Anaerobic Populations Research Articles

Variation in Energy Available to Populations of Subsurface Anaerobes in Response to Geological Carbon Storage

Microorganisms can strongly influence the chemical and physical properties of the subsurface. Changes in microbial activity caused by geological CO(2) storage, therefore, have the potential to influence the capacity, injectivity, and integrity of CO(2) storage reservoirs and ultimately the environmental impact of CO(2) injection. This analysis uses free energy calculations to examine variation in energy available to Fe(III) and SO(4)(2-) reducers and methanogens because of changes in the bulk composition of brine and shallow groundwater following subsurface CO(2) injection. Calculations were performed using data from two field experiments, the Frio Formation experiment and an experiment at the Zero Emission Research and Technology test site. Energy available for Fe(III) reduction increased significantly during CO(2) injection in both experiments, largely because of a decrease in pH from near-neutral levels to just below 6. Energy available to SO(4)(2-) reducers and methanogens varied little. These changes can lead to a greater rate of microbial Fe(III) reduction following subsurface CO(2) injection in reservoirs where Fe(III) oxides or oxyhydroxides are available and the rate of Fe(III) reduction is limited by energy available prior to injection.

In-house windrowing of a commercial broiler farm during the summer months and its effect on litter composition

SUMMARY In-house windrowing of broiler litter between flocks has been suggested as an effective method to reduce pathogens and improve the quality of the house environment in which new chicks are placed. In this study, different methods of in-house windrowing were tested in commercial broiler houses during the summer months of July and August to determine the effects each would have on litter composition. Three windrowing treatments and 1 control treatment were applied to 2 commercial broiler houses containing litter that was used over multiple flock grow-outs. Windrowing treatments included windrows with either the addition of water, turning of the windrow, or leaving the windrow undisturbed for 8 d. Each commercial broiler house was divided into 16 plots, each 6.1 × 6.1 m, with each treatment being applied to 4 plots per house. Litter from each plot was analyzed for moisture content, pH, NH3, and microbes over a 17-d period, with d 17 representing 7 d after chick placement. Moisture content was significantly lower in the control treatment compared with all other treatments. Litter in all treatments had decreased pH from d 0 to 17. Ammonia decreased from d 10 to 17. Litter windrowed without the addition of water or turning resulted in a decrease in anaerobic bacteria on d 17 compared with the control treatment. In conclusion, in-house windrowing may be beneficial for newly placed chicks by changing the litter environment and reducing the population of pathogenic anaerobes, which can be detrimental to bird health and production.

Consumption of Agaricus bisporus mushroom affects the performance, intestinal microbiota composition and morphology, and antioxidant status of turkey poults

We investigated the consequences of consumption of Agaricus bisporus mushroom on turkey poults fed ad libitum a basal diet supplemented with the dried mushroom at levels of 0, 10 or 20 g/kg feed (treatments: CON, M10, M20) to 10 wk of age. Body weight, feed intake and feed conversion ratio were monitored weekly. The populations of total aerobes and anaerobes, Lactobacillus spp., Bifidobacterium spp., Escherichia coli, Bacteroides spp. and Enterococci spp. were enumerated in ileum and caecum. Morphological examinations of the intestine were carried out on segments from small intestine. To evaluate the antioxidant status of turkeys, refrigerated liver, breast and thigh muscle were assayed for levels of glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), glutathione reductase (GR), reduced glutathione (GSH), and malondialdehyde (MDA). Dietary mushroom inclusion improved (P≤0.05) both growth performance and feed efficiency. In the ileum, lactobacilli counts were higher (P≤0.05) in supplemented turkeys at both inclusion levels compared to controls; E. coli counts were lower (P≤0.05) in M20 compared to M10 group. In the caecum, the lactobacilli and bifidobacteria counts were higher (P≤0.05) in M20 compared to controls. The ratio of E. coli to lactobacilli was lower (P≤0.05) in M20 birds compared to controls in both ileum and caecum. Morphometrical examinations revealed an increase in villus height in all three intestinal sections of supplemented birds. Mushroom supplementation elevated (P≤0.05) GSH-Px, GR, and GST activity and reduced (P≤0.05) MDA production in tissues compared to controls. Dietary mushroom inclusion beneficially affected performance and exerted changes in intestinal microbial communities, intestinal integrity and antioxidant protective activity, consistent with the improvements in performance.

