhizoids were observed. This is the first time that anaerobic fungi have been isolated from saliva and faeces of sheep. The ability of these organisms to survive in saliva and faeces, at reduced temperatures, suggests two possible routes of transfer of anaerobic fungi between animals.

Growth and survival of rumen fungi

The life cycle and growth kinetics of an anaerobic rumen fungus (Neocallimastix R1) in liquid and solid media are described, together with its response to light, temperature and oxygen. These results are discussed in relation to the survival of rumen fungi in saliva and faeces of sheep, and the possible routes for the transfer of anaerobic fungi between ruminants. The thallus and life cycle of Neocallimastix R1 are compared with those of aerobic chytrids.

Biocatalysis in Anaerobic Extremophiles

By and large most studies on biocatalysis deal with enzymes derived from animal, plant, or microbial sources that grow under “normal physiological” conditions, that is, those environmental conditions that would describe the discovery of physiological biochemistry in the 1930–1950s when normal enzyme environmental conditions (e.g., pH 7.0, 1.5% NaCl, 37°C) were representative of an animal cell as the model system of the times. In the 1970–1980s the vast physiobiochemical diversity of microbes has been extended by microbiologists who can now recognize microbes as living in both normal environments (e.g., human body flora) or in environments (e.g., thermal springs, hypersaline lakes, acidic peat bogs) that represent extreme conditions in relation to the origins of physiological chemistry and a normal animal or plant cell. This chapter will report on our laboratory’s efforts to understand biocatalysis in both obligate anaerobes and their enzymes that have adapted to extreme environmental conditions of pH, salinity, or temperature.