Jean Christophe Balouet

Joel G Burken

Frank Karg

Don Vroblesky

Kevin T Smith

Hakan Grudd

Anders Rindby

Francois Beaujard

Michel Chalot

Dendrochemistry of Multiple Releases of Chlorinated Solvents at a Former Industrial Site

Trees can take up and assimilate contaminants from the soil, subsurface, and groundwater. Contaminants in the transpiration stream can become bound or incorporated into the annual rings formed in trees of the temperate zones. The chemical analysis of precisely dated tree rings, called dendrochemistry, can be used to interpret past plant interactions with contaminants. This investigation demonstrates that dendrochemistry can be used to generate historical scenarios of past contamination of groundwater by chlorinated solvents at a site in Verl, Germany. Increment cores from trees at the Verl site were collected and analyzed by energy-dispersive X-ray fluorescence (EDXRF) line scanning. The EDXRF profiles showed four to six time periods where tree rings had anomalously high concentrations of chlorine (Cl) as an indicator of potential contamination by chlorinated solvents.

■ INTRODUCTION

Annual growth rings can preserve past environmental changes within the biological requirements of tree survival. [START_REF] Schweingruber | Tree Rings and Environment: Dendroecology; Swiss Federal Institute for Forest[END_REF][START_REF] Smith | An organismal view of dendrochronology[END_REF] The relationship of one ring representing 1 year of tree growth was recognized by Theophrastus in ancient Greece, [START_REF] Briand | Tree rings and the aging of trees: A controversy in 19th century America[END_REF] and Leonardo da Vinci [START_REF] Da Vinci | Treatise on Painting (Codex Urbinas Latinus[END_REF] in Renaissance Italy acknowledged year-to-year changes in rings as being related to climatic changes. The assignment of absolute calendar dates for the year of wood formation, the hallmark of dendrochronology, enables the dating of wood samples and artifacts as long as an unbroken record from the current time exists. [START_REF] Speer | Fundamentals of Tree-Ring Research[END_REF] Chiment and Chiment [START_REF] Chiment | Analytic bibliography of basic and applied dendrochemistry[END_REF] reviewed over 300 research articles on dendrochemical records derived from elemental isotopes covalently bound to wood constituents; essential "fertilizer elements" associated with ionexchange sites in wood such as nitrogen (N), potassium (K), and magnesium (Mg); semivolatile or volatile organic compounds, including halogenated volatile organic compounds (SVOCs, VOCs, and HVOCs, respectively). Inferring changes in the external environment from dendrochemical patterns requires an understanding of tree biology, tree physiology, and tree infections. [START_REF] Smith | Tree biology and dendrochemistry[END_REF] Dendrochemical interpretations are strengthened when combined with traditional analyses of wood chemistry and dendrochronology and can be used effectively to date environmental events.

Dendrochemistry, the chemical analysis and interpretation of precisely dated tree rings, can provide a date-specific record of chemical changes [START_REF] Balouet | Emerging Forensic Techniques[END_REF][START_REF] Balouet | Use of dendrochronology and dendrochemistry in environmental forensics: Does it meet the Daubert criteria?[END_REF] and expands on phytoscreening, in which the chemical analysis of plants is used to monitor and detect plant-chemical exposure. [START_REF] Burken | Dendrochemistry, and Phytoscreening: New Green Tools for Delineating Contaminants from Past and Present[END_REF] Phytoscreening methods have documented the presence of contaminants or their metabolic products from chlorinated solvents and fossil fuels. [START_REF] Vroblesky | User's Guide to the Collection and Analysis of Tree Cores to Assess the Distribution of Subsurface Volatile Organic Compounds[END_REF] When combined, phytoscreening and dendrochemical investigation can increase the understanding of current and past environmental events, termed phytoforensics. [START_REF] Burken | Dendrochemistry, and Phytoscreening: New Green Tools for Delineating Contaminants from Past and Present[END_REF] Dendrochemistry can indicate a chemical change in the annual rings, and this information can be combined with groundwater data and modeling, knowledge of contaminant source type and location, and knowledge of tree species physiology to collectively generate scenarios of past events leading to contaminant release and transport and also potential exposure pathways.

Dendrochemistry has successfully utilized several analytical techniques. Examples include energy-dispersive X-ray fluorescence (EDXRF), proton-induced X-ray emission (PIXE), analysis of wood extracts or digests (e.g., inductively coupled plasma-mass spectroscopy, or ICPMS), and atomic emission spectroscopy (AES). EDXRF methods tend to be favored largely because of the capacity for simultaneous analysis of 31 target or marker elements and compounds at a high spatial resolution of 50 μm across the tree-ring sample. [START_REF] Chiment | Analytic bibliography of basic and applied dendrochemistry[END_REF][START_REF] Balouet | Applied dendroecology and environmental forensics. Characterizing and age dating environmental releases: Fundamentals and case studies[END_REF] Regardless of analytical method, it is important that the dendrochemist determine which observations are related to processes involved with tree physiology and species relative to the interaction with pollutants. [START_REF] Balouet | Use of dendrochronology and dendrochemistry in environmental forensics: Does it meet the Daubert criteria?[END_REF][START_REF] Balouet | Applied dendroecology and environmental forensics. Characterizing and age dating environmental releases: Fundamentals and case studies[END_REF][START_REF] Cutter | Anatomical, Chemical, and Ecological Factors Affecting Tree Species Choice in Dendrochemistry Studies[END_REF] In this study, we describe the dendrochemical investigation methods and results in a groundwater pollution case, where the tree chemistry appears to reflect four to five asynchronous releases of HVOCs, along with other environmental influences and physiological anomalies. For this study, four trees were used that were located along a plume (see Figures 12345). These trees (see Table 1) belong to three genera (two oaks, one sycamore, and one fir).

■ EXPERIMENTAL SECTION

Study Area. Verl is a relatively small city in Germany with an area of 73 km 2 and a population of about 25000. The aquifer at Verl consists of heterogeneous glacial deposits, resulting in uncertainties in determining the aquifer characteristics. The aquifer contains an elongated plume of groundwater contaminated by chlorinated aliphatic solvents (Figure 1). An earlier investigation using phytoscreening methods demonstrated that the trees take up groundwater contamination and could be used to map the plume. [START_REF] Burken | Dendrochemistry, and Phytoscreening: New Green Tools for Delineating Contaminants from Past and Present[END_REF][START_REF] Limmer | Phytoscreening for Chlorinated Solvents Using Rapid in Vitro SPME Sampling: Application to Urban Plume in Verl, Germany[END_REF] The dominant contaminants in the groundwater are tetrachloroethene (PCE), trichloroethene (TCE), and cis-1,2-dichloroethene (cDCE). TCE and cDCE are known to be dechlorination products of PCE. Much of the contamination is believed to originate from a former mechanical plant that used PCE in dipping baths from 1948 to 1969 (Figure 1). Groundwater contamination by PCE was discovered in 1972. Up-filling of PCE drums continued in the 1970s, and a private garage shop that operated at the site between 1980 and 1999 also used PCE. Phytoscreening was successfully used to map the configuration of the chlorinated solvent plume of groundwater. [START_REF] Burken | Dendrochemistry, and Phytoscreening: New Green Tools for Delineating Contaminants from Past and Present[END_REF][START_REF] Limmer | Phytoscreening for Chlorinated Solvents Using Rapid in Vitro SPME Sampling: Application to Urban Plume in Verl, Germany[END_REF] The phytoscreening analysis also provided evidence of multiple TCE sources, subsequently supported by the discovery that fill material at the site was also contaminated by HVOCs. [START_REF] Limmer | Phytoscreening for Chlorinated Solvents Using Rapid in Vitro SPME Sampling: Application to Urban Plume in Verl, Germany[END_REF] Sample Preparation. Increment cores (10 mm in diameter) were extracted from nine trees (Figures 1 and2) on February 22, 2010. After 5 days of drying in ambient air between grooved wooden blocks, cores were surfaced with a graded series of silicon carbide sandpapers (50, 100, 200, and 400 grit). The sanded cores were observed under a digital microscope (40-100× magnification) to evaluate the presence of branch anomalies, infection, and tree response to injury and infection that could have affected the dendrochemical pattern. Cores extracted from trees 2, 5, 6, and 7 passed the preliminary screening and were analyzed by EDXRF at Dendrolab, Stockholm University, using an Itrax Multiscanner (www. coxsys.se). All sample processing and EDXRF analyses were conducted under conditions of constant air temperature (20 °C) and relative humidity (50%). The sample cores were first cut along their full length parallel to the transverse wood surface with a twin-blade saw to produce 2-mm-thick laths with planar surfaces. The laths were then mounted vertically in the Itrax Multiscanner to allow for radiographic density and EDXRF analyses along the length of the planar transverse surface of the samples. EDXRF Analysis. The Itrax Multiscanner is a "flat-beam" Xray scanner where samples are exposed to a precisely defined, thin X-ray beam with a vertical dimension of 50 μm. An X-ray line camera records the transmitted radiation that passes through the sample. Hence, a full-scale two-dimensional radiographic image with 50-μm resolution is obtained by moving the sample relative to the beam in 50-μm steps. At each step, a silicon drift chamber detector (SDD) records the characteristic fluorescent X-ray radiation (XRF) specific alkaline earth and other metal elements with the atomic weight of Mg and higher. The collimator system within the SDD confines XRF recording to a sample area of 0.05 × 2 mm 2 . The SDD functions as an energy-dispersive X-ray detector, with a capacity of 100000 counts per second (cps) and a resolution of about 135 eV at 5.9 keV. Equipped with the standard 1.9-kW chromium-tube X-ray source, the SDD records about 20000 cps for wood tissue. An exposure time of 10 s is normally sufficient to record element concentrations above 10 ppm. [START_REF] Lindeberg | X-ray based tree ring analyses[END_REF] With the resolution of 50 μm, a tree ring with a width of 1 cm provides 200 XRF data points for elemental concentrations. High-resolution profiles of elemental concentrations were established over a large number of rings within a few hours (about 3.3 min/mm) and in a nondestructive way. The raw EDXRF data, made available as an Excel file, provide relative concentrations in counts for 31 elements along with physical parameters (locations, counts, mean squared errors). The raw data were computerized using an xls template, called Scanchem, that automatically generates density-corrected concentrations and time-weighted centered moving averages (TWCMAs). [START_REF] Balouet | Use of dendrochronology and dendrochemistry in environmental forensics: Does it meet the Daubert criteria?[END_REF][START_REF] Balouet | Applied dendroecology and environmental forensics. Characterizing and age dating environmental releases: Fundamentals and case studies[END_REF] Influences on Tree Chemistry. Dendrochemical profiles can have characteristic intra-ring patterns that show some level of annual repetition. In this study, dendrochemical anomalies are considered to be parts of the tree core where the chemical concentration differs from the characteristic pattern by at least 30% of the regular concentration minima, maxima, or annual averages, extending over one or more rings. In the Verl study, the anomalies represent time periods when relatively large amounts of chloride were taken up by the trees.

