Fütterungsbedingte mikrobielle Zusammensetzung von Rinderkot ...

4. Determination of microbial biomass and fungal and bacterial distribution in cattle faeces 13
highly dynamic community of bacteria, archaea and fungi that has yet to be sufficiently
quantified by the methods currently available. Most probable number and plate count
approaches discriminate archaea and other non-cultivable microorganisms, which
contribute more than 80% to the total number of species (Ouwerkerk and Klieve, 2001).
Direct microscopic approaches often severely underestimate fungi (Joergensen and
Wichern, 2008). The extraction of DNA followed by analysis of its composition
provides important information on the microbial community composition of faeces (van
Vliet et al., 2007; Sekhavati et al., 2009). However, DNA data usually provides no
information on the biomass, due to losses during extraction, due to unknown or highly
variable concentrations within different microbial species (Leckie et al., 2004;
Joergensen and Emmerling, 2006) or due to its occurrence in dead microorganisms
(Pisz et al., 2007; Bae and Wuertz, 2009). ATP would be a good candidate for the
differentiation of living and dead microorganisms in faecal samples (Wolstrup and
Jensen, 2008). However, in the anaerobic or micro-aerobic environment of faeces
samples, the AEC and thus the ATP concentration within the microbial biomass is more
variable than in soil (Jenkinson, 1988; Dyckmans et al., 2006).
The chloroform fumigation extraction (CFE) method has been applied in a wide
range of substrates over the past two decades and is able to estimate the biomass of all
microorganisms, surrounded by an intact cell membrane, i.e. bacteria, fungi, and
archaea. The integrity of cell membranes is destroyed by chloroform, and especially the
cytoplasmic part of the microbial constituents is further degraded by enzymatic
autolysis and transformed into extractable components. This enables the CFE method to
differentiate accurately between living and dead microbial tissue. The CFE method has
also been successfully used to determine microbial biomass in a variety of substrates
high in organic matter, such as litter layers (Joergensen and Scheu, 1999), peat (Brake et
al., 1999), compost samples (Gattinger et al., 2004), and also pig manure (Aira et al.,
2006), but not cattle manure. The strong comminution of the diet by cattle, followed by
fermentation processes in the rumen in combination with a high water content and a
highly dispersible crude protein fraction results in a pasty structure of cattle faeces,
which may cause severe problems for the CFE method.
The first objective of the present study was to test the applicability of the CFE
method for determining microbial biomass in cattle faeces. Thus, different extractants
were tested on faeces to obtain stable extracts and reproducible microbial biomass C
and N values. The second objective was to determine ergosterol as an index for fungal