Evaluation of the effect of fat content of sunflower meal on rumen fungi growth and population by direct (quantitative competitive polymerase chain reaction) and indirect (dry matter and neutral detergent fibre disappearance) methods

The major aim of this study was to evaluate the effect of fat content of sunflower meal (150 and 30 g fat /kg dry matter, high and low fat, respectively) on population, growth and activity of rumen anaerobic fungi by using direct (quantitative competitive polymerase chain reaction, QC-PCR) and indirect (dry matter (DM) and neutral detergent fibre (NDF) disappearance in rumen fungi media culture for 12 days) methods. The results of QC-PCR showed that rumen anaerobic fungi population in the medium containing high fat sunflower meal was greater as compared to low fat sunflower meal (+0.14 vs. +0.10) (P 0.05). High fat of sunflower meal caused increase in natural detergent fibre disappearance 12 days after culturing as compared to low fat sunflower meal (145.2 vs 139.2 mg/g dry matter, respectively) (P<0.05). Therefore, it appears that fat content of sunflower meal does not negatively affect the population, growth and activity of rumen fungi. Key words: Fat, sunflower meal, rumen fungi, quantitative competitive polymerase chain reaction, disappearance.

Two respiratory enzyme systems in Campylobacter jejuni NCTC 11168 contribute to growth on l‐lactate

Campylobacter jejuni, a major food-borne intestinal pathogen, preferentially utilizes a few specific amino acids and some organic acids such as pyruvate and L- and D-lactate as carbon sources, which may be important for growth in the avian and mammalian gut. Here, we identify the enzymatic basis for C. jejuni growth on L-lactate. Despite the presence of an annotated gene for a fermentative lactate dehydrogenase (cj1167), no evidence for lactate excretion could be obtained in C. jejuni NCTC 11168, and inactivation of the cj1167 gene did not affect growth on lactate as carbon source. Instead, L-lactate utilization in C. jejuni NCTC 11168 was found to proceed via two novel NAD-independent L-LDHs; a non-flavin iron-sulfur containing three subunit membrane-associated enzyme (Cj0075c-73c), and a flavin and iron-sulfur containing membrane-associated oxidoreductase (Cj1585c). Both enzymes contribute to growth on L-lactate, as single mutants in each system grew as well as wild-type on this substrate, while a cj0075c cj1585c double mutant showed no L-lactate oxidase activity and did not utilize or grow on L-lactate; D-lactate-dependent growth was unaffected. Orthologues of Cj0075c-73c (LldEFG/LutABC) and Cj1585c (Dld-II) were recently shown to represent two novel families of L- and D-lactate oxidases; this is the first report of a bacterium where both enzymes are involved in L-lactate utilization only. The cj0075c-73c genes are located directly downstream of a putative lactate transporter gene (cj0076c, lctP), which was also shown to be specific for L-lactate. The avian and mammalian gut environment contains dense populations of obligate anaerobes that excrete lactate; our data indicate that C. jejuni is well equipped to use L- and D-lactate as both electron-donor and carbon source.

The Sulfate-Rich and Extreme Saline Sediment of the Ephemeral Tirez Lagoon: A Biotope for Acetoclastic Sulfate-Reducing Bacteria and Hydrogenotrophic Methanogenic Archaea

Our goal was to examine the composition of methanogenic archaea (MA) and sulfate-reducing (SRP) and sulfur-oxidizing (SOP) prokaryotes in the extreme athalassohaline and particularly sulfate-rich sediment of Tirez Lagoon (Spain). Thus, adenosine-5′-phosphosulfate (APS) reductase α (aprA) and methyl coenzyme M reductase α (mcrA) gene markers were amplified given that both enzymes are specific for SRP, SOP, and MA, respectively. Anaerobic populations sampled at different depths in flooded and dry seasons from the anoxic sediment were compared qualitatively via denaturing gradient gel electrophoresis (DGGE) fingerprint analysis. Phylogenetic analyses allowed the detection of SRP belonging to Desulfobacteraceae, Desulfohalobiaceae, and Peptococcaceae in ∂-proteobacteria and Firmicutes and SOP belonging to Chromatiales/Thiotrichales clade and Ectothiorhodospiraceae in γ-proteobacteria as well as MA belonging to methylotrophic species in Methanosarcinaceae and one hydrogenotrophic species in Methanomicrobiaceae. We also estimated amino acid composition, GC content, and preferential codon usage for the AprA and McrA sequences from halophiles, nonhalophiles, and Tirez phylotypes. Even though our results cannot be currently conclusive regarding the halotolerant strategies carried out by Tirez phylotypes, we discuss the possibility of a plausible “salt-in” signal in SRP and SOP as well as of a speculative complementary haloadaptation between salt-in and salt-out strategies in MA.

