The impact of supplemental dietary methionine sources on volatile compound concentrations in broiler excreta.

The impact of different Met sources on broiler fecal odor volatiles was determined by evaluating the types of sulfur compounds produced in broiler excreta. Two experiments were conducted using straight-run broiler chicks randomly distributed in battery cages, with 3 replicate pens of 16 birds each. The treatment groups were 1) dry Met hydroxy analogue (dry MetHA), 2) sodium methioninate aqueous solution (NaMet), 3) liquid Met hydroxy analogue (Liq MetHA), 4) D,L- Met, and 5) no supplemental Met (control group). The Met activities of each Met source were 52, 45.9, 88, and 98%, respectively. All diets were formulated to contain either 0.8% (experiment 1) total Met activity or 0.5% Met activity in the starter and 0.38% Met activity in the grower (experiment 2) (except the control group, 0.35% Met activity), but otherwise met NRC nutrient requirements (NRC, 1994). Diets were fed ad libitum from d 1 to 6 wk of age. There were no significant differences in BW among the treatments. All excreta were collected in litter pans lined with aluminum foil. In experiment 1, at wk 6, broiler excreta were collected for a 24-h period, and 4.5 g of broiler excreta from each treatment group was collected into 15-mL headspace vials. Samples were analyzed by gas chromatography/mass spectrometry (GC/MS). The volatile sulfur compounds that were identified and quantified in the broiler excreta were H2S, carbonyl sulfide (COS), methyl mercaptan (CH3SH), dimethyl disulfide (CH3SSCH3), and dimethyl trisulfide (CH3SSSCH3). The NaMet treatment group had significantly higher concentrations of H2S, COS, and CH3SSCH3 compared with all other treatment groups. The Liq MetHA group had significantly lower concentrations of H2S, COS, CH3SH, and CH3SSCH3 compared with the other treatment groups. The dry MetHA group significantly had the highest concentration of CH4SH. The D,L-Met treatment group had the significantly highest concentration of CH3SSSCH3 and the lowest concentration of H2S. The control group had the significantly lowest concentrations of CH3SH, CH3SSCH3, and CH3SSSCH3 compared with the other treatment groups. In experiment 2, at wk 6, an electronic nose was used to evaluate 15 air samples per treatment group. In addition, 15 air samples (containing 6 to 8 L of air in a Tedlar bag, 3 samples per treatment group) were collected for odor evaluation by a sensory panel. Electronic nose sensor data revealed that volatile compounds in broiler excreta from the control group were significantly different from the other 4 treatment groups. Evaluation of the air samples by a sensory panel determined that there was a statistically significant difference in odor threshold detection between the control group and the other treatment groups. The dilutions to threshold of control group, NaMet, dry MetHA, Liq MetHA, and D,L-Met were 350, 492, 568, 496, and 526 odor units, respectively. These findings demonstrate that dietary Met sources significantly influenced odorous volatile concentrations in broiler excreta.

Membrane-associated quinoprotein formaldehyde dehydrogenase from Methylococcus capsulatus Bath.

A membrane-associated, dye-linked formaldehyde dehydrogenase (DL-FalDH) was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The enzyme was the major formaldehyde-oxidizing enzyme in cells cultured in high (above 1 micromol of Cu per mg of cell protein) copper medium and expressing the membrane-associated methane monooxygenase. Soluble NAD(P)(+)-linked formaldehyde oxidation was the major activity in cells cultured in low-copper medium and expressing the soluble methane monooxygenase (Tate and Dalton, Microbiology 145:159-167, 1999; Vorholt et al., J. Bacteriol. 180:5351-5356, 1998). The membrane-associated enzyme is a homotetramer with a subunit molecular mass of 49,500 Da. UV-visible absorption, electron paramagnetic resonance, and electrospray mass spectrometry suggest the redox cofactor of the DL-FalDH is pyrroloquinoline quinone (PQQ), with a PQQ-to-subunit stochiometry of approximately 1:1. The enzyme was specific for formaldehyde, oxidizing formaldehyde to formate, and utilized the cytochrome b(559/569) complex as the physiological electron acceptor.

Abatement of ammonia and hydrogen sulfide emissions from a swine lagoon using a polymer biocover.

The purpose of this research was to determine the efficiency of a polymer biocover for the abatement of H2S and NH3 emissions from an east-central Missouri swine lagoon with a total surface area of 7800 m2. The flux rate of NH3, H2S, and CH4 was monitored continuously from two adjacent, circular (d = 66 m) control and treatment plots using a nonintrusive, micrometeorological method during three independent sampling periods that ranged between 52 and 149 hr. Abatement rates were observed to undergo a temporal acclimation event in which NH3 abatement efficiency improved from 17 to 54% (p = < 0.0001 to 0.0005) and H2S abatement efficiency improved from 23 to 58% (p < 0.0001) over a 3-month period. The increase in abatement efficiency for NH3 and H2S over the sampling period was correlated with the development of a stable anaerobic floc layer on the bottom surface of the biocover that reduced mass transfer of NH3 and H2S across the surface. Analysis of methanogenesis activity showed that the biocover enhanced the rate of anaerobic digestion by 25% when compared with the control. The biocover-enhanced anaerobic digestion process was shown to represent an effective mechanism to counteract the accumulation of methanogenic substrates in the biocovered lagoon.

Functional classification of swine manure management systems based on effluent and gas emission characteristics.

Gaseous emissions from swine (Sus scrofa) manure storage systems represent a concern to air quality due to the potential effects of hydrogen sulfide, ammonia, methane, and volatile organic compounds on environmental quality and human health. The lack of knowledge concerning functional aspects of swine manure management systems has been a major obstacle in the development and optimization of emission abatement technologies for these point sources. In this study, a classification system based on gas emission characteristics and effluent concentrations of total phosphorus (P) and total sulfur (S) was devised and tested on 29 swine manure management systems in Iowa, Oklahoma, and North Carolina in an effort to elucidate functional characteristics of these systems. Four swine manure management system classes were identified that differed in effluent concentrations of P and S, methane (CH4) emission rate, odor intensity, and air concentration of volatile organic compounds (VOCs). Odor intensity and the concentration of VOCs in air emitted from swine manure management systems were strongly correlated (r2 = 0.88). The concentration of VOC in air samples was highest with outdoor swine manure management systems that received a high input of volatile solids (Type 2). These systems were also shown to have the highest odor intensity levels. The emission rate for VOCs and the odor intensity associated with swine manure management systems were inversely correlated with CH4 and ammonia (NH3) emission rates. The emission rates of CH4, NH3, and VOCs were found to be dependent upon manure loading rate and were indirectly influenced by animal numbers.

Correlation of human olfactory responses to airborne concentrations of malodorous volatile organic compounds emitted from swine effluent.

Direct multicomponent analysis of malodorous volatile organic compounds (VOCs) present in ambient air samples from 29 swine (Sus scrofa) production facilities was used to develop a 19-component artificial swine odor solution that simulated olfactory properties of swine effluent. Analyses employing either a human panel consisting of 14 subjects or gas chromatography were performed on the air stream from an emission chamber to assess human olfactory responses or odorant concentration, respectively. Analysis of the olfactory responses using Fisher's LSD statistics showed that the subjects were sensitive to changes in air concentration of the VOC standard across dilutions differing by approximately 16%. The effect of chemical synergisms and antagonisms on human olfactory response magnitudes was assessed by altering the individual concentration of nine compounds in artificial swine odor over a twofold concentration range while maintaining the other 18 components at a constant concentration. A synergistic olfactory response was observed when the air concentration of acetic acid was increased relative to the concentration of other VOC odorants in the standard. An antagonistic olfactory response was observed when the air concentration of 4-ethyl phenol was increased relative to the other VOC odorants in the standard. The collective odorant responses for nine major VOCs associated with swine odor were used to develop an olfactory prediction model to estimate human odor response magnitudes to swine manure odorants through measured air concentrations of indicator VOCs. The results of this study show that direct multicomponent analysis of VOCs emitted from swine effluent can be applied toward estimating perceived odor intensity.

Rapid method to estimate the presence of secondary metabolites in microbial extracts.

Screening microbial secondary metabolites is an established method to identify novel biologically active molecules. Preparation of biological screening samples from microbial fermentation extracts requires growth conditions that promote synthesis of secondary metabolites and extraction procedures that capture the secondary metabolites produced. High-performance liquid chromatography (HPLC) analysis of fermentation extracts can be used to estimate the number of secondary metabolites produced by microorganisms under various growth conditions but is slow. In this study we report on a rapid (approximately 1 min per assay) surrogate measure of secondary metabolite production based on a metabolite productivity index computed from the electrospray mass spectra of samples injected directly into a spectrometer. This surrogate measure of productivity was shown to correlate with an HPLC measure of productivity with a coefficient of 0.78 for a test set of extracts from 43 actinomycetes. This rapid measure of secondary metabolite productivity may be used to identify improved cultivation and extraction conditions by analyzing and ranking large sets of extracts. The same methods may also be used to survey large collections of extracts to identify subsets of highly productive organisms for biological screening or additional study.

Use of direct-infusion electrospray mass spectrometry to guide empirical development of improved conditions for expression of secondary metabolites from actinomycetes.

A major barrier in the discovery of new secondary metabolites from microorganisms is the difficulty of distinguishing the minor fraction of productive cultures from the majority of unproductive cultures and growth conditions. In this study, a rapid, direct-infusion electrospray mass spectrometry (ES-MS) technique was used to identify chemical differences that occurred in the expression of secondary metabolites by 44 actinomycetes cultivated under six different fermentation conditions. Samples from actinomycete fermentations were prepared by solid-phase extraction, analyzed by ES-MS, and ranked according to a chemical productivity index based on the total number and relative intensity of ions present in each sample. The actinomycete cultures were tested for chemical productivity following treatments that included nutritional manipulations, autoregulator additions, and different agitation speeds and incubation temperatures. Evaluation of the