A variety of factors can cause differential annual uptake of solutes, including differences in rainfall, depth to groundwater or contaminants, locations of the root systems, and groundwater solute concentrations. During years with comparatively large amounts of rainfall, trees are likely to contain lower concentrations of groundwater constituents, such as contamination, because the roots intercept infiltrating rainwater. In addition, local recharge to the aquifer dilutes the shallow groundwater and can force plumes into deeper zones where they are farther from the root system. Differences in annual precipitation can also influence the position of the root system. The optimum root depth for most hardwood trees in areas of shallow groundwater is neither submerged beneath the water table nor out of reach of the capillary fringe. In areas such as at tree 2 where the groundwater is only about 50 cm below the land surface, it is possible that even small changes in the watertable depth could result in some level of root adjustment by the trees. Adjustment of root location also can occur to position the roots in a less toxic zone, which can result in a shift in the zone from which the plant derives water (root avoidance and microroot turnover). The historical groundwater contaminant concentrations are not known, but the presence of greater than 1000 ppb of HVOCs at the downgradient part of the plume (Figure 1) and evidence of HVOC decay (dechlorination products) imply that historical concentrations might have been substantially higher than are now. Thus, root avoidance issues also might have occurred. Therefore, the chemical signature in tree rings from groundwater contamination could change from year to year. Some natural events are known to leave characteristic dendrochemical signatures, such as calcium (Ca), Mg, and K for infections, including those introduced by wounds from pruned and naturally shed branches; [START_REF] Smith | Tree biology and dendrochemistry[END_REF] and zinc (Zn) and iron (Fe) [START_REF] Dewalle | Tree-ring cation response to experimental watershed acidification in West Virginia and Maine[END_REF] or Ca, Mg, and manganese (Mn) [START_REF] Dewalle | Wood Chemistry Response to Fertilization at Two Ozark Sites[END_REF] for soil acidification. Similarly, pollution effects can have characteristic dendrochemical fingerprints. [START_REF] Smith | Tree biology and dendrochemistry[END_REF] For example, a dendrochemical anomaly from dechlorination of chlorinated solvents might be expected to consist only of Cl, whereas a dendrochemical anomaly from road salt might consist of increased concentrations of multiple common constituents of road salt. Additional constituents besides Cl that might be present in groundwater affected by road salt include sulfur (S), K, and Ca. S and Cl can be associated with fossil fuels, and petroleum fuels can be associated with trace metals such as vanadium (V) or nickel (Ni), versus lead (Pb) for leaded gasoline. Fertilizers contain phosphorus (P) and K, [START_REF] Smith | Tree biology and dendrochemistry[END_REF] and groundwater affected by sewage leakage can be characterized by elevated P. EDXRF analysis provides the capability to analyze for these constituents in tree cores on a sub-ring scale, potentially allowing interpretation of the environmental or physiological events associated with the ring chemistry.

For the Verl site, Cl associated with chlorinated solvent degradation was used as a monoelemental marker that was detectable by XRF. [START_REF] Balouet | Applied dendroecology and environmental forensics. Characterizing and age dating environmental releases: Fundamentals and case studies[END_REF] HVOCs are naturally degraded by bacteria within groundwater and soils in reducing environments in reductive dechlorination or "dehalorespiration" (i.e., PCE to TCE to cDCE) and can generate large plumes of Cl ions that can be taken up by root systems of trees. [START_REF] Wiedemeier | Natural Attenuation of Fuels and Chlorinated Solvents in the Subsurface[END_REF][START_REF] Yanosky | Use of tree rings to investigate the onset of contamination of a shallow aquifer by chlorinated hydrocarbons[END_REF] The parent HVOC can also be taken up by trees. HVOCs within trees can spread through the porous wood [START_REF] Baduru | Direct Measurement of VOC Diffusivities in Tree Tissues: Impacts on Tree-Based Phytoremediation[END_REF] and can partly evaporate through the stem [START_REF] Ma | Diffusion of TCE to the Atmosphere in Phytoremediation Applications[END_REF] and leaf crown. [START_REF] Graber | Competitive Uptake of Trichloroethene and 1,1,1-Trichloroethane by Eucalyptus camaldulensis Seedlings and Wood[END_REF] HVOCs can also degrade in plant tissues if reducing conditions dominate (i.e., in a decaying tree) or through aerobic plant defense pathways to form trichloroacetic acid and other metabolites that can be further degraded. [START_REF] Newman | Uptake and biotransformation of trichloroethylene by hybrid poplars[END_REF][START_REF] Vroblesky | Groundwater chlorinated ethenes in tree trunks: Case studies, influence of recharge, and potential degradation mechanism[END_REF] At relatively low concentrations, Cl is usually stable in woody tissues after fixation, presumably through ionic binding, and is not readily remobilized/retranslocated. [START_REF] Balouet | Use of dendrochronology and dendrochemistry in environmental forensics: Does it meet the Daubert criteria?[END_REF][START_REF] Balouet | Applied dendroecology and environmental forensics. Characterizing and age dating environmental releases: Fundamentals and case studies[END_REF] The potential for HVOC movement across ring boundaries and for HVOC dechlorination within trees leads to the possibility that Cl generated from in-tree degradation might be fixated in growth rings not corresponding to the year of HVOC uptake. In situations where there is no substantial dechlorination activity in the groundwater and soils, the Cl signature in tree cores is unlikely to provide information on the timing of HVOC uptake. When there is substantial underground dechlorination within the capture zone of the root system, however, it is likely that the groundwater Cl signature would overwhelm in-tree effects and would predominantly be located in the growth ring corresponding to the year of uptake. In the tree cores examined at Verl, there was no distinctive change in Cl chemistry from one side of the heartwood/sapwood boundary to the other, implying that there was no significant translocation across the boundary, as is sometimes seen for translocated elements. [START_REF] Vroblesky | Increased Concentrations of Potassium in Heartwood of Trees in Response to Groundwater Contamination[END_REF]

■ RESULTS AND DISCUSSION

The EDXRF procedure allows for the direct comparison of two or more elements at once (Figures 3 and4). Bielemental profiles for Ca (in white) and Cl (in green) are superimposed on an X-ray image of the core from tree 7 (Figure 3). The direction of growth is from left to right, with the transition between annual growth rings marked by the contrast between the more physically dense late wood (light-colored in the X-ray image) and the less dense early wood of the following year (dark-colored in the X-ray image). Note the characteristic annual, intra-ring patterns of Cl, which usually are at their lowest during the early wood growth period, or the summer. The Cl-only anomalies are interpreted as being associated with the chlorinated solvent plume. Also, in the 1990/1991 rings of tree 7, the Cl uptake appears to have occurred during the late wood growth period. Consistent with this interpretation, the minimum Cl concentration in the susbsequent early wood growth period was substantially higher than in previous rings (Figure 3).

EDXRF shows distinct bielemental anomalies for Ca and Cl in tree 2 (Figure 4), peaking in 1908, 1918, 1931, and 1976. The dual Ca and Cl signature was interpreted as possibly being the result of drought or other climatic influences related to contamination; the 1976 drought had a similar Ca/Cl signature in tree 2 as the three earlier anomalies. Tree 2 also shows four to five asynchronous Cl-only impacts (Table 1), potentially indicating the presence of HVOCs. [START_REF] Balouet | Emerging Forensic Techniques[END_REF][START_REF] Balouet | Use of dendrochronology and dendrochemistry in environmental forensics: Does it meet the Daubert criteria?[END_REF][START_REF] Balouet | Applied dendroecology and environmental forensics. Characterizing and age dating environmental releases: Fundamentals and case studies[END_REF] The 1948/1949 Cl peak in tree 2 does not have a directly corresponding Ca peak, but there are other Ca anomalies in close proximity (Figure 4), imparting some level of uncertainty as to whether this was a Clonly event. It is of interest, however, that, while searching for a potential cause of the 1948/1949 Cl anomaly in tree 2, investigators found that the industrial plant had been active since 1945 and that two buildings containing PCE were destroyed in 1948 and on April 22, 1949, potentially resulting in a contamination release.

It is highly likely that the chemistry in tree 2 is influenced by groundwater chemistry because the depth to groundwater at tree 2 is only about 50 cm. Cl anomalies in tree 2 are present in growth rings from two years that the PCE-containing buildings were destroyed and might represent HVOCs related to those events. If so, then the HVOCs would have been rapidly transported from the source area to tree 2. A potential explanation for rapid transport of HVOCs is that they could have moved by overland flow through a drainage ditch or creek. There is, in fact, the remnant of a former ditch or stream near trees 1 and 2, over 15 m in length and oriented in the direction of groundwater flow (the ditch is shown in the background of Figure 2). Construction and landscaping have eliminated much of the stream and obscured its original path; however, proximity to trees 1 and 2 and orientation in the direction toward the source area imply the possibility that the ditch once provided surface drainage from the source area to the vicinity of tree 2. The subsequent Cl anomaly in tree 2, 11-12 years later (1960/1961), could potentially be explained by groundwater transport of contamination and delayed impact from the same 1948 and 1949 releases. Modeling simulations (MODFLOW) produced time-of-travel estimates consistent with groundwater transport of contaminants to the four trees at dates corresponding to the Cl anomalies (see Figure 5).