Real-Time PCR Assays for Monitoring Anaerobic Fungal Biomass and Population Size in the Rumen

The relationship between copy numbers of internal transcribed spacer 1 (ITS1) and biomass or zoospore count of anaerobic fungi was studied to develop a quantitative real-time PCR-based monitoring method for fungal biomass or population in the rumen. Nine fungal strains were used to determine the relationship between ITS1 copy number and fungal biomass. Rumen fluid from three sheep and a cow were used to determine the relationship between ITS1 copy number and fungal population. ITS1 copy number was determined by real-time PCR with a specific primer set for anaerobic fungi. Freeze-dried fungal cells were weighed for fungal biomass. Zoospore counts were determined by the roll-tube method. A positive correlation was observed between both ITS1 copy number and dry weight and ITS1 copy number and zoospore counts, suggesting that the use of ITS1 copy numbers is effective for estimating fungal biomass and population density. On the basis of ITS1 copy numbers, fluctuations in the fungal population in sheep rumen showed that although the values varied among individual animals, the fungal population tended to decrease after feeding. In the present study, a culture-independent method was established that will provide a powerful tool for understanding the ecology of anaerobic fungi in the rumen.

Analysis of Biostimulated Microbial Communities from Two Field Experiments Reveals Temporal and Spatial Differences in Proteome Profiles

Stimulated by an acetate-amendment field experiment conducted in 2007, anaerobic microbial populations in the aquifer at the Rifle Integrated Field Research Challenge site in Colorado reduced mobile U(VI) to insoluble U(IV). During this experiment, planktonic biomass was sampled at various time points to quantitatively evaluate proteomes. In 2008, an acetate-amended field experiment was again conducted in a similar manner to the 2007 experiment. As there was no comprehensive metagenome sequence available for use in proteomics analysis, we systematically evaluated 12 different organism genome sequences to generate sets of aggregate genomes, or "pseudo-metagenomes", for supplying relative quantitative peptide and protein identifications. Proteomics results support previous observations of the dominance of Geobacteraceae during biostimulation using acetate as sole electron donor, and revealed a shift from an early stage of iron reduction to a late stage of iron reduction. Additionally, a shift from iron reduction to sulfate reduction was indicated by changes in the contribution of proteome information contributed by different organism genome sequences within the aggregate set. In addition, the comparison of proteome measurements made between the 2007 field experiment and 2008 field experiment revealed differences in proteome profiles. These differences may be the result of alterations in abundance and population structure within the planktonic biomass samples collected for analysis.

Relation between the activity of anaerobic microbial populations in oil sands tailings ponds and the sedimentation of tailings

Oil sands tailings ponds contain a variety of anaerobic microbes, including methanogens, sulfate- and nitrate-reducing bacteria. Methanogenic activity in samples from a tailings pond and its input streams was higher with trimethylamine (TMA) than with acetate. Methanogens closely affiliated to Methanomethylovorans hollandica were found in the TMA enrichments. Tailings sedimentation increased with methanogenic activity, irrespective whether TMA or acetate was used to stimulate methanogenesis. Increased sedimentation of autoclaved tailings was observed with added pure cultures under methanogenic, as well as under nitrate-reducing conditions, but not under sulfate-reducing conditions. Scanning electron microscopy and energy-dispersive X-ray spectroscopy indicated the presence of microbes and of extracellular polymeric substances in tailings particle aggregates, especially under methanogenic and nitrate-reducing conditions. Hence different classes of microorganisms growing in tailings ponds contribute to increased tailings aggregation and sedimentation. Because addition of nitrate is known to lower methane production by methanogenic consortia, these observations offer the potential to combine lower methane emissions with improved microbially-induced tailings sedimentation.

Colour Removal and the Effect of Reactive Dyes on Growth Substrate Utilization by an Unacclimated Ethanol-Enriched Anaerobic Culture

Abstract The cultures from an ethanol-enriched anaerobic master culture reactor were dosed with 0, 50, 100, 200, 400, 800, and 1600 mg/L of a mixture of three Procion reactive dyes: Red MX-8B, Red MX-5B, and Orange MX-2R, in order to study the colour removal and the inhibitory effect of dyes on the methanogenic degradation of ethanol that was added as a cosubstrate. The reduction in colour intensity, measured at 514 nm, was found to be similar to a trial using an inactivated culture. These results indicate that removal of colour by the unacclimated anaerobic population is due to adsorption of the dyes on the biomass. The dyes caused a competitive inhibition at doses above 200 mg/L on utilization of ethanol by the anaerobic bacteria.

