K. Fliegerová

Classical and Molecular Approaches as a Powerful Tool for the Characterization of Rumen Polycentric Fungi

RibosomalITS1 andITS2 fragments from 8 isolates of polycentric rumen anaerobic fungi were PCR-amplified and sequenced; the sequences obtained were aligned with published data and phylogenetic analyses were performed. Analysis of theITS1 fragment clearly differentiated between the two polycentric generaOrpinomyces andAneromyces and this classification is supported by morphological observation. A multi-order phylogram based onITS2 sequences proved that anaerobic rumen fungi are separated from aerobic chytrids, which form a well-supported monophylum with the highest possible bootstrap proportion values of 100%. Sequence analysis of ITS regions is a powerful tool for classification of anaerobic fungi but morphological description of strains is still necessary because some genera of rumen fungi display a high genetic heterogeneity.

Differentiation of anaerobic polycentric fungi by rDNA PCR-RFLP.

The suitability of restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA cluster for discriminating two genera of anaerobic polycentric fungi,Orpinomyces andAnaeromyces, was determined. Three PCR-amplified DNA fragments — nuclear small subunit (SSU; 18S rDNA), the nuclear large subunit (LSU; 28S rDNA) and internal transcribed spacer (ITS) — were restricted with endonucleasesAluI,DraI,HinfI andMboI. Although the SSU DNA fragment could be restricted successfully by all four enzymes, no differences were observed between restriction patterns ofOrpinomyces andAnaeromyces. The most polymorphic restriction pattern betweenOrpinomyces andAnaeromyces resulted from cleavage of LSU rDNA fragments cut byAluI andHinfI and ITS fragment cut byDraI andHinfI. Genus-specific RFLP patterns were determined forOrpinomyces andAnaeromyces genera; the results showed that the PCR-RFLP analysis of rDNA offers an easy and rapid tool for differentiation of two polycentric genera of anaerobic fungi, which could be hardly separated on the basis of morphology.

Anaerobic Fungi and Their Potential for Biogas Production

Plant biomass is the largest reservoir of environmentally friendly renewable energy on earth. However, the complex and recalcitrant structure of these lignocellulose-rich substrates is a severe limitation for biogas production. Microbial pro-ventricular anaerobic digestion of ruminants can serve as a model for improvement of converting lignocellulosic biomass into energy. Anaerobic fungi are key players in the digestive system of various animals, they produce a plethora of plant carbohydrate hydrolysing enzymes. Combined with the invasive growth of their rhizoid system their contribution to cell wall polysaccharide decomposition may greatly exceed that of bacteria. The cellulolytic arsenal of anaerobic fungi consists of both secreted enzymes, as well as extracellular multi-enzyme complexes called cellulosomes. These complexes are extremely active, can degrade both amorphous and crystalline cellulose and are probably the main reason of cellulolytic efficiency of anaerobic fungi. The synergistic use of mechanical and enzymatic degradation makes anaerobic fungi promising candidates to improve biogas production from recalcitrant biomass. This chapter presents an overview about their biology and their potential for implementation in the biogas process.

Effect of soluble maillard reaction products on rumen microorganisms

After incubation of Maillard reaction polymers (MRPs) with rumen fluid from wethers neither volatile fatty acids nor lactate were produced. Soluble polymeric products of the Maillard reaction were nonmetabolizable by a mixed culture of rumen microorganisms. MRPs added at 0.5 and 2 g/L inhibited the growth of seven ruminal Gram-negative bacteria by 20 and 30%, respectively. In four strains of Gram-positive bacteria, MRPs lowered the cell concentration by 11% (0.5 g/L) and 25% (2 g/L). The rumen fungusOrpinomyces jojonii also did not metabolize soluble MRPs.

Multiresistant strains ofEscherichia coli isolated from the rumen of young calves

Five multiresistant strains ofEscherichia coli were isolated from the rumen fluid of young calves. Resistance to tetracycline and ampicillin was found to be associated with the transfer of a 6.4 kbp plasmid present in two investigated strains.

