Particle-associated Microorganisms Research Articles

Trace Mineral Source Influences Trace Mineral Solubility in Water and Mineral Binding Strength to Ruminal Digesta.

Two experiments were conducted to examine the impact of trace mineral (TM) source on in vitro and in vivo solubility characteristics. Experiment 1: Hydroxy TM (HTM) and sulfate TM (STM) sources of Cu, Mn, and Zn were incubated separately in water for 24 h. Immediately after mixing, initial pH of each solution was greater (P < 0.03) for HTM compared to STM for all elements. Final pH tended to be greater for Cu (P = 0.09) and Zn (P = 0.07) from HTM compared to STM. Water solubility of Cu, Mn, and Zn from STM was greater (P < 0.01) than HTM sources. Experiment 2: Eight steers fitted with rumen cannula were blocked by body weight and randomly assigned to treatments. Treatments consisted of 10 mg Cu, 40 mg Mn, and 60 mg Zn/kg DM from either STM or HTM sources. Steers were individually fed a cracked corn-corn silage-based diet. Treatments were top-dressed daily. Rumen contents were collected at 0, 2, and 4 h post-feeding on d 1 and 14. On d 15, strained ruminal fluid and particle-associated microorganisms were obtained. Zinc was more tightly bound (P = 0.01) to the digesta in HTM-supplemented steers compared to STM on d 14. These data indicate that TM source influences pH and solubility of Cu, Mn, and Zn in water and may affect rumen soluble Cu concentrations and binding strength of Zn to solid digesta.

Trace mineral sources influence in vitro fermentation characteristics and trace mineral solubility.

Two experiments were conducted to determine: 1) the impact of strained rumen fluid (SRF) alone or SRF with particle-associated microorganisms (PAO) included and dilution on in vitro dry matter digestibility (DMD) and 2) the impact of trace mineral (TM) source on in vitro fermentation characteristics and TM solubility under simulated abomasal and intestinal conditions. In experiment 1, 3 cannulated steers were adapted to a diet formulated to meet the nutrient requirements for lactating dairy cows. Strained RF was obtained by straining rumen content through 2 layers of cheesecloth. Half of the remaining digesta was washed with McDougall's buffer and filtered through 2 layers of cheesecloth to obtain PAO. Both SRF and PAO were filtered again through 8 layers of cheesecloth. Strained RF was mixed with either McDougall's buffer (SRF) or PAO (SRF+ + PAO) at a ratio of 1:2 or 1:4 and incubated at 39 °C for 12h using the ground basal diet as the substrate. Digestibility of DM was greater in digestion tubes containing SRF and SRF + PAO at a 1:2 ratio. In experiment 2, 8 steers fitted with a ruminal cannula were blocked by body weight and assigned to 1 of the 2 treatment groups. Treatments consisted of 10mg Cu, 40mg Mn, and 60mg Zn/kg DM from either: 1) sulfate (STM) or 2) hydroxychloride (HTM) sources. Steers were housed in individual pens and fed the same diet as described in experiment 1. Dietary TM treatments were mixed with dried distiller grains and mixed in the diet, by hand, immediately after basal diet delivery. Dietary treatments were fed for 14 d. On day 15, SRF + PAO was collected from each steer (STM-RF and HTM-RF) and used in a series of in vitro crossover experiments. In vitro substrates (S) used were the ground diets consumed by the animals on each treatment (STM-S and HTM-S). Incubations containing HTM-S had greater (P < 0.01) total volatile fatty acid (VFA) concentration and propionic acid molar proportions, but lesser (P < 0.01) acetic acid molar proportions than STM-S. Rumen fluid from steers supplemented with HTM had a greater (P < 0.03) total VFA than STM-RF at 24h post incubation. After 12h post incubation, the molar proportion of propionic acid in HTM-RF was lesser (P = 0.04) than in STM-RF. After simulated abomasal digestion, soluble Mn concentration in HTM-S was greater (P < 0.01) than in STM-S. These data indicate that the source of TM can influence in vitro rumen fermentation characteristics and Mn solubility under simulated abomasal conditions.