Characterization of an anaerobic population digesting a model substrate for maize in the presence of trace metals

The influence of a defined trace metal solution and additionally Ni2+ on anaerobic digestion of biomass was investigated. A novel synthetic model substrate was designed consisting of cellulose, starch and urea as carbon and nitrogen source in a ratio mimicking the basic composition of maize silage. Two independent batch fermentations were carried out over 21d with the synthetic model substrate in the presence of the trace metal solution. Particularly an increase in nickel concentrations (17 and 34μM) enhanced methane formation by up to 20%. This increased activity was also corroborated by fluorescence microscopy measurements based on cofactor F420. The eubacterial and methanogenic population was characterized with the single strand conformational polymorphism analysis and the amplified 16S rDNA restriction analysis of 16S rRNA genes amplified by different primer systems. Nearly the half of the analyzed bacteria were identified as Firmicutes while 70% in this phylum belonged to the class of Clostridiales and 30% to the class of Bacilli. Bacteroides and uncultured bacteria represented each a quarter of the analyzed community. Methanogenic archaea were investigated with ARDRA, too. The hydrogenotrophic Methanoculleus sp. was the dominant genus which is commonly described for maize digestion thus confirming the value of the model substrate.

Periodontal parameters and BANA test in patients with chronic renal failure undergoing hemodialysis

ObjectivesThe aim of this study was to analyze the periodontal parameters of patients with chronic renal failure.Material and MethodsThe periodontal status of 16 Brazilian patients aged 29 to 53 (41.7±7.2) years with chronic renal failure (CRF) and another matched group of 14 healthy controls with periodontitis was assessed clinically and microbiologically. Probing pocket depth (PPD), gingival recession (GR), dental plaque index (PLI), gingival index (GI), and dental calculus index (CI) were the clinical parameters recorded for the entire dentition (at least 19 teeth), while the anaerobic periodontopathogen colonization in four sites with the highest PPD was evaluated using the BANA test (“PerioScan”; Oral B).ResultsThe results for the CRF group and control group, respectively were: PPD: 1.77±0.32 and 2.65±0.53; GR: 0.58±0.56 and 0.51±0.36; PLI: 1.64±0.56 and 1.24±0.67; GI: 0.64±0.42 and 0.93±0.50; CI: 1.17±0.54 and 0.87±0.52. Comparison between groups using the "t" test revealed a significantly increased PPD (p<0.001) in the control group. Comparison of the other clincial parameters by the Mann-Whitney test showed differences only for PLI, which was significantly higher (p<0.05) in the CRF group. Spearman's test applied to each group showed a positive correlation among all clinical parameters, except for GR (p<0.05). None of the groups showed any correlation between GR and GI, while a significant negative correlation between GR and PPD was observed for the CRF group. The percentage of BANA-positive sites was 35.9% for the CRF group and 35.7% for the control group. The BANA test correlated positively with PPD only in the control group and with GR only in the CRF group.ConclusionsIn spite of a higher PLI and dense anaerobic microbial population even in shallow PPD, patients with CRF exhibited better periodontal conditions than periodontitis patients, which is an evidence of altered response to local irritants.

Effect of mannan oligosaccharides on rabbit performance, digestibility and rectal bacterial anaerobic populations during an episode of epizootic rabbit enteropathy.

The effect of three levels of mannan oligosaccharides (MOS at 0.5, 1.0 and 1.5 g/kg diet) compared to dietary antibiotic supplementation (ANT, colistin sulphate 144, tylosin 100 and oxytetracycline 1000 ppm) and to a diet without MOS or medication (control group) were studied on 5 groups of rabbits (n=342 cages/group and 2 rabbits/cage) from weaning (35 d) to 60 d of age in a farm with an anamnestic history of Epizootic Rabbit Enteropathy (ERE). Mortality rate was recorded daily. Thirty two cages per group were randomly chosen to record growth performance and rectal anaerobic microbial population. Live weight and feed intake were recorded weekly. At 49 d of age rectal swabs were collected with cotton swabs from 8 rabbits per group and the anaerobic microbial population was counted. Feed digestibility was determined per cage using acid insoluble ash as an internal marker, collecting faeces from 57 to 59 d of age. An episode of ERE occurred during the second and the third week of the trial. The control group showed a high mortality rate during the fattening period (78%) and was not considered for further measurements. Total mortality rate was higher (P

Diversity of anaerobic microbes in spacecraft assembly clean rooms.

