Pure Culture
Studies of Methanogenic Bacteria
PAUL H. SMITH, Assistant Professor
University of Florida
Gainesville, Florida
INTRODUCTION
Dissimilation of organic material occurs during anaerobic sludge digestion.
The dissimilation is accompanied by growth of an anaerobic microflora. The digestion process is determined by environmental conditions, and the micro-organisms present. Kluyver (1) elucidated the biological principle that the metabolism
of living organisms involves a continuous and directed flow of electrons from electron donors to electron acceptors. Metabolic processes terminate when electron
transfers cease. Anaerobic dissimilation therefore ends with the formation of
methane and carbon dioxide, since these are terminal products of anaerobic electron transfer. Possible exceptions could be the transfer of electrons from methane
to some inorganic molecule such as sulfate, or a transfer of electrons from methane involving reactions of photosynthesis.
Little is known about the bacteria which produce methane, or the other bacteria involved in the dissimilation process. Lack of information in this area of
microbiology has been recently emphasized by van Niel (2).
During the past twenty years the concept of quantitative bacterial ecology has
been developed in the writings of Hungate who recently summarized (3) the goals
of quantitative microbial ecology. Relating to sludge digestion, the goals include:
determination of the number and kinds of organisms present, the magnitude of
their activities, and the factors influencing this magnitude. Little information is
available relating to these questions. It is generally recognized (4, 5) that the
lack of understanding is due to the inadequacy of existing methods for the isolation
and study of the anaerobic bacteria involved in sludge digestion. The methanogenic bacteria are among the most difficult to isolate and study. It is probable
that methods adequate for the study of these organisms are also adequate for the
study of other sludge anaerobes. The methods to be described were developed
from the basic procedures of Hungate (6) and have been successfully applied by the
author to the isolation and study of methanogenic bacteria from domestic sewage
sludge.
MATERIALS
The equipment used in the procedure is shown in Figure 1. The copper column consists of an 80 by 5 cm glass tube, packed with fine copper turnings, and
containing a 400-watt immersion heating element. The outlet from the copper
column is connected by rubber tubing to 4-in., 20-gauge syringe needles. The
tubes preceding the needles are packed with two ins. of sterile cotton. The
syringe pipetting assembly has a five-ml capacity. The automatic pipetting
syringe has a two-ml capacity and is fitted with a 1.5-in. number-19 needle having a htuoer point. Nine automatic pipetting syringes are required. The 150 by
16 mm culture tubes have reinforced lips to reduce the danger of finger lacerations. The tubes can be sealed with number 0 rubber stoppers. The press is 19 by
25 cm and will hold a test tube support containing 72 tubes.    The inoculating
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