The effect of maize silage as co-substrate for swine manure on the bacterial community structure in biogas plants

The qualitative and quantitative changes in the bacterial community composition in two mesophilic, commercially used biogas plants were monitored by denaturing gradient gel electrophoresis (DGGE) and real-time PCR. The main objective was to evaluate the influence of the co-substrate maize silage on total bacteria and some selected bacterial groups by comparing full-scale reactors fed solely with pig manure or additionally with maize silage. DGGE fingerprints reflected shifts in the bacterial community structure associated with maize silage as co-substrate and the real-time PCR results showed clear changes in the quantitative composition of the bacterial consortia of each fermenter. A clear dominance of Clostridia in all surveyed fermenters and considerably lower abundance of Bacteroidetes in the biogas plant fed with maize silage was shown.

Plasmids ofSelenomonas ruminantium and development of host-vector system

A high frequency of plasmids was detected in the rumen bacteriumSelenomonas ruminantium. Plasmids 0.9–20 kb in size were detected in more than 50% tested strains. Densitometric analysis indicated that plasmid DNA could represents more than 25% of total cellular DNA. Up to six plasmids were detected in strainS. ruminantium 18. Two smallest cryptic plasmids pSRD181 and pSRD182 from this strain were cloned intoEscherichia coli vector pBluescriptSK+ and partially characterized. The plasmid pSRD181 is 1.4 kb and pSRD182 is 2.0 kb. While computer analysis of pSRD181 sequence data showed high homology with replication protein ofStaphylococcus aureus plasmids, the pSRD182 sequence showed no significant homology in GenBank data. StrainS. ruminantium 28 was successfully transformed with pJW1 derived plasmid pJ1B1 using ampicillin resistance gene as marker. This is the first report on transformation of selenomonads with foreign DNA.

Laying performance, blood profiles, nutrient digestibility and inner organs traits of hens fed an insect meal from Hermetia illucens larvae

Given probable the increment in the nutritional needs of both humans and animals, animal production will have increased dramatically by 2050. Insect meals could be an alternative protein source for livestock, and they would also be able to reduce the environmental problems related to intensive animal production system. The aim of this study was to evaluate productive performance, blood analysis, nutrient digestibility, and changes in the internal organs of laying hens fed Hermetia illucens larvae meal (HI) at two different levels in substitution (25 or 50%) of soybean meal (SBM). A total of 162 Hy-line Brown hens (sixteen weeks old) were equally divided into three experimental groups and fed isoprotein and isoenergetic diets. Egg weight, feed intake, and feed conversion rate were not affected by the soybean meal substitution at both inclusion levels of insect meal. Egg mass was positively affected by the insect meal diets, as was the lay percentage, although only at the lowest inclusion level. Dry matter, organic matter, and crude protein digestibility coefficients were lower for the HI50 diet, probably due to the negative effect of chitin. A reduction in serum cholesterol and triglycerides was observed in both insect-meal fed groups, while serum globulin level increased only at the highest level of insect meal inclusion, and, consequently, the albumin to globulin ratio decreased. Overall, a protein replacement of 25% with an insect meal from Hermetia illucens larvae in the diet of laying hens seems to be more suitable and closer to the optimal level.

A comparison of methanogens of different regions of the equine hindgut

The diversity of the methanogenic archaea associated with the six segments of the horse and donkey hindgut (caecum, right ventral colon, left ventral colon, left dorsal colon, right dorsal colon, and rectum) was analyzed using 16S rDNA gene clone library. A total of 641 positive clones, 321 from the horse and 320 from the donkey hindgut, were examined by the RFLP, revealing 9 different ribotypes, 8 in the horse and 5 in the donkey hindgut. In both the animals Methanobacteriales (Methanobrevibacter-like sequences) and Methanomicrobiales (Methanocorpusculum-like sequences) were detected as the dominant orders followed by the uncultured Methanomassiliicoccales. The composition of the equine archaeal community was found to be dependent on the gut region. In both the two animals no Methanobrevibacter-like clones were detected in the caeca, which were instead inhabited by the Methanocorpusculum-like archeons. The Methanosarcinales were found only in distal regions of the horse hindgut.