Although the cultivable and noncultivable microbial diversity of spacecraft assembly clean rooms has been previously documented using conventional and state-of-the-art molecular techniques, the occurrence of obligate anaerobes within these clean rooms is still uncertain. Therefore, anaerobic bacterial communities of three clean-room facilities were analyzed during assembly of the Mars Science Laboratory rover. Anaerobic bacteria were cultured on several media, and DNA was extracted from suitable anaerobic enrichments and examined with conventional 16S rRNA gene clone library, as well as high-density phylogenetic 16S rRNA gene microarray (PhyloChip) technologies. The culture-dependent analyses predominantly showed the presence of clostridial and propionibacterial strains. The 16S rRNA gene sequences retrieved from clone libraries revealed distinct microbial populations associated with each clean-room facility, clustered exclusively within gram-positive organisms. PhyloChip analysis detected a greater microbial diversity, spanning many phyla of bacteria, and provided a deeper insight into the microbial community structure of the clean-room facilities. This study presents an integrated approach for assessing the anaerobic microbial population within clean-room facilities, using both molecular and cultivation-based analyses. The results reveal that highly diverse anaerobic bacterial populations persist in the clean rooms even after the imposition of rigorous maintenance programs and will pose a challenge to planetary protection implementation activities.

Influence of dietary mushroom Agaricus bisporus on intestinal morphology and microflora composition in broiler chickens

In this study, we evaluated the intestinal morphology and bacteria populations in broiler chickens fed for six weeks diets that contained different amount of the mushroom Agaricus bisporus. Ninety day-old female chicks were randomly divided into three dietary treatments, each with three replicates kept in floor pens and fed a basal diet supplemented with the dried mushroom at levels of 0, 10 or 20 g/kg fresh feed. Feed and water were offered to birds ad libitum. The morphological examinations of the intestine were carried out on 1-cm long excised segments from duodenum, jejunum and ileum. The populations of total aerobes, total anaerobes, Lactobacilli spp., Bifidobacteria spp., Escherichia coli, Bacteroides spp. and Enterococci were enumerated in ileum and caecum by conventional microbiological techniques using selective agar media. The results of the study showed that dietary mushroom supplementation did not significantly affect intestinal morphology at either level of inclusion. Morphometrical parameters of depth of duodenum, jejunum and ileum crypt and height of villi revealed no differences amongst dietary treatments. In the ileum, Lactobacilli spp. were higher in birds supplemented at the level of 20 g/kg compared to controls; however, other measurements of bacteria loads were similar amongst the three dietary treatments. In the caecum, Lactobacilli spp. and Bifidobacteria spp. loads were higher in birds supplemented at either level of inclusion compared to control birds, although these did not differ between the two levels of supplementation. In conclusion, dietary mushroom supplementation may beneficially affect intestinal health of broiler chickens.

Development of microorganisms in the chernozem under aerobic and anaerobic conditions

A microbial succession was studied under aerobic and anaerobic conditions by means of experiments with microcosms in different horizons of a chernozem. It was revealed that, under aerobic conditions, all the microorganisms grow irrespective of the soil horizon; fungi and bacteria grow at the first succession stages, and actinomycetes grow at the last stages. It was shown that, in the case of a simulated anaerobiosis commonly used to study anaerobic populations of bacteria, the mycelium of micromycetes grows in the upper part of the chernozem’s A horizon. Under anaerobic conditions, the peak of the mycelium development is shifted from the 3rd to 7th days (typical for aerobic conditions) to the 7th to 15th days of incubation. The level of mycelium length’s stabilization under aerobic and anaerobic conditions also differs: it is higher or lower than the initial one, respectively. Under anaerobic conditions, the growth of fungal mycelium, bacteria, and actinomycetes in the lower part of the A horizon and in the B horizon is extremely weak. There was not any observed growth of actinomycetes in all the chernozem’s horizons under anaerobic conditions.

Identification of facultative anaerobic bacteria isolated from the intestine of the minke whale Balaenoptera acutorostrata by 16S rRNA sequencing analysis

There are few reports in the literature about the isolation of bacteria from whale intestine. In this report, we counted colony-forming units in the feces obtained from three female common minke whales (Balaenoptera acutorostrata). The number of colony-forming units ranged from (2.2 ± 0.4) × 105 to (8.9 ± 2.0) × 108 per gram (wet weight) of excrement. 16S rRNA gene sequences of 141 isolates were determined. These strains were identified as Enterococcus faecalis, Enterococcus sp., Enterobacter cloacae, Enterobacter sp., Escherichia coli, Edwardsiella ictaluri or Clostridium sp. The data suggested that the facultative anaerobic population of the intestinal bacterial flora of the minke whale was similar to that of ground mammals.

Propagation of anaerobic bacteria within an aerobic multi-species chronic wound biofilm model

Most chronic wound biofilms have been shown to have significant populations of anaerobes. In order to better screen antimicrobial and antibiofilm therapeutics, we evaluated the ability of key anaerobes to incorporate and propagate within our aerobic chronic wound biofilm. We had previously developed a rapid model to simulate polymicrobial chronic wound biofilms. This model incorporated meticillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE) and Pseudomonas aeruginosa. The model was used along with a variety of anaerobes to determine whether this biofilm model would support propagation of anaerobes similar to that we have identified in chronic wounds. Using our previously defined Lubbock Chronic Wound Biofilm (LCWB) model combined with quantitative PCR, anaerobic bacteria were shown to proliferate through integration into the biofilm under aerobic conditions. Using electron microscopy we show close association between aerobes and anaerobes within the biofilm suggesting a synergistic relationship. We have expanded the utility of the LCBW to show the ability of clinically significant anaerobic bacteria to thrive in aerobic conditions. The expansion of this model can further simulate the functional characteristics of chronic wound pathogenic biofilms and the species that dwell within them allowing improved ability to evaluate therapeutics that target anaerobes.

Behaviour of different anaerobic populations on the biodegradation of textile chemicals

The anaerobic biodegradation of textile chemicals was evaluated with inocula grown under mesophilic (37 ± 2° C) or thermophilic (55 ± 2° C) conditions, on glucose (glucose-grown) or acetate (acetate-grown) as sole carbon sources. Wool dyebath chemicals (acetic acid, a liposomal surfactant, a synthetic amphoteric surfactant), single or as binary acetate–surfactant mixtures, were used as test carbon sources, in the presence or absence of Acid Orange 7 as model dye. First, the two mesophilic inocula (glucose- or acetate-grown) were compared relatively to lag-phase durations, specific biogas production rates, biogas yields and overall COD removal yields. In some runs, sulphide and/or the model dye were included, to test for inhibition effects. Then, the two glucose-grown inocula (mesophilic and thermophilic) were assessed in batch biodegradation tests with the same carbon feeds. The kinetics for substrate-COD and dye colour removal were described and quantified using a pseudo-first order model. The presence of dye had no effect on performance parameters for all substrates tested. Acetoclastic methanogens seemingly played an important role in biogas production from the liposomal additive, but less so from the synthetic surfactant. The association of acetate and surfactants apparently introduced mutual inhibitory effects on the rates of biogas production, substrate uptake and dye decolourisation.

Development and use of quantitative competitive PCR assays for relative quantifying rumen anaerobic fungal populations in both in vitro and in vivo systems

This paper describes the use of a quantitative competitive polymerase chain reaction (QC-PCR) assay; using PCR primers to the rRNA locus of rumen fungi and a standard-control DNA including design and validation. In order to test the efficiency of this method for quantifying anaerobic rumen fungi, it has been attempted to evaluate this method in in vitro conditions by comparing with an assay based on measuring cell wall chitin. The changes in fungal growth have been studied when they are grown in in vitro on either untreated (US) or sodium hydroxide treated wheat straw (TS). Results showed that rumen fungi growth was significantly higher in treated samples compared with untreated during the 12 d incubation ( P < 0.05) and plotting the chitin assay's results against the competitive PCR's showed high positive correlation ( R 2 ≥ 0.87). The low mean values of the coefficients of variance in repeatability in the QC-PCR method against the chitin assay demonstrated more reliability of this new approach. And finally, the efficiency of this method was investigated in in vivo conditions. Samples of rumen fluid were collected from four fistulated Holstein steers which were fed four different diets (basal diet, high starch, high sucrose and starch plus sucrose) in rotation. The results of QC-PCR showed that addition of these non-structural carbohydrates to the basal diets caused a significant decrease in rumen anaerobic fungi biomass. The QC-PCR method appears to be a reliable and can be used for rumen samples.