Trees 5-7 also show, depending on their age and location, a minimum of two to four asynchronous Cl anomalies (Figures 5 and6). Potentially, the anomalies might represent the transport Figure 3. Relative concentrations of chlorine (green) and calcium (white) determined by energy-dispersive X-ray fluorescence overlaid on an X-ray image of the tree 7 core (Verl, Germany). Also, note that, at the 1990/1991 rings, the Cl impact occurred during the period of late wood formation, as evidenced by the peak having its highest amplitude in the 1990 late wood, and the subsequent early wood recorded a Cl minima significantly higher than in previous rings. Note the characteristic annual, intra-ring patterns, often showing the lowest Cl concentrations in the early wood. of additional releases of contaminants. Trees 5 and 6 are at the edge of the present groundwater plume boundary (Figure 1); however, the historical extent of contamination might have been different than what is now present. Knowledge of groundwater and potential contamination sources and events is needed to provide corroborating lines of evidence.

Measured groundwater flow rates and model (MODFLOW) estimated plume migration rates (consulting firm HPC Envirotec, written communication, 2010) showed the probable plume migration rate to be about 20-30 m/year in the southern part and 2-3 times higher in the northern part of the plume. The plume migration rate, as estimated from a comparison of Cl anomalies in trees to the distance separating the trees, was about 30 m/year in the southern part of the plume and 70-120 m/year toward the northern half of the plume. The average plume migration rate based on the tree cores was about 54 m/year over the 820 m separating the source from the most distant tree, tree 5 (see Figure 6). The close agreement between the estimates of the plume migration rate supports the hypothesis that the Cl anomalies represent movement of groundwater contamination. Agreement between model-derived and tree-chemistry-derived groundwater flow rates wasalso observed in a previous study. [START_REF] Vroblesky | Use of tree-ring chemistry to document historical ground-water contamination events[END_REF] It should be noted that there are uncertainties in both the groundwater-and tree-derived estimates of contaminant transport. The uncertainties in the groundwater-derived estimates involve uncertainties in both modeling formulations and porosity estimates, particularly in these heterogeneous glacial deposits. Uncertainties in the tree-derived estimates involve a possible half-year lag time during the nongrowing season when a dendrochemical signal might not be effectively recorded. If, however, dendrochemical signals can be shown to be a reasonable estimate of contaminant arrival dates for a particular site, then the information could have great supportive value as a complementary approach for estimating contaminant transport rates.

The black, red, blue, and yellow lines in Figures 5 and6 identify the potential asynchronous releases of Cl, labeled A-E, respectively, and correspond to the suspected releases listed in Table 2. Based on the plume migration rates through groundwater estimated from the dendrochemical record and the distance between the southernmost trees and those closest to the source and historical records, the timing of the releases were estimated to be 1948-1949 (release A), 1975 (release B), 1985 (release C), 1990 (release D), and 1999 (release E), as reported in Table 2. Releases that took place after industry ceased its production activities in 1969 might be associated with 2. Note that tree 2 is the only tree old enough to have reflected potential releases identified from 1948/1949 and the subsequent 1960/1961 impact via groundwater; tree 6 is too recent to show the impact found in tree 7 in 1980 and later found in trees 2 and 5 (red arrow). The recent 2004 Cl anomaly found in tree 7 would not have impacted tree 6 in 2010. The orange arrows represent the location of the heartwood/sapwood boundary (HSB). The lack of a distinctive change in Cl concentrations across the HSB provides evidence that there is no significant chlorine lateral translocation in these trees.

progressive plant dismantling and remediation activities or with activities at a garage shop that began operation in 1980.

The data provided herein demonstrate that the high resolution of the EDXRF method allows comparisons of wood chemistry at a scale that can differentiate changes within a single growth ring. The data can be used to examine the timing of environmental events with approximately annual precision. Although the past can never be reconstructed with complete accuracy, the Verl study area shows the potential of dendrochemical methods to indicate multiple historical and asynchronous environmental impacts to trees, along with corresponding plume migration rates, and to age-date releases from the source. The combination of the dendrochemical data obtained using today's methodologies, available information on historical activities at the industrial locations, and groundwater modeling data potentially can be used to project back possible scenarios of events that occurred in the past.

Figure 1 .

 1 Figure 1. Location of study area, groundwater sampling, and trees sampled for dendrochemistry in 2010. Groundwater concentrations were measured in 2010 by the consulting firm HPC Envirotec. The plume map used in figure is for the total of PCE, TCE and cDCE.

Figure 2 .

 2 Figure 2. Sampling of tree 2 using a 10-mm Pressler borer.

Figure 4 .

 4 Figure 4. Detail of bielemental profiles for Ca in white and Cl in green, superimposed on an X-ray image of the tree 2 core. The direction of growth is from left to right, expressed by the transition from dark early wood (with low density) to light late wood (with high density). The chlorine-only anomalies are interpreted as a potential response to a chlorinated solvent plume.

Figure 5 .

 5 Figure 5. EDXRF data for the four analyzed cores from Verl, Germany, showing elemental profiles for Cl. They correspond, from top to bottom, to trees 7, 6, 2, and 5 and are listed in order of their distance from the plume. The red, blue, and yellow lines correspond to interpreted events B, C, and D, respectively, in Table2. Note that tree 2 is the only tree old enough to have reflected potential releases identified from 1948/1949 and the subsequent 1960/1961 impact via groundwater; tree 6 is too recent to show the impact found in tree 7 in 1980 and later found in trees 2 and 5 (red arrow). The recent 2004 Cl anomaly found in tree 7 would not have impacted tree 6 in 2010. The orange arrows represent the location of the heartwood/sapwood boundary (HSB). The lack of a distinctive change in Cl concentrations across the HSB provides evidence that there is no significant chlorine lateral translocation in these trees.

Figure 6 .

 6 Figure 6. Interpretation of the contaminant releases at source, plume migration, and impact times, based on the parsimony principle. The scenarios show five potential asynchronous releases impacting the four trees within the time needed for the plume to migrate from the source to each tree. The left vertical arrow shows the plume migration direction, with distance from the source; the four cores are arranged by proximity to the source. The connecting lines correspond to the interpreted events in Table2. The colors of the descriptive boxes represent the analytical response in counts (cts). The term "salt" is used for coincident Ca and Cl anomalies observed in tree 2. Orange arrows show the location of the heartwood/sapwood boundary (HSB).

Table 1 .

 1 Tree Species Sampled for Dendrochemical Analysis in Verl, Germany

	tree		oldest
	number	tree species	ring	years of unique Cl anomalies
	2	Quercus sp.	1896	1948/1949, 1961, 1987, 1997,
				2002
	5	Quercus sp.	1978	1989, 2000
	6	Platanus	1983	1993, 1996
		occidentalis
	7	Abies sp.	1975	1980, 1990, 1995, 2004

Table 2 .

 2 Estimated Times of Pollutant Releases at the Site in Verl, Germany

	plume	time of contaminant release
	A	1948, 1949
	B	1975
	C	1985
	D	1990
	E	1999

■ AUTHOR INFORMATION

Corresponding Author *Phone: +33344397312; e-mail: balouet.jean-christophe@neuf. fr.

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The site investigation was funded by HPC Envirotec R&D division. Field, premapping, and important historical research were conducted by Ralf Sutka, HPC Envirotec AG. We are also deeply indebted to PIT (Pollution Investigation by Trees), an international program funded by Ademe (French Agence de l'Environnement et de la Mai ̂trise de l'Energie). We are also indebted to James Landmeyer of the U.S. Geological Survey and the many others who contributed to the review of this article. The use of trade names in this document does not imply endorsement on the part of the organizations represented by the authors or by the U.S. Government.


D P Morgavi
email: diego.morgavi@clermont.inra.fr

C Martin

H Boudra

Fungal secondary metabolites from Monascus spp. reduce rumen methane production in vitro and in vivo 1

Keywords: enteric methane, fungal metabolite, inhibitor, rumen methanogenic archaea

ABSTRACT: Decreasing methanogenesis without affecting fermentation and digestion of feeds in the rumen can reduce the environmental impact of ruminant production and have a benefi cial effect on feed conversion effi ciency. In this work, metabolites produced by Monascus spp. molds were assayed for their antimethanogenic activity in vitro and in vivo. The capacity of 7 strains of Monascus to produce secondary metabolites was assessed in solid media. Monitored metabolites included the statins monacolin K, pravastatin, and mevastatin, and the mycotoxin citrinin. Ethanolic extracts from 5 different solid media from 2 selected strains were tested in vitro. Fermentation was not negatively affected by any treatment, but one extract decreased methane production (P < 0.05). This extract was further assayed in 3 consecutive batch incubations where a marked decrease in methane was observed in the third batch (P < 0.05). In contrast, methane produced in fl asks with pure monacolin K was not different from controls (P > 0.05). Rice on which the selected Monascus sp. was grown also decreased methane production when used as substrate for in vitro incubations (P < 0.05). The effect of Monascus-fermented rice on methane production was then assayed in vivo. Six wethers were adapted to a diet containing rice grain and hay (1:1 ratio). Rice was then replaced by fermented rice and given to animals for nearly 2 wk. Animals were monitored for a further 2 wk after the treatment. Daily methane emissions decreased (P < 0.05) by 30% after 2 to 3 d into the treatment and remained low throughout the administration period. This change was associated with reduced ruminal acetate to propionate ratio and decreased numbers of methanogens as detected by quantitative PCR (P < 0.05). In contrast, no changes in the methanogenic community were observed by denaturing gradient gel electrophoresis (DGGE). Total bacteria numbers increased (P < 0.05) with changes in the DGGE profi le community, whereas protozoa were not affected by the treatment. Methane emissions and the acetate to propionate ratio remained numerically less in the 2 wk posttreatment as compared with measures before treatment. Metabolites produced by Monascus appear to have an inhibitory effect on methanogens and decreased methanogenesis in vitro and in short-term in vivo without any apparent negative effect on rumen fermentation. This strategy deserves to be further explored and could be an abatement option under certain feeding situations.

INTRODUCTION

A major criticism of modern ruminant production is its emission of greenhouse gases and its impact on natural resources [START_REF] Steinfeld | Livestock's long shadow: Environmental issues and options[END_REF]. About onehalf of the equivalent CO 2 required for producing a kilogram of milk or beef meat comes from enteric methane.