Comparison of Community Composition and Function of Particle-associated Microorganisms and Free-living Microorganisms in the Mariana Trench

Comparison of Community Composition and Function of Particle-associated Microorganisms and Free-living Microorganisms in the Mariana Trench

Lacustrine plastisphere: Distinct succession and assembly processes of prokaryotic and eukaryotic communities and role of site, time, and polymer types

Microplastics as a carrier can promote microbial diffusion, potentially influencing the ecological functions of microbial communities in aquatic environments. However, our understanding of the assembly mechanism of microbial communities on different microplastic polymers in freshwater lakes during succession is still insufficient, especially for the eukaryotes. Here, the colonization time, site, and polymer types of microplastics were comprehensively considered to investigate the composition and assembly of prokaryotic and eukaryotic communities and their driving factors during the lacustrine plastisphere formation. Results showed that the particle-associated microorganisms in water were the main source of the plastisphere prokaryotes, while the free-living microorganisms in water mainly accounted for the plastisphere eukaryotes. The response of prokaryotic communities to different microplastic polymers was stronger than eukaryotic communities. The assembly of plastisphere prokaryotic communities was dominated by homogenizing processes (mainly homogenous selection), while the assembly of eukaryotic communities was dominated by differentiating processes (mainly dispersal limitation). Colonization time was an important factor affecting the composition of prokaryotic and eukaryotic communities during the formation of the plastisphere. The Chao1 richness of prokaryotic communities in the plastisphere increased with the increase of colonization time, whereas the opposite was true in eukaryotic communities. This differential response of species diversity and composition of prokaryotic and eukaryotic communities in the plastisphere during dynamic succession could lead to their distinct assembly processes. Overall, the results suggest that distinct assembly of microbial communities in the plastisphere may depend more on specific microbial sub-communities and colonization time than polymer types and colonization site.

Stochasticity-driven weekly fluctuations distinguished the temporal pattern of particle-associated microorganisms from its free-living counterparts in temperate coastal seawater

Microbial community dynamics directly determine their ecosystem functioning. Despite the well-known annual recurrence pattern, little is known how different lifestyles affect the temporal variation and how community assembly mechanisms change over different temporal scales. Here, through a high-resolution observation of size fractionated samples over 60 consecutive weeks, we investigate the distinction in weekly distribution pattern and assembly mechanism between free-living (FL) and particle-associated (PA) communities in highly dynamic coastal environments. A clear pattern of annual recurrence was observed, which was more pronounced in FL compared to PA, resulting in higher temporal specificity in the former samples. Both the two size fractions displayed significant temporal distance-decay patterns, yet the PA community showed a higher magnitude of community variation between adjacent weeks, likely caused by sudden, drastic and long-lived blooms of heterotrophic bacteria. Generally, determinism (environmental selection) had a greater effect on the community assembly than stochasticity (random birth, death, and dispersal events), with significant contributions from temperature and inorganic nutrients. However, a clear shift in the temporal assembly pattern was observed, transitioning from a prevalence of stochastic processes driving short-term (within a month) fluctuations to a dominance of deterministic processes over longer time intervals. Between adjacent weeks, stochasticity was more important in the community assembly of PA than FL. This study revealed that stochastic processes can lead to rapid, dramatic and irregular PA community fluctuations, indicating weak resistance and resilience to disturbances, which considering the role of PA microbes in carbon processing would significantly affect the coastal carbon cycle. Our results provided a new insight into the microbial community assembly mechanisms in the temporal dimension.

Metagenomic Insights Into the Microbial Assemblage Capable of Quorum Sensing and Quorum Quenching in Particulate Organic Matter in the Yellow Sea.