The reduction of enteric methane is being explored through different angles at the farm, animal, and microbial level [START_REF] Martin | Methane mitigation in ruminants: From microbe to the farm scale[END_REF]. The use of feed additives and supplements as modulators of rumen methanogenesis is a common area of study. There is also a demand by consumers, or imposed by the legislation, for the development of additives issued from natural sources [START_REF] Jouany | Use of 'natural' products as alternatives to antibiotic feed additives in ruminant production[END_REF]. Plant secondary metabolites fi t well into this classifi cation and are being intensively studied (reviewed in [START_REF] Hart | Plant extracts to manipulate rumen fermentation[END_REF][START_REF] Patra | Meta-analyses of effects of phytochemicals on digestibility and rumen fermentation characteristics associated with methanogenesis[END_REF]Benchaar and Greathead, 2011). In contrast, there are fewer studies on other natural alternatives like feed enzymes, probiotics, and the use of molecules produced by bacteria and fungi [START_REF] Lee | The effect of bovicin HC5, a bacteriocin from Streptococcus bovis HC5, on ruminal methane production in vitro[END_REF][START_REF] Chung | Differing effects of 2 active dried yeast (Saccharomyces cerevisiae) strains on ruminal acidosis and methane production in nonlactating dairy cows[END_REF][START_REF] Grainger | Can enteric methane emissions from ruminants be lowered without lowering their production?[END_REF]. Monascus spp. are molds used in food production and medicine and are known to produce a number of biologically active substances, among them 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) inhibitors [START_REF] Shi | Benefi cial effects of Monascus purpureus NTU 568-fermented products: A review[END_REF] that have been described to inhibit the growth of rumen methanogens in vitro [START_REF] Miller | Inhibition of growth of methane-producing bacteria of the ruminant forestomach by hydroxymethylglutaryl-SCoA reductase inhibitors[END_REF]. However, not all secondary metabolites produced by Monascus have a benefi cial effect, as some strains can also produce mycotoxins, particularly citrinin under some conditions [START_REF] Liu | Evaluation of citrinin occurrence and cytotoxicity in Monascus fermentation products[END_REF]. We hypothesized that secondary metabolites of Monascus could modulate rumen fermentation and methane production. We monitored the production of secondary metabolites of fi eld isolates and type strains under various culture conditions and conducted a series of in vitro and in vivo experiments that, taken together, showed an inhibitory effect of secondary metabolites of Monascus on methanogenesis.

MATERIALS AND METHODS

Procedures with animals were conducted in accordance with the guidelines for animal research of the French Ministry of Agriculture and applicable European guidelines and regulations for experimentation with animals (www2.vet-lyon.fr/ens/expa/acc_regl.html; verifi ed January 24, 2013).

Monascus Strains and Culture

Seven strains of Monascus sp. were tested. Three isolates, unidentifi ed at the species level were obtained from corn silage in our laboratory (Strains 1 to 3); a Monascus sp. and a Monascus ruber strain were a gift from INSA Toulouse (Strains 4 and 5); and the last 2 strains of Monascus purpureus 1603 isolated from Chinese anka and Monascus ruber 62748 isolated from corn silage, Strains 6 and 7, respectively, were purchased through the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). The media used to grow the strains were 2 rice-based solid media and 2 liquid media. Rice media was prepared by soaking rice overnight with tap water on a 1:1 ratio. The next day, free water was drained, 100 g of wet rice were transferred to 250-mL Erlenmeyer fl asks and autoclaved (120°C, 20 min). The sterilized wet rice was used as is (i.e., without any additional component), or enriched by aseptically adding 1 g of ammonium sulfate, reported to increase monacolin K production [START_REF] Su | Production of the secondary metabolites gamma-aminobutyric acid and monacolin K by Monascus[END_REF]. Liquid medium favoring monacolin K production was prepared according to [START_REF] Su | Production of the secondary metabolites gamma-aminobutyric acid and monacolin K by Monascus[END_REF]. The medium contained per liter 60 g glucose, 25 g peptone, 5 g corn-steep liquor, and 5 g ammonium chloride. Liquid medium favoring citrinin production was based on a YES medium according to [START_REF] Blanc | Characterization of monascidin A from Monascus as citrinin[END_REF]. Fifty milliliters of liquid media were dispensed in culture fl asks and autoclaved (120°C, 20 min).

Fungal inocula were prepared by culturing the strains in potato dextrose agar medium, except for number 6 that was maintained in Yeast protein Soluble starch (YpSs, M190 medium of DSMZ) agar medium. After 5 d of incubation at 25°C, spores were collected by washing the surface of cultures with 0.05% Tween 80 and enumerated in a Malassez hemocytometer. Each fl ask was inoculated with 3.5 × 10 5 spores, incubated at 30°C for 2 d and then at 25°C for 2 wk. Cultures were manually agitated every 24 h for the fi rst 3 d of incubation. Each medium-strain combination was incubated in duplicate. At the end of the incubation, 100 mL of 67% ethanol were added to the solid media, stirred in a rotary agitator for 90 min, and fi ltered through fi lter paper (0.45 μm, Sartorius, Aubagne, France). Aliquots (20 μL) of fi ltrates were then injected into the HPLC system for analysis and the remaining volumes were stored at 4°C until use in in vitro assays. Several ethanolic extracts from cultures were prepared as above and used in the different in vitro experiments described below.

For the in vivo trial, wet rice was prepared in the same way as above, but the incubation was done in 1.2-L plastic jars (SACO2, Eke, Belgium) that were inoculated with a piece of a Monascus culture grown on agar (1/8 of a 100 mm Petri dish) and incubated at 30°C for 3 wk with periodic manual mixing. At the end of the 3-wk incubation, cultured rice was taken out of jars, spread in trays, and partially dried at 45°C for 2 d to eliminate excess humidity. Contents of all jars were thoroughly mixed to produce a homogenous lot and stored at -20°C in plastic bags until use. The amount needed to feed experimental animals for 1 d was thawed at room temperature overnight before use.

Metabolite Analysis

Separation and quantifi cation of Monascus metabolites were performed at room temperature by HPLC, using UV detection for monacolin K, mevastatin, and pravastatin; and fl uorometry for citrinin. The HPLC system consisted of a P1000XR pump (SpectraSYSTEM, San Jose, CA) and an automatic sampler (SpectraPhysics, Santa Clara, CA). Separation was performed on a C18 Gravity Nucleodur reversedphase column: 150 × 4.6 mm, 5 μm (Macherey-Nagel, Hoerdt, France) using a gradient solvent (Solvent A: 0.5% acetic acid, and Solvent B: 0.5% acetic acid/ acetonitrile) at a fl ow rate of 1 mL/min. The gradient was started at 40% of B, which increased to 75% in 15 min; maintained at 75% for 4 min, and then returned to the initial condition in 1 min. The detection was set at 238 nm in UV and at 336 nm excitation and 440 nm emission in fl uorescence mode. The retention times were 3, 8, 19, and 20 min for pravastatin, citrinin, mevastatin, and monacolin K, respectively. Standards were obtained from Sigma (St. Louis, MO). Working solutions at adequate concentrations were prepared by dissolving in ethanol, except for pravastatin, which was dissolved in methanol. Aliquots were stored at -20°C until use.

In Vitro

Rumen fl uid was obtained from 3 rumen-cannulated Texel wethers fed a hay diet twice daily, at 0800 and 1600 h. Wethers had unrestricted access to water and a mineral salt block supplement. Whole rumen contents were collected before the morning feeding and strained through a polyester monofi lament fabric (250-μm mesh aperture) to remove solids. Equal volumes of rumen fl uid from each animal were pooled and then mixed in a 1:5 ratio with an anaerobic buffer solution [START_REF] Weller | Passage of protozoa and volatile fatty acids from the rumen of the sheep and from a continuous in vitro fermentation system[END_REF] kept at 39°C under oxygen-free carbon dioxide gas. This rumen fl uid-buffer mixture was used immediately after preparation for fermentation experiments.

A total of 5 mL of the rumen fl uid-buffer mixture were transferred to Hungate tubes containing 100 ± 2 mg alfalfa hay (470 g NDF, 156 g CP per kg DM) followed by the addition of 100 μL of Monascus extracts. Control tubes contained 100 μL of extract from unfermented rice prepared as per Monascus extracts. Extracts were adjusted so as to have a fi nal concentration of monacolin K in the fermentation media of 5 and 20 μg/mL. Pure monacolin K and citrinin were also used as treatments. The tubes were capped, keeping the anaerobic conditions in the head space and incubated horizontally at 39°C in a shaking water bath for 18 or 24 h. At the end of the incubation period gas production was measured with the aid of a pressure transducer and samples were collected for analysis of constituents by gas chromatography. For VFA, 0.8 mL fermentation liquid were transferred to a microcentrifuge tube containing 0.5 mL of a 0.5 N HCl solution containing 2% (wt/vol) metaphosphoric acid and 0.4% (wt/vol) crotonic acid, kept at 4°C for 2 h and centrifuged (16,500 × g, 10 min, 4°C). Supernatants were stored at -20°C until analysis. All treatments were assayed in triplicate and the experiment was repeated twice (3 × 2).

In a second series of experiments, the effect of Monascus extracts was tested using a consecutive batch culture technique [START_REF] Theodorou | Effect of phenolic acids and phenolics from plant cell walls on rumen like fermentation in consecutive batch culture[END_REF]. The fi rst culture was prepared as described above and incubated for 48 h in triplicate. Two milliliters of each replicate were mixed and 1.5 mL was used to inoculate 3 Hungate tubes containing alfalfa hay substrate, extracts, to have a fi nal concentration of monacolin in the fermentation media of 20 μg/mL, and 3.5 mL of anaerobic buffer and incubated for another 48 h. Including the fi rst incubation, a total of 3 serial cultures were performed.

In addition to extracts, Monascus-fermented rice was also used as a substrate for in vitro incubations. Mixed rumen fl uid was prepared as above and 10 mL were immediately inoculated into 120-mL vials containing 30 mL of an anaerobic buffer solution kept at 39°C, and 500 mg of ground (1-mm sieve) Monascus-fermented rice (868 g starch, 10 g NDF, 94 g CP per kg DM-values before fermentation from INRA Tables 2007;Beaumont et al., 2007) alone (or combined at a 1:1 ratio (250 mg each) with alfalfa hay (470 g NDF, 156 g CP per kg DM). Monascus-fermented rice was replaced by unfermented rice in controls. Vials were incubated anaerobically at 39°C for up to 24 h. At the end of the incubation period gas production was measured with the aid of a pressure transducer and samples were collected for analysis of constituents by gas chromatography. Samples for VFA were prepared as above. All treatments were assayed in triplicate and the experiment was repeated twice. The consecutive batch culture technique was also used on the Monascus-fermented rice using 3 consecutive series transferred every 48 h. For the second and third series, at the end of each 48-h incubation, 15 mL of culture were transferred into vials containing 250 mg fermented or unfermented (control) substrate and 15 mL of anaerobic buffer.

In Vivo

The experiment was conducted at the animal experimental facilities of the INRA's Herbivores Research Unit (Saint-Genès Champanelle, France). Six adult Texel wethers fi tted with rumen cannulae were used in this study. Animals were housed in individual pens and had unrestricted access to water and a mineral salt block supplement. They were weighed regularly during the study; at the start and end of the experiment, average BW was 63.7 ± 3.7 kg and 63.3 ± 4.5 kg, respectively. Wethers were restricted fed on a DM basis 600 g of rice and 600 g of a poor quality prairie hay (NDF 730 g/kg DM and ADF 399 g/kg DM) twice a day at 0800 and 1600 h during 10 wk. Wet rice was prepared daily by adding 40% (wt/vol) of warm water and letting it soak overnight. In wk 7 and 8, wet rice was replaced by an equal amount of Monascus-fermented rice (60% DM). Methane production measurements were done as before [START_REF] Martin | Methane output and diet digestibility in response to feeding dairy cows crude linseed, extruded linseed, or linseed oil[END_REF][START_REF] Morgavi | Changes in methane emission and rumen fermentation parameters induced by refaunation in sheep[END_REF] on wk 3 and 4 and daily from wk 7 to 10 (Fig. 1). Rumen content samples for fermentation and microbiological parameters were taken through the cannulae before the morning feeding at the end of each methane measurement week (5 times). The pH was measured immediately after collection. A portion of the sample (~150 g) was strained through a polyester monofi lament fabric (250-μm mesh aperture) and samples of the liquid fi ltrate were taken and stored as described for in vitro experiments for VFA analysis. For protozoa counts, strained rumen fl uid was sampled, stored, and analyzed as described below. Another portion of the whole rumen sample (~50 g) was homogenized for three 1-min cycles with 1-min intervals on ice using a Polytron grinding mill (Kinematica GmbH, Steinhofhalde, Switzerland). Approximately 0.5 g were transferred to 2-mL Eppendorf tubes and stored at -80°C for molecular biology analyses.

Measurements

Volatile fatty acids were analyzed by gas chromatography (CP 9002 Chrompack, Middelburg, Germany) using a wall-coated open-tubular fused silica column (0.25 mm i.d. × 25 m) coated with CP-wax 58 (FFAP)-CB [START_REF] Morgavi | Prevention of patulin toxicity on rumen microbial fermentation by SHcontaining reducing agents[END_REF]. Fermentation gases were analyzed by gas-chromatography (Micro GC 3000A, Agilent Technologies, Les Ulis, France). Individual gas molar concentration was calibrated using a certifi ed standard (relative accuracy of 2%, ALPHAGAZ No. 073562.00).

Methane production in vivo was determined using the sulfur hexafl uoride (SF 6 ) tracer technique [START_REF] Johnson | Measurement of methane emissions from ruminant livestock using a SF 6 tracer technique[END_REF] as described by [START_REF] Martin | Methane output and diet digestibility in response to feeding dairy cows crude linseed, extruded linseed, or linseed oil[END_REF]. A calibrated permeation tube containing ultrapure SF 6 was introduced into the rumen of each sheep through the rumen cannulae 2 wk before the initiation of measures. The expected useful life, which is the length of time when SF6 release is constant (i.e., zero-order release of each permeation tubes), was calculated based on the diffusion rates of SF6 (1115 ± 149 ng/min, mean ± SD). As a precaution, only tubes that had a calculated useful life that was at least 8 wk longer than the expected end of the experiment were used.

The protozoal, bacterial, and archaeal communities present in rumen contents were characterized by different methods. For protozoa, strained rumen fl uid samples were mixed with methylgreen-formalin solution in a 1:1 ratio and stored at room temperature in the dark until used for protozoal counting. Samples were diluted in methylgreen-formalin solution if necessary and enumeration was done using a Neubauer counting chamber. Bacteria and archaea were evaluated by quantitative (q) PCR and PCR-denaturing gradient gel electrophoresis (DGGE). Total genomic DNA was extracted using the method described by [START_REF] Yu | Improved extraction of PCRquality community DNA from digesta and fecal samples[END_REF] and quantifi ed by spectrophotometry using a NanoQuant Plate on an Infi nity spectrophotometer (TECAN, Männedorf; Switzerland).

The qPCR for methanogens was done using primers targeting the methyl coenzyme-M reductase (mcrA) gene of the methanogenesis pathway as described by [START_REF] Denman | Quantitation and diversity analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane[END_REF] and total bacteria were quantifi ed using primers targeting the rrs gene [START_REF] Edwards | Characterization of the dynamics of initial bacterial colonization of nonconserved forage in the bovine rumen[END_REF]. Assays were run in triplicate using the SYBR Premix Ex Taq kit (Lonza, Levallois-Perret, France) on a StepOnePlus system (Applied Biosystems, Foster City, CA). Negative controls without DNA template were run with every assay to assess the overall specifi city. The PCR amplifi cation effi ciency was checked as described before [START_REF] Mosoni | Longterm defaunation increases the abundance of cellulolytic ruminococci and methanogens but does not affect the bacterial and methanogen diversity in the rumen of sheep[END_REF] using standard curves, 10 8 to 10 3 copies prepared from mcrA or rrs DNA fragments amplifi ed from genomic DNA of Methanobrevibacter smithii DSM861 and a mixture of 11 rumen bacterial species, respectively. The slope and effi ciency for mcrA and rrs primers were: -3.74 and 85% and -3.58 and 90%, respectively, with R 2 being greater than 0.99 in both cases. The PCR effi ciency with DNA samples was confi rmed to be similar to that obtained with the standard curves. For each rumen content sample, the results were expressed as the mean of 3 replicates in rrs or mcrA copies per gram of rumen content. In addition the relative abundance of methanogens relative to the abundance of total bacteria considered as the reference gene was calculated using the comparative C T method (2 -ΔC T ; [START_REF] Denman | Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen[END_REF][START_REF] Schmittgen | Analyzing real-time PCR data by the comparative C(T) method[END_REF].

For PCR-DGGE, the mcrA gene was also targeted using primers

5 ´-G G T G G T G T M G G AT T C A C A C A RTAY G C - WACAGC-3´
and 5´-TTCATTGCRTAGTTWGGR-TAGTT-3´, forward and reverse, respectively [START_REF] Luton | The mcrA gene as an alternative to 16S rRNA in the phylogenetic analysis of methanogen populations in landfi ll[END_REF]. The use of mcrA as a target in DGGE analysis has been used before [START_REF] Galand | Depth related diversity of methanogen Archaea in Finnish oligotrophic fen[END_REF][START_REF] Wilms | Methane and sulfate profi les within the subsurface of a tidal fl at are refl ected by the distribution of sulfate-reducing bacteria and methanogenic archaea[END_REF]. The forward primer had a 40 bp GC clamp added at its 5´ end [START_REF] Muyzer | Profi ling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplifi ed genes coding for 16S rRNA[END_REF]. The PCR mixture (50 μL) contained 1 × PCR buffer (Qiagen GmbH, Hilden, Germany), 4.5 mM MgCl 2 , 0.25 μM of each primer, 200 μM of each dNTP, 2.5 U HotStar Taq polymerase (Qiagen) and 50 ng extracted DNA. The PCR conditions were those used by [START_REF] Luton | The mcrA gene as an alternative to 16S rRNA in the phylogenetic analysis of methanogen populations in landfi ll[END_REF], with a 5-cycle slow ramp protocol to allow the extension of mismatched primers, except for the initial denaturation and fi nal extension steps. The initial hotstart denaturation was done for 15 min at 95°C and the fi nal extension step at 72°C was done for 30 min to eliminate artifactual double bands [START_REF] Janse | A simple remedy against artifactual double bands in denaturing gradient gel electrophoresis[END_REF]. For bacteria, the V3 variable region of the rrs gene was targeted with primers 341f-(5´-CCTACGGGAGGCAGCAG-3´) and 534r (5´-ATTACCGCGGCTGCTGG-3´; [START_REF] Muyzer | Profi ling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplifi ed genes coding for 16S rRNA[END_REF] as described by [START_REF] Sadet | PCR-DGGE analysis reveals a distinct diversity in the bacterial population attached to the rumen epithelium[END_REF].

The DGGE was performed as described by [START_REF] Sadet | PCR-DGGE analysis reveals a distinct diversity in the bacterial population attached to the rumen epithelium[END_REF]. Briefl y, the gel contained 8% (wt/vol) polyacrylamide in 0.5 × Tris-acetate-EDTA buffer (TAE) with a denaturant gradient of 20 to 55% [100% denaturant is 7 M urea and 40% (vol/vol) formamide]. Electrophoresis was done for 5 h at 200 V at 60°C. Gels were silver stained using a commercial kit (Bio-Rad Laboratories, Hercules, CA) and images were acquired using an optical density calibrated scanner (ImageScanner, GE Healthcare, Piscataway, NJ) at a spatial resolution of 400 dpi. Images were analyzed using GelCompar II version 4.0 package (Applied Maths, Kortrijk, Belgium). GelCompar II was used to normalize and compare all the DGGE patterns using hierarchical clustering to join similar patterns into groups [START_REF] Fromin | Statistical analysis of denaturing gel electrophoresis (DGE) fi ngerprinting patterns[END_REF]. To this end, all the images of DGGE gels were matched using the internal control sample and the bands were quantifi ed after a local background subtraction. A tolerance in the band position of 1% was applied. The similarity among patterns was calculated with the Pearson product-moment correlation coeffi cient, recommended for the analysis of this type of profi les [START_REF] Savelkoul | Amplifi ed-fragment length polymorphism analysis: The state of an art[END_REF], and the clustering was done using the unweighted pair-group method with arithmetic averages (UPGMA). The lowest percentage of similarity between standards on the gel was 77.8 and 90.4 for bacteria and methanogens, respectively.

Statistical Analysis

In vitro data were statistically analyzed by 1-way ANOVA using the General Linear procedures (SAS Inst. Inc., Cary, NC) and fermentation vials as experimental units. Differences among means were tested against a unique control using the Dunnet adjustment option in the fi rst in vitro experiment. In the other experiments containing more than 1 control treatment, the Tukey adjustment option was used. In vivo data were analyzed in repeated measures using the MIXED procedure of SAS. The model included the fi xed effect of sampling period and the random effect of wether. Best fi tting covariance structure was compound symmetry for all variables studied. Signifi cance was declared at the P < 0.05 probability level, and trends were discussed at P < 0.10 probability level.

RESULTS

Production of Metabolites by Different Monascus Strains

All strains grew well on the media used to monitor metabolite production. The concentration in different media of the 4 metabolites monitored is shown in Table 1. In liquid media the only metabolite detected was monacolin K, but only for some strains and at less concentrations than that found in solid media (data not shown). Monacolin K was also the only metabolite produced by all strains although the differences in concentration were large, ranging from 7 up to 570 mg/ kg. Addition of a nitrogen source to increase production [START_REF] Su | Production of the secondary metabolites gamma-aminobutyric acid and monacolin K by Monascus[END_REF] was effective in Strains 1, 2, and 3 only. Strains 4 and 5 did not produce or were low producers of metabolites. Strain 6 was the largest producer of monacolin K and mevastatin but also of citrinin and was not further considered because the concentration of this toxic metabolite was considered too high. Maximum acceptable levels of citrinin proposed in the EU for feeds and foods is 100 ng/g whereas in Japan the legal limit has been set at 200 ng/g [START_REF] Shi | Benefi cial effects of Monascus purpureus NTU 568-fermented products: A review[END_REF]. Strain 7 cultured on rice, which had the greatest monacolin K to citrinin ratio, and Strain 1, which had a lower monacolin K to citrinin ratio were chosen for in vitro studies with rumen fl uid.

In Vitro Experiments

Monascus extracts, particularly those from Strain 7, reduced the production of methane in 18-h incubations without negatively affecting fermentations as assessed by total gas and VFA production (Table 2). When used at the concentration of 20 μg of monacolin equivalent (Monascus 7 high), the production of gas and VFA were even stimulated. Pure secondary metabolites gave contrasting results. Citrinin had a marked negative effect on methane production that was reduced to trace amounts but also reduced fermentation. In contrast, monacolin K did not affect either gas or methane production. γ-Aminobutyric acid, another common secondary metabolite with biological activity produced by Monascus did not negatively affect fermentations.

Monascus extracts and citrinin increased the proportion of propionate and butyrate and reduced that of acetate.

As the original extract preparations were all consumed in the in vitro tests replications, new extracts from Monascus Strains 1 and 7 cultured in different media as described in the Materials and Methods section were prepared. The effect of the different extracts obtained on gas production and methane production were tested as before, and whereas most extracts stimulated gas production, the effect on methane was disappointing as none of them had a reducing effect (data not shown). Monascus cultures were regenerated and in vitro batch experiments repeated, but similar results were obtained.

In a second series of experiments, we used the consecutive batch culture technique [START_REF] Theodorou | Effect of phenolic acids and phenolics from plant cell walls on rumen like fermentation in consecutive batch culture[END_REF]. Only extracts from Monascus Strain 7 were used in this sequence of experiments. In the fi rst batch, no major differences were observed between Monascus extract and control incubations (Table 3). In the second and third batch there was a shift in VFA proportions, with a decrease in acetate and an increase in propionate. Methane production decreased by 86% in the third batch with a smaller reduction, about 10%, in the amount of gas and VFA produced, although changes in VFA were not signifi cant. The recovery of hydrogen estimated from the amount of acetate, propionate, butyrate, and methane formed [START_REF] Demeyer | Quantitative aspects of microbial metabolism in the rumen and hindgut[END_REF] was 100% in controls, but only 60% in Monascus extracts. The effect of Monacolin K was intermediate with methane production reduced by 35% in the third batch (P = 0.041).

In vitro experiments were also performed with Monascus-fermented rice without extraction. The rice employed as the solid media for culturing Monascus Strain 7 was dried and used as substrate in 72-h batch incubations and in consecutive batch technique experiments. In batch incubations, fermented rice was used as the sole substrate or combined with hay. Results at the end of the 72-h incubation are shown in Table 4. Gas production kinetics throughout the incubation for rice alone are shown in Fig. 2A. Monascusfermented rice was more rapidly degraded at early incubation times, but gas production was the same between treatments at the end of the incubation time. Monascus-fermented rice substrate, when used alone, increased VFA production (P = 0.040). Fermented rice also tended to increase molar proportions of propionate 1 Ethanolic extracts from rice cultures of Monascus Strains 1 and 7 adjusted to an equivalent concentration of monacolin K of 5 (low) or 20 μg/mL (high). Pure monacolin and citrinin treatments were used at a concentration of 5 (low) or 20 μg/mL (high). Control contained the same amount of ethanol than treatments.

2 Total gas and methane produced in 5 mL fermentation volume containing 100 mg alfalfa hay substrate at the end of the incubation period.

3 Valerate. 4 Means values (n = 6) within a column differ from control (P < 0.10).

5 ND: not detected, less than 100 mg/m 3 .

6 Main effect of treatment. (P = 0.074) and decrease that of acetate and the acetate to propionate ratio (P = 0.088). Methane production, although not signifi cantly, was numerically less with fermented rice as substrate, particularly when used alone (Table 4 and Fig. 2B). Amount of methane per milliliter of gas produced was less than control at 8 h, and was numerically reduced after 72-h incubation times (Fig. 2B inset). In consecutive incubations, Monascus-fermented rice differed from the control in the molar proportion of VFA, with increases in propionate and butyrate and decreases in acetate (Table 5). In the fi rst 24-h batch, the amount of gas and VFA produced was greater than control. Methane production did not differ from control in the fi rst batch, but decreased in the third batch by up to 60% of control (P = 0.006). At the same time, an increase (P = 0.029) (n = 3) within a column and for each batch differ from control (P < 0.05). Signifi cant main effect of treatment with no differences between control and Monascus metabolites are not indicated by * symbol.

Signifi cant main effect of treatment with no differences between control and Monascus metabolites are not indicated by * symbol.

1 Ethanolic extracts from rice culture of Monascus Strain 7 adjusted to an equivalent concentration of monacolin K of 20 μg/mL. Pure monacolin was used at a concentration of 20 μg/mL. Control contained the same amount of ethanol than treatments.

2 Total gas and methane produced in 5 mL fermentation volume containing 100 mg alfalfa hay substrate at the end of the incubation period.

3 Valerate and caproate. 4 Means values (n = 3) within a column and for each batch followed by differ from control (P < 0.10).

5 Main effect of treatment. ) within a column differ from corresponding, nonfermented substrate control (P < 0.05). 1 Total gas and methane produced in 40-mL fermentation vials containing 500 mg substrate at the end of the incubation period.

in the amount of dihydrogen accumulation, which was not observed in the other in vitro experiments (data not shown), was detected.

In Vivo Trial

Methane emissions in adult wethers decreased when rice was replaced by Monascus-fermented rice (P < 0.001; Table 6). The abatement was up to 30% compared with measures before the treatment and, as shown in Fig. 3, the effect was relatively quick to take place and was clearly observed on the third day of feeding. Figure 3 boxplot also shows that values 1.5 times lower and higher of the interquartile range were often attributed to the same individuals. Wethers were on restricted intake during the 10-wk-long experiment, and results are expressed as grams of methane per day, as no refusals were recorded. Body weight was measured 5 times at regular intervals and was stable over the whole experiment (P > 0.1; 63.5 ± 3.92 kg, mean ± SD). Rumen concentrations of VFA varied throughout the trial. During the treatment (S2 and S3) they were less than at the beginning of the trial (S1), but did not differ from the values obtained at the end of the trial (S5). Concentrations of VFA posttreatment were greater than in the previous periods (Table 6). Compared with the pretreatment period, the molar proportion of acetate to total VFA decreased (P < 0.001) and that of propionate (S1 vs. S2; P = 0.077) and butyrate increased numerically during treatment. The acetate to propionate ratio decreased as well.

The treatment stimulated slightly but in a signifi cant manner the concentration of bacteria in the rumen (rrs copies/g DM rumen content) and decreased the concentration of methanogens (mcrA copies/g DM rumen contents; P = 0.006 and 0.012, respectively). The proportion of methanogens relative to bacteria decreased threefold during treatment as compared with values before treatment (P < 0.001; Table 6). In contrast, no changes were observed in the number and proportion of main protozoal groups. Protozoa populations were composed of 97.1% small entodiniomorphs, 1.6% large entodiniomorphs (>100 μm), and the rest composed by Holotrichs Dasytricha ruminantium and Isotricha spp., 1.23 and 0.12%, respectively.

The profi les of the bacterial community from individual wethers obtained by PCR-DGGE and analyzed by clustering of the whole densitometric curves showed that samples obtained in Sampling Periods 2 and 3 formed 3 small clusters at the fi rst, second, and third nodes, whereas samples taken before (S1) and after the treatment (S4 and S5) grouped together and exhibited a greater percentage of similarity between them (Fig. 4). Wether Number 6 had an unusual, highly different profi le in S2, with only 26% similarity with the rest of the samples (data not shown). As it was not possible to exclude a technical problem with this sample, Wether 6 was not included in the analysis. In contrast, methanogens (mcrA gene) profi les were not affected by the treatment (data not shown).

DISCUSSION

Secondary metabolites from Monascus reduced rumen methane production in vitro and in vivo without any apparent negative effect on fermentation variables. However, not all the strains tested were effective for decreasing methanogenesis. In addition, the culture conditions also seem important for the production of the active molecules. Screening of strains is important, as we show that among the secondary compounds monitored, citrinin had a marked negative effect on fermentations. Strains producing this mycotoxin have also to be avoided for feed safety reasons [START_REF] Shi | Benefi cial effects of Monascus purpureus NTU 568-fermented products: A review[END_REF] and for reducing toxicity incidents in ruminants (Griffi ths and Done, 1991). Culture conditions favoring 24, 48, and 72 h, respectively; 1.42, 2.71, 2.56, and 1.91 for A; 22.03, 32.19, 41.49, and 32.92 for B; and1.19, 0.68, 0.77, and1.13 for inset. *Signifi cant (P < 0.05) between treatments at equal incubation time.

citrinin production in Monascus are known and should be used to screen out any citrinin-producing strain before testing them on animals. It should be noted that Monascus spp. produce many secondary metabolites [START_REF] Shi | Benefi cial effects of Monascus purpureus NTU 568-fermented products: A review[END_REF] but only 4 were monitored as they were commercially available to us and, at this stage, we cannot affi rm that these metabolites were responsible of the antimethanogenic activity.

There is a large body of literature describing the effect of natural compounds, particularly plant- Protozoa,7 log 10 cells/mL rumen fl uid 5.9 5.8 6.0 5.8 5.8 0.08 0.68 a-c Within a row, means without a common superscript letter differ (P < 0.05).

1 Sampling time, see Fig. 1.

2 Main effect of treatment.

3 Mol/100 mol. 4 Signifi cant main effect of treatment with no differences between sampling time means compared by Tukey's test. 5 Estimated by quantitative PCR. 6 Relative quantifi cation assay using methanogens' mcrA gene and total bacteria rrs gene for normalization. derived products, as antimethanogenic agents on rumen fermentations in vitro [START_REF] Calsamiglia | Essential oils as modifi ers of rumen microbial fermentation[END_REF]Benchaar and Greathead, 2011). However, the assessment in animals of compounds selected in vitro is still scarce. Animal trials performed using some of these in vitro promising compounds failed to show a practical methane mitigation effect under the conditions studied (e.g., [START_REF] Holtshausen | Feeding saponin-containing Yucca schidigera and Quillaja saponaria to decrease enteric methane production in dairy cows[END_REF]Klevenhusen et al., 2011b;[START_REF] Van Zijderveld | Dietary inclusion of diallyl disulfi de, yucca powder, calcium fumarate, an extruded linseed product, or medium-chain fatty acids does not affect methane production in lactating dairy cows[END_REF]. In the work presented here, the traditional in vitro batch system was marginal or ineffective at detecting an antimethanogenic activity (except for the extracts used in 1 experiment; discussed below). In vitro effects were clearly observed using a consecutive batch culture approach. Whether this method is representative of the rumen function could be a matter of debate, as the composition of the microbiota at the end of the incubation period is certainly not the same than the initial inoculum. However, in this work the method was useful, as it gave a better indication of the antimethanogenic potential of different strains and solid state fermentation conditions (H. Boudra and D. Morgavi, unpublished data) than traditional batch fermentations. In contrast to other products reported in the literature, reduction in methane by metabolites of Monascus was more apparent in our in vivo than in our in vitro studies. In the proof of concept, sheep trial, an important 30% enteric methane reduction effect was observed. An observation emanating from the trial was that methane production values placed outside the whiskers in the box plot graph might suggest different animal susceptibility to the treatment and perhaps also differences in the capacity to reestablish the methanogenesis function after the disturbance to the microbial community produced by the treatment, akin to that described after antibiotic treatment [START_REF] Dethlefsen | Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation[END_REF]. It should be mentioned that there was a confounding effect of time in the experimental design used that could also have an effect on methane emissions. This effect, if any, should be minor under the controlled conditions of the assay but certainly need to be considered in future trials. We were limited in the amount of Monascus-fermented rice available and that was the reason of the relatively short duration of the animal experiment. We are working on improving the solid state fermentation process and on the use of other feed substrates to conduct other research on this subject.

A hypothesis was that monacolin K was the active molecule. This compound has been cited as one of the most biologically active in red mold rice [START_REF] Shi | Benefi cial effects of Monascus purpureus NTU 568-fermented products: A review[END_REF]. Monacolin K and other polyketide compounds belonging to the statin group are HMGCR inhibitors and have cholesterol-lowering effect in mammals. The HMGCR inhibitors, such as lovastatin and mevastatin, have been shown to suppress the growth of rumen methanogens in pure culture [START_REF] Miller | Inhibition of growth of methane-producing bacteria of the ruminant forestomach by hydroxymethylglutaryl-SCoA reductase inhibitors[END_REF]. In mixed rumen cultures, lovastatin was not effective in short-term batch incubations [START_REF] Busquet | Effect of garlic oil and four of its compounds on rumen microbial fermentation[END_REF], but reduced methane production by ~40% in a semicontinuous rumen fermentor (Rusitec; [START_REF] Soliva | Ruminal methane inhibition potential of various pure compounds in comparison with garlic oil as determined with a rumen simulation technique (Rusitec)[END_REF]. The incubation and adaptation of the microbiota to the treatment were different between these 2 reports but also the doses used; 5 mg/L in the study of [START_REF] Busquet | Effect of garlic oil and four of its compounds on rumen microbial fermentation[END_REF] and 150 mg/L in that of [START_REF] Soliva | Ruminal methane inhibition potential of various pure compounds in comparison with garlic oil as determined with a rumen simulation technique (Rusitec)[END_REF]. Lovastatin was not effective in sheep in the only in vivo trial that we are aware of (Klevenhusen et al., 2011a). Monacolin K was not previously assayed in vitro or in vivo on the rumen and, based on our results, it is not possible to confi rm or refute the hypothesis that monacolin K was responsible for the effect on fermentation and methane. Extracts used in in vitro experiments were all adjusted to have equal concentration of monacolin K, yet their activity was not the same. In addition, monitoring monacolin K during solid state fermentations is not clearly correlated to the antimethanogenic activity (H. Boudra and D. Morgavi, unpublished data). We also used LC-MS to detect monacolin K and analogs produced by Monascus spp., but until now it is diffi cult to correlate metabolic profi les with biological activities (H. Boudra and D. Morgavi, unpublished data). The lack of an effect reported with pure lovastatin (Klevenhusen et al., 2011a) might suggest that the activity of secondary metabolites produced by Monascus spp. is due to a mix of compounds and not only HMGCR inhibitors. In contrast, Monascusfermented rice reduced the number of methanogens in wethers, whereas bacterial populations were increased. In addition, the results obtained with the consecutive batch method suggest a microbiostatic and specifi c effect against methanogens. Taken together, these effects could be ascribed to the inhibition of HMGCR enzymes involved in the mevalonate pathway, which is present in archaea methanogens but absent in most prokaryotes [START_REF] Friesen | The 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductases[END_REF]. The mechanism of action of Monascus metabolites, however, remains to be validated. It is worth noting that there was a signifi cant effect on numbers and structure (evidenced with PCR-DGGE) of the rumen bacterial community in treated wethers, indicating that reduction in methane emissions are not exclusively due to changes in methane producers, but also to other members of the rumen microbiota [START_REF] Morgavi | Microbial ecosystem and methanogenesis in ruminants[END_REF].

This work showed that commonly used in vitro batch cultures were not adequate to detect an antimethanogenic activity that was observed in vivo. This result challenges the principle that if it does not work in vitro, it is not worth testing in vivo. We have recourse to a consecutive batch technique that was capable of detecting the antimethanogenic activity in vitro. Although consecutive batch cultures might be less representative of the rumen ecosystem than other in vitro techniques, it could be useful for improving the chances of fi nding promising candidates using a battery of relatively inexpensive in vitro methods.

Monascus metabolites stimulated in vitro fermentation, suggesting that they do not negatively affect digestibility and feed utilization, an essential aspect for a potential adoption of the technology. The effect on digestibility needs to be confi rmed in vivo though.

Future works for establishing the economic viability and sustainability of the technology should be directed to the improvement of the solid fermentation process, the evaluation of an optimal dose, and the long-term effi cacy.

Conclusions

Secondary fungal metabolites from Monascus spp. reduced enteric methane emissions in sheep by 30% in a short-term trial. Reduction of methane was accompanied, both in vitro and in vivo, by a shift in VFA pathways, decreasing the acetate to propionate ratio. The main microbial modifi cations observed were a reduction in methanogens numbers, suggesting a specifi c and toxic effect on this microbial group and a concomitant increase in bacterial numbers, with marked changes in the structure of the bacterial community.

LITERATURE CITED

Figure 1 .

 1 Figure 1. Diagram showing methane measurement days and rumen content sampling (S1 to S5) on wethers (n = 6) before (pretreatment), during (treatment), and after (posttreatment) feeding with Monascus-fermented rice. Rumen samples were taken before feeding.

Figure 2 .

 2 Figure 2. Production of (A) gas and (B) methane by mixed rumen microbes in the presence of rice (diamonds) or Monascus Strain 7 fermented rice (squares) used as substrate. Inset shows methane production in μmol per mL of gas produced at each incubation time. Values are means (n = 6); SEM at 8,24, 48, and 72 h, respectively; 1.42, 2.71, 2.56, and 1.91 for A; 22.03, 32.19, 41.49, and 32.92 for B; and 1.19, 0.68, 0.77, and 1.13 for inset. *Signifi cant (P < 0.05) between treatments at equal incubation time.

Figure 3 .

 3 Figure 3. Daily methane emissions of wethers (n = 6) before, during, and after feeding with Monascus-fermented rice. Each box gives the mean values (diamonds), the median (line across the box), the upper and lower quartiles, and the maximum and minimum value (bars) within 1.5 interquartile range of the higher and lower quartile, respectively. Outlier symbols are followed by wether number. Unfi lled arrows indicate the start and end of the treatment and gray arrows indicate rumen content sampling (S1 to S5).

Figure 4 .

 4 Figure 4. Denaturing gradient gel electrophoresis profi les and unweighted pair-group method using arithmetic averages dendrogram generated from rumen bacteria (rrs gene). Rumen content samples from wethers (n = 5) taken before (open square, S1), during (fi lled square, S2 and S3), and after (open square, S4 and S5) feeding with Monascus-fermented rice.

Table 2 .

 2 Effect of Monascus spp. metabolites on rumen fermentation characteristics of 18-h in vitro incubations

					VFA
	Treatment 1	Gas 2	Methane 2	Total	Acetate (Ac)	Propionate (Pr)	Butyrate	Iso-acids	Minor 3	Ac:Pr ratio
		mL	μmol	mmol/L	----------mol per 100 mol ----------
	Control	5.8	61.2	96.1	71.2	22.1	4.8	1.0	0.9	3.2
	Monascus 1 low	5.9	64.4	98.5	70.6	22.6	5.0	0.9	0.9	3.1
	Monascus 1 high	5.5	45.1	93.5	63.6*	28.1*	6.2*	0.9	1.2 4	2.3*
	Monascus 7 low	5.7	28.4*	102.1	63.2*	29.3*	5.9*	0.6*	1.0	2.2*
	Monascus 7 high	7.4*	33.6 4	111.3*	54.8*	35.3*	8.3*	0.4*	1.2	1.6*
	Monacolin low	6.1	60.3	99.0	70.8	22.2	4.9	1.0	1.0	3.2
	Monacolin high	6.4	67.4	100.6	72.2	21.2	4.6	1.0	0.9	3.4
	Citrinin low	4.7*	ND* 5	82.0*	63.7*	28.0*	5.9*	1.2*	1.2	2.3*
	Citrinin high	3.1*	2.4*	64.4*	63.6*	28.4*	5.8 4	1.4*	0.9	2.2*
	SEM	0.19	6.59	2.99	0.76	0.70	0.27	0.04	0.10	0.08
	P-value 6	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
	*Means values (n = 6) within a column differ from control (P < 0.05).

Table 1 .

 1 Metabolite production of various Monascus strains grown on different culture media

						Strain
	Metabolite	Medium 1	1	2	3	4	5	6	7
			-------mg/kg -------
	Monacolin K	rice	260 265 239	7	8 570 240
		N-enriched rice 322 349 297	ND 2 1 331 14
	Citrinin	rice	0.15 0.16 0.12 ND 0.41 18.50 0.06
		N-enriched rice 0.17 0.20 0.15 ND 0.19 0.56 ND
	Pravastatin	rice	151 139	84	ND ND 56	ND
		N-enriched rice 39	32	43	ND ND ND ND
	Mevastatin	rice	ND 1	1	ND ND 78	1
		N-enriched rice ND 1	1	ND ND ND ND
	1 Media composition described in Materials and Methods section. Data not
	shown for liquid media.
	2 ND, not detected.

Table 3 .

 3 Effect of Monascus Strain 7 metabolites on rumen fermentation characteristics of 48-h consecutive batch cultures

					VFA
	Treatment 1	Gas 2	Methane 2	Total	Acetate (Ac) Propionate (Pr)	Butyrate	Iso-acids	Minor 3	Ac:Pr ratio
		mL	μmol	mmol/L	---------mol per 100 mol ---------
	First batch
	Control	10.5	155.9	98.3	72.2	17.3	6.7	2.4	1.5	4.2
	Monascus 7 extract	10.0	162.0	106.5	71.9	18.0	7.1 4	1.6*	1.4 4	4.0
	Monacolin K	10.3	162.5	96.3	72.6	17.3	6.4	2.3	1.5	4.2
	SEM	0.54	10.70	3.0	0.24	0.33	0.11	0.12	0.02	0.08
	P-value 5	0.79	0.85	0.11	0.23	0.30	0.012 4	0.011	0.056	0.28
	Second batch
	Control	7.2	57.8	86.3	68.1	22.2	7.2	1.6	0.9	3.1
	Monascus 7 extract	5.3	24.6	77.8	62.1*	30.0*	6.4	0.7*	0.8	2.1*
	Monacolin K	6.2	16.2	76.5	64.3*	25.7*	7.7	1.4	0.9	2.5*
	SEM	0.59	21.12	6.52	0.66	0.56	0.25	0.08	0.09	0.08
	P-value 5	0.15	0.40	0.55	0.002	<0.001	0.027 4	<0.001	0.77	<0.001
	Third batch
	Control	8.7	90.2	101.1	64.2	25.4	6.8	1.6	1.9	2.5
	Monascus 7 extract	6.7*	12.8*	90.6	56.0*	35.4*	6.0*	0.5*	2.1	1.6*
	Monacolin K	7.6*	59.1*	95.1	60.2*	30.0*	6.9	1.0*	1.9	2.0*
	SEM	0.18	4.79	3.99	0.53	0.45	0.12	0.02	0.09	0.05
	P-value 5	0.001	<0.001	0.25	<0.001	<0.001	0.003	<0.001	0.41	<0.001
	*Means values

Table 4 .

 4 Effect of Monascus Strain 7 with fermented rice used as substrate on rumen fermentation characteristics of 72-h in vitro incubations

								VFA
	Substrate	DMD		Methane 1	Gas 1	Total	Acetate (Ac) Propionate (Pr) Butyrate Iso-acids Ac:Pr ratio
		%	μmol	μmol/100 mg DMD	mL	mmol/L	------mol per 100 mol-------
	Rice	63.7	852.2	267.7	103.6	74.8	50.6	27.1	20.6	0.6	1.9
	Fermented rice	66.2	754.4	229.1	104.8	78.4*	48.8	30.0 2	19.1	0.7	1.6 2
	Rice-hay (1:1)	64.7	824.8	256.6	98.0	74.9	59.6	25.1	11.0	2.2	2.4
	Fermented rice-hay (1:1) 65.8	796.8	242.8	93.2	74.2	57.5 2	25.8	12.4	2.2	2.2
	SEM	1.31	32.92	12.62	1.91	0.86	0.59	0.79	0.93	0.10	0.07
	P-value 3	0.55	0.22	0.19	0.001 4	0.009	<0.001 4	0.002 4	<0.001 4 <0.001 4 <0.001 4
	*Means values (n = 6

Table 5 .

 5 Effect of Monascus Strain 7 with fermented rice used as substrate on rumen fermentation characteristics of 24-h consecutive batch cultures Total production in 40-mL fermentation vials containing 500 mg substrate at the end of the incubation period.

							VFA
	Substrate	Gas 1	Methane 1	H 2	1	Total	Acetate (Ac) Propionate (Pr)	Butyrate	Minor 2	Ac:Pr ratio
		mL	---μmol ---	mmol/L	----mol per 100 mol -----
	First batch
	Rice	54.5	616.6	2.7	49.3	57.0	25.7	15.3	1.2	2.2
	Fermented rice	63.6	640.6	8.3	53.2	51.9	30.7	15.3	1.5	1.7
	SEM	1.61	33.99	1.84	1.41	0.87	0.61	0.44	0.06	0.08
	P value 3	0.003	0.63	0.06	0.08	0.002	<0.001	0.97	0.014	<0.001
	Second batch
	Rice	41.2	377.4	2.0	49.7	52.6	27.9	17.9	1.2	1.9
	Fermented rice	43.7	342.1	9.8	51.3	46.9	31.5	19.9	1.4	1.5
	SEM	0.91	22.04	2.88	0.96	0.89	0.86	0.28	0.09	0.08
	P value 3	0.08	0.28	0.09	0.26	0.001	0.015	<0.001	0.08	0.005
	Third batch
	Rice	29.4	208.5	3.7	35.6	46.2	32.2	20.1	0.9	1.4
	Fermented rice	29.7	83.2	46.6	32.4	39.7	37.2	22.3	1.2	1.1
	SEM	0.84	25.64	12.8	1.56	1.32	1.60	0.35	0.14	0.09
	P-value 3	0.93	0.028	0.029	0.12	0.004	0.049	0.001	0.20	0.018
	1

2 Valerate and caproate.

3 Main effect of treatment.

Table 6 .

 6 In vivo rumen fermentation characteristics and abundance of bacteria, archaea methanogens, and protozoa in the rumen of wethers (n = 6) before, during, and after feeding with Monascus-fermented rice

		Pretreatment	Treatment	Posttreatment
		S1 1	S2	S3	S4	S5	SEM	P-value 2
	Fermentation products
	Methane, g/kg DMI	35.2 a	24.9 b	25.8 b	33.5 a	25.8 ab	2.75	<0.001
	Total VFA, mmol/L	73.4 b	61.8 c	75.3 b	94.4 a	89.6 a	3.84	<0.001
	Acetate (A), 3 %	70.8 a	60.0 b	63.8 bc	66.2 ac	67.5 ac	1.25	<0.001
	Propionate (P), %	12.5	18.0	16.4	15.6	15.7	1.41	0.06
	Butyrate, %	13.1	16.3	15.7	13.7	12.8	1.13	0.013 4
	Iso-acids, %	2.6 ab	3.0 a	1.9 b	3.0 a	2.5 ab	0.28	0.39
	A:P	5.8 a	3.5 b	4.0 b	4.6 ab	4.3 ab	0.38	0.002
	Microbial numbers
	Bacteria, 5 log 10 rrs copies/g DM rumen content	11.1 b	11.3 ab	11.4 a	11.2 ab	11.2 ab	0.04	0.006
	Archaea, 5 log 10 mcrA copies/g DM rumen content	9.9 a	9.5 b	9.6 ab	9.6 ab	9.7 ab	0.07	0.012
	% archaea 6	6.2 a	1.7 b	1.9 b	2.9 b	3.3 ab	0.79	<0.001

  Baumont, R., J. Dulphy, D. Sauvant, F. Meschy, J. Aufrère, and J. Peyraud. 2007. Valeur alimentaire des fourrages et des matières premières: tables et prévision. In: J. Agabriel, editor, Alimentation des bovins, ovins et caprins. Besoins des animaux. Valeurs des Aliments. Tables INRA, Editions Quae, Versailles. p 149-179. Benchaar, C., and H. Greathead. 2011. Essential oils and opportunities to mitigate enteric methane emissions from ruminants. Anim. Feed Sci. Technol. 166-167:338-355.

at INRA Institut National de la Recherche Agronomique on March 13, 2013 www.journalofanimalscience.org Downloaded from

Means values (n = 6) within a column differ from corresponding, nonfermented substrate control (P < 0.10).4 Signifi cant main effect of treatment with no differences between non-fermented control substrate and fermented substrate are not indicated by * symbol.

Main effect of treatment. at INRA Institut National de la Recherche Agronomique on March 13, 2013 www.journalofanimalscience.org Downloaded from

Estimated by microscopy. Protozoa were composed of 97.1% small entodiniomorphs, 1.6% large entodinimorphs (> 100 μm), 1.23% Dasytricha ruminantium, and 0.12% Isotricha spp. at INRA Institut National de la Recherche Agronomique on March 13, 2013 www.journalofanimalscience.org Downloaded from

1 This work has been partially fi nanced by INRA's internal valorization project fund and by the French National Research Agency project CREDIT (ANR-09-EBIO-009), Paris, France. The authors are grateful to C. Provenchère who performed the fi rst culture and in vitro experiment; D. Alvarez, D.