Quorum sensing (QS) is a density-dependent communicating mechanism that allows bacteria to regulate a wide range of biogeochemical important processes and could be inhibited by quorum quenching (QQ). Increasing researches have demonstrated that QS can affect the degradation of particulate organic matter (POM) in the photic zone. However, knowledge of the diversity and variation of microbial QS and QQ systems in sinking POM is scarce. Here, POM samples were collected from surface seawater (SW), bottom seawater (BW), and surficial sediment (SS) in the Yellow Sea of China. 16S rRNA gene amplicon and metagenome sequencing were performed to analyze the community structure of particle-associated microorganisms and distribution of QS genes [acylated homoserine lactone (AHL) synthesizing gene luxI and AHL sensing gene luxR] and QQ genes (genes encoding for AHL lactonase and acylase) in POM. Shifting community structures were observed at different sampling depths, with an increase of microbial abundance and diversity from SW to BW. Along with the variation of microbial communities, the abundances of luxI and luxR decreased slightly but were restored or even exceeded when POM arrived at SS. Comparatively, abundances of AHL lactonase and acylase remained constant during the transportation process from SW to BW but increased dramatically in SS. Correlation tests indicated that abundances of luxI and luxR were positively correlated with temperature, while those of AHL acylase were positively correlated with depth, SiO42–, PO43–, and NO3–, but negatively correlated with temperature and pH. According to phylogenetic analyses, the retrieved QS and QQ genes are more diverse and distinctive than ever experimentally identified. Besides, the vertical transmission of QS and QQ genes along with POM sinking was observed, which could be one of the key factors leading to the prevalence of QS and QQ genes in marine ecosystems. Overall, our results increase the current knowledge of QS and QQ metabolic pathways in marine environment and shed light on the intertwined interspecies relationships to better investigate their dynamics and ecological roles in POM cycling.

Tracing particulate matter and associated microorganisms in freshwaters

Sediment resuspension represents a key process in all natural aquatic systems, owing to its role in nutrient cycling and transport of potential contaminants. Although suspended solids are generally accepted as an important quality parameter, current monitoring programs cover quantitative aspects only. Established methodologies do not provide information on origin, fate, and risks associated with uncontrolled inputs of solids in waters. Here we discuss the analytical approaches to assess the occurrence and ecological relevance of resuspended particulate matter in freshwaters, with a focus on the dynamics of associated contaminants and microorganisms. Triggered by the identification of specific physical–chemical traits and community structure of particle-associated microorganisms, recent findings suggest that a quantitative determination of microorganisms can be reasonably used to trace the origin of particulate matter by means of nucleic acid-based assays in different aquatic systems.

Influence of ultrasound enhancement on chlorine dioxide consumption and disinfection by-products formation for secondary effluents disinfection

Chlorine dioxide (ClO2) has been promoted as an alternative disinfectant because of its high disinfection efficiency and less formation of organic disinfection by-products (DBPs). However, particle-associated microorganisms could be protected during the disinfection process, which decreases the disinfection efficiency or increases the required dosage. Besides, the formation of inorganic disinfection by-products is a significant concern in environment health. Ultrasound (US)-combined disinfection methods are becoming increasingly attractive because they are efficient and environmentally friendly. In this study, US was introduced as an enhancement method to identify its influence on ClO2 demand reduction and to minimize the production of potential DBPs for secondary effluents disinfection. Fecal coliform was used as an indicator, and DBPs, including trichloromethane (TCM), dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), chlorite (ClO2−), and chlorate (ClO3−), were analyzed to observe the potential DBPs formation. Results show that US pretreatment could reduce half of ClO2 dosage compared with ClO2 disinfection alone for the same disinfection efficiency, and that an input power density of 2.64kJ/L pretreatment with the 1.5mg/L ClO2 was enough to meet the discharge requirement in China (i.e., fecal coliform below 1000CFU/L for Class 1A) for secondary effluent disinfection, and the ClO2− concentration in the disinfection effluent was only 1.37mg/L at the same time. Furthermore, the different effects of US on the two processes (US as pretreatment and simultaneous US/ClO2 disinfection) were also analyzed, including deagglomerating, cell damage, and synergistic disinfection as well as degasing/sonolysis. It was proved that the production of TCM, DCAA, and TCAA was insignificantly influenced with the introduction of US, but US pretreatment did reduce the production of ClO2− and ClO3− effectually. In general, US pretreatment could be a better option for disinfection enhancement methods combined with ClO2 in terms of both disinfection efficiency and disinfection by-product formation.

Assessment of E. coli partitioning behavior via both culture-based and qPCR methods

Quantitative polymerase chain reaction (qPCR) offers a rapid, highly sensitive analytical alternative to the traditional culture-based techniques of microbial enumeration typically used in water quality monitoring. Before qPCR can be widely applied within surface water monitoring programs and stormwater assessment research, the relationships between microbial concentrations measured by qPCR and culture-based methods must be assessed across a range of water types. Previous studies investigating fecal indicator bacteria quantification using molecular and culture-based techniques have compared measures of total concentration, but have not examined particle-associated microorganisms, which may be more important from a transport perspective, particularly during the calibration of predictive water quality models for watershed management purposes. This study compared total, free-phase, and particle-associated Escherichia coli concentrations as determined by the Colilert defined substrate method and qPCR targeting the uidA gene in stream grab samples partitioned via a calibrated centrifugation technique. Free-phase concentrations detected through qPCR were significantly higher than those detected using Colilert although total concentrations were statistically equivalent, suggesting a source of analytical bias. Although a specimen processing complex was used to identify and correct for inhibition of the qPCR reaction, high particle concentrations may have resulted in underestimation of total cell counts, particularly at low concentrations. Regardless, qPCR-based techniques will likely have an important future role in stormwater assessment and management.

Effects of essential oils on in vitro ruminal fermentation and ammonia release

Ruminal inoculum enriched with particle-associated microorganisms was collected from two lactating dairy cows fed an alfalfa hay/cereal silage/concentrate diet 1 h before feeding and used to evaluate effects of essential oils (EO) on ruminal fermentation in short-term in vitro incubations. Ruminal ammonia N was labeled with 15N and native and hydrolyzed casein were provided as sources of amino acids. Forty EO were tested at 10 and 100 mg/l final medium concentration. Monensin-Na, and sodium laurate were also incubated at 5 and 2000 mg/l, respectively. Compared with blanks ( i.e., no addition of EO), sodium laurate increased medium pH and a number of EO reduced medium pH. Both sodium laurate and monensin reduced ammonia concentrations compared to the blank. Only one of the tested EO ( i.e., Caraway) slightly reduced ammonia concentration, by 8%, compared with the blank. Monensin and sodium laurate resulted in higher ( i.e., 9–34%, monensin, and 29–47%, sodium laurate) 15N enrichment of ammonia N, an indication of reduced deamination of amino acids in these treatments versus the blank. Several EO ( i.e., FrankMyrrh, Gardenia, Hibiscus, Eucaliptus, and Peppermint) had similar effects, but of a smaller magnitude ( i.e., 5–12%). Some EO increased medium total VFA concentration, primarily through an increase in acetate concentration. Overall, effects of EO on fermentation were subtle, and it is unlikely that these moderate in vitro effects would correspond to any substantive impact on ruminal fermentation in vivo.

Inactivation of particle-associated microorganisms in wastewater disinfection: Modeling of ozone and chlorine reactive diffusive transport in polydispersed suspensions

Occlusion of microorganisms in wastewater particles often governs the overall performance of a disinfection system, and the associated health risks of post-disinfected effluents. Little is currently known on the penetration of chemical oxidants into particles developed in wastewater treatment. In this work, a reactive transport model that incorporates intra- and extra-particle chemical decay, radial intra-particle diffusion, mass transfer resistance at particle surfaces, and non-linear reaction kinetics within a competitive multi-particle size aqueous environment, was used to analyze the penetration of ozone and chlorine into wastewater particles. Individual characteristics from two secondary wastewater treatment facilities were used in model calibration. Simulations revealed that significant ozone transport within particles greater than 6 μm required large initial concentrations to exhaust the preferential reaction with aqueous soluble matter. Chlorinated samples exhibited apparently slower reactions and thus deeper penetration (22–40 μm). Chlorine penetration was less sensitive to variations in the extra-particle reaction and disinfectant concentration than ozone. Model simulations that considered elevated initial concentrations of chemical disinfectants revealed that complete inactivation of all particle size domains was not possible with current disinfection practices (e.g., contact times). Reduction in the health risks associated with wastewater particles requires treatment that efficiently balances particle removal (filtration) and particle inactivation (disinfection).

Comparison of Physical and Chemical Methods for Extraction of Coliform from Wastewater Particles and Flocs

Disinfection processes are hindered by the presence of particles that protect the micro-organisms from the disinfectant. To understand better the extent of particle association and its effect on disinfection, it is necessary to efficiently release and enumerate particle-associated micro-organisms. Using a wastewater from a single treatment facility, various physical and chemical methods were evaluated to extract micro-organisms from particles present in wastewater, and a method was recommended. Increases in coliform bacteria were used as the indicator to evaluate the effectiveness of extraction methods. Physical extraction methods evaluated were blending, homogenization, stirring, sonication, and centrifugation, and chemical extraction methods were EDTA and EGTA extraction together with a combination of Zwittergent 3-12, Tris buffer, EGTA, and peptone. The results of this study indicate that high shear forces are not needed to extract particle-associated coliform in wastewater, as measured by increases in bacterial counts. Lowspeed blending (3,500 rpm) or high-speed stirring (1,000 rpm) were both effective in extracting coliform from wastewater particles and flocs. Homogenization, high-speed blending, and sonication at typical operational speeds and durations used in soil and activated carbon studies decreased the coliform counts in wastewater. Among the methods tested in this study, blending with EGTA, EDTA, and a combination of Zwittergent 3-12, Tris buffer, EGTA, and peptone at 3,500 rpm were found most effective in extracting particle-associated coliform from wastewater particulates.

Evaluation of techniques to detach particle-associated microorganisms from rumen contents

Effects of the most commonly used treatments to detach particle-associated microorganisms from rumen contents were investigated using rumen particles of different sizes. Particles were obtained before feeding from ruminally cannulated sheep. The extent of microorganism dissociation was determined using 15N as an external marker. The first experiment studied the effect of anaerobiosis on efficiency of 1 g/l methylcellulose, pH 8, and chilling (4 °C). Due to poor detachment, the anaerobic procedure was discarded. The following factors, separately or in combination, were then examined in aerobic conditions on two classes of particles obtained from whole ruminal contents (large: >400 μm; small: 100–400 μm) being: (a) stomacher pummelling (5 min); (b) Waring blender homogenisation (3×1 min); (c) chilling (4 °C); (d) pH 8; (e) 1 g/l methylcellulose; (f) 10 ml/l methanol and 10 ml/l tertiary butanol. Samples were incubated for 5 h, except for treatments (a) and (b), and washed after treatment for 2 min under running tap water in a 25 μm gauze. Blending proved to be the most effective treatment (from 50 to 57% removal). Combinations of treatments did not improve detachment. N losses from treated samples were linearly related to 15N removal. The percentage of particles removed was calculated using incubated and/or washed particles as the control, according to treatments. Results suggest that caution is needed when evaluating the effectiveness of treatments, because results are dependent on the type of particles chosen as the control.

Ultraviolet and ionizing radiation for microorganism inactivation

The impacts of UV irradiation, gamma irradiation, and a combination of both on Escherichia coli inactivation in primary and secondary wastewater effluents were investigated. UV doses of 35 and 62J/m2 were required for a 1-log inactivation of E. coli in the primary and secondary wastewater samples, respectively. A gamma dose of 170Gy (J/kg) was required for a 1-log inactivation of E. coli in both wastewater samples. Variation in gamma radiation dose rates did not have a significant impact on the extent of inactivation at a given total dose. Gamma irradiation of previously UV-irradiated samples indicated that particle-associated microorganisms, which are protected from UV, can be inactivated by ionizing radiation at a rate similar to that for free microorganism inactivation. An estimation of the energy required for disinfection indicated that, in general, the required energy and the energy cost for E. coli inactivation using ionizing radiation are considerably higher than those for UV radiation.

A simple method for differential isolation of freely dispersed and particle-associated peat microorganisms

Differential isolation of physiologically and ecologically diverse microbial groups facilitates evaluation of their activities in the environment. Here we describe a new method for differential isolation of freely dispersed and particle-associated peat microorganisms. The method is based on sequential elution of peat. Two types of natural peat, “young” sphagnum peat and “old” reed-based peat that have significantly different molecular composition as revealed by solid-state 13C NMR, were used for the method development. Various combinations of elution parameters (pH values, surfactant concentration, and sonication) were tested. Protein yield, CFU counts, 16S rDNA gene sequence, and T-RFLP analyses were used as criteria for evaluating the effectiveness of the elution protocols. The optimal method consisted of sequential elutions with (i) pH 6.2, 0.1 M potassium phosphate buffer, (ii) the same buffer amended with 0.01% of Tween 80, and (iii) treatment (ii) amended with sonication for 2 min. This method allowed differential isolation of freely dispersed and particle-associated microorganisms with (i) highest yield, (ii) highest diversity within the each microbial fraction, and (iii) best discrimination between the freely dispersed and particle-associated microbial communities. The method was applied for characterization of peat microbial communities and revealed different taxonomic composition and similar spatial organization of sphagnum and reed-sage peat microorganisms.

Diet and procedures used to detach particle-associated microbes from ruminal digesta influence chemical composition of microbes and estimation of microbial growth in Rusitec fermenters.

Four different detachment methods were evaluated for their ability to remove particle-associated microorganisms (PAM) from ruminal digesta in semicontinuous fermenters fed two diets differing in their forage:concentrate ratio (80:20 [C20] and 20:80 [C80]). In the methylcellulose method, ruminal digesta was incubated at 38 degrees C for 15 min with saline solution containing 0.1% methylcellulose before being stored at 4 degrees C for 24 h. In the other procedures, samples were incubated with 0.1% methylcellulose before storage for 24 h at 4 degrees C in different solutions (pH = 2): 1) saline solution with 0.1% Tween 80; 2) saline solution with 0.1% Tween 80 and 1% tertiary butanol; and 3) saline solution with 0.1% Tween 80, 1% methanol, and 1% tertiary butanol. Common to all treatments was subsequent homogenization, followed by filtration and resuspension of the residue five times in the treatment solutions. Microbial removal was estimated indirectly by measuring removal of 15N. There were no differences (P > 0.05) among detachment procedures, neither in the detaching efficiency (mean values of 79.7 and 88.1% for C20 and C80 diets, respectively) nor in the total recovery of PAM (54.9 and 34.9% for C20 and C80, respectively). There were no differences (P > 0.05) among PAM pellets obtained by the different detachment procedures in their N content, purine bases (PB) concentration, or PB:N ratio. For the C80 diet, 15N enrichment was greater (P < 0.05) in PAM pellets obtained with methylcellulose than in those obtained by the other methods. However, there were no differences (P > 0.05) due to the detachment procedure in the values of daily microbial growth estimated using as reference the different PAM pellets. The PAM pellets for diet C20 presented greater (P < 0.01) N content and lower (P < 0.01) PB concentration than those for diet C80 (mean values of 74.3 vs 49.1 mg of N/g of dry matter, and 22.8 vs 26.0 micromol PB/mg of dry matter, respectively). Daily microbial growth was greater (P < 0.05) for the C80 diet than for the C20 diet (121 vs 114 mg of microbial N, respectively). Results suggest that the treatment of ruminal digesta with a saline solution with 0.1% methylcellulose at 38 degrees C for 15 min combined with homogenizing and chilling at 4 degrees C for 24 h removed a major proportion of PAM, although further research is needed to decrease microbial losses during the isolation process.

Particle-associated microorganisms in stormwater runoff

This research investigated the effects of blending and chemical addition before analysis of the concentration of microorganisms in stormwater runoff from a single summer storm to determine whether clumped or particle-associated organisms play a significant role. The standard membrane filtration method was used to enumerate the microorganisms. All organisms, except for Escherichia coli, showed an increase in the measured concentration after blending samples at 22,000 rpm with or without the chemical mixture. Other than fecal streptococci, the organism concentrations decreased with the addition of the Camper's solution in both blended and unblended samples before analyses. There was a statistically significant interaction between the effects of Camper's solution and the effects of blending for all the organisms tested, except for total coliform. Blending did not alter the mean particle size significantly. The results show no correlation between increased total coliform, fecal coliform, and fecal streptococcus concentrations and the mean particle size.

Effect of barley and its amylopectin content on ruminal fermentation and bacterial utilization of ammonia-N in vitro

The objective of this study was to determine the effect of variety and amylopectin content of barley on ruminal fermentation and ammonia-N utilization by mixed ruminal bacteria in vitro. Three barley varieties (WestBred Gustoe, Nebula, Baronesse) and their waxy counterparts were tested. The control grain was corn. All barley varieties were examined at three substitution levels: 25, 50, and 75% with the remaining being corn. Ammonia-N was labeled with 15 N -ammonium sulfate. Samples were collected at 0, 2, 4, 6, and 8 h. Barley inclusion enhanced ( P<0.05) propionate production and total VFA at the 75% substitution level and, at 8 h, increasing the proportion of barley in the grain mix increased ( P<0.05, linear effect) propionate concentration and decreased ( P<0.05, linear effect) acetate to propionate ratio. Across sampling points, the waxy variety of WestBred Gustoe resulted in higher ( P<0.05) fluid bacteria incorporation of ammonia-N than corn (by 7.6 and 11.9%, 25 and 75% substitution levels, respectively). At the 75% substitution level, waxy barleys resulted in an 8% higher ( P<0.01) ammonia- 15 N incorporation into bacterial N than their non-waxy counterparts. At the end-point of the incubation, barley treatments and waxy barleys had higher ( P<0.05) 15 N -enrichment of the fluid bacterial N compared with corn and non-waxy barleys, respectively. The combined (fluid- and particle-associated bacteria and protozoa) microbial ammonia-N uptake, however, was not different between corn control and treatments with barley. Results suggest that ruminal responses to barley supplementation may vary depending on its variety and starch composition and differ between fluid- and particle-associated microorganisms.

Fibrolytic activities and cellulolytic bacterial community structure in the solid and liquid phases of rumen contents.

Four sheep were fed an alfalfa hay diet. Rumen content samples were collected three hours after feeding in order to total microorganism population (TP), solid attached population (SAP) and solid attached firmly population (SAFP). Fibrolytic specific activities (xylanase, CMCase and beta-glycosidases) were estimated by the amount of reducing sugars or p-nitrophenol released from the appropriate substrate. The distribution of the three main cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens) was quantified by dot-blot hybridisation using specific 16S-rRNA-targeting probes. Specific activities of polysaccharidase enzymes were higher in SAP than in TP, and in SAFP than in SAP. The sum of RNA of the three cellulolytic bacterial species represented on average 9% of the total bacterial RNA, and increased after filtration. In all samples, the relative population size of F. succinogenes was higher than that of R. albus and of R. flavefaciens. These results demonstrate that the most active enzymes are secreted by the particle-associated microorganisms. The differences in composition of the microflora between the solid and liquid phase suggest that bacteria are not equally distributed throughout the rumen content: the cellulolytic species are present in a higher proportion in the solid phase of rumen contents.

Improved techniques for dissociating particle-associated mixed ruminal microorganisms from ruminal digesta solids.

Combinations of physical and chemical methods were evaluated for their ability to remove particle-associated microorganisms (PAM) from saline-washed ruminal digesta solids (SWRDS). Physical methods included chilling and storage, homogenization, multiple extraction, and agitation with marbles. Chemical methods included use of low pH, Tween 80, formaldehyde, methanol, tertiary butanol, and methylcellulose. Microbial removal from SWRDS was determined directly by using epifluorescence microscopy and indirectly by measuring removal of diaminopimelic acid and total purines. Different combinations of methods resulted in removals of 46 to 82% for particle-associated bacteria (PAB), 52 to 98% for particle-associated protozoa (PAP), and 60 to 83% for PAB plus PAP. Two methods were considered most effective, based on microscopy; both removed similar amounts of PAB (79 to 82%) and PAB plus PAP (80 to 83%). In one method, SWRDS were stored for 24 h at 4 degrees C in a solution of pH 2 saline, .1% Tween 80, 1.0% methanol, and 1.0% tertiary butanol. In the other method, SWRDS were incubated for 30 min in .1% methylcellulose before storage for 24 h at 4 degrees C in pH 2 saline, .1% Tween 80, and 1.0% methanol. Common to both treatments was subsequent homogenization of the suspensions for 15 s followed by washing the digesta solids seven times with the treatment solutions. Both methods resulted in values that exceeded those reported previously for removal of PAM from ruminal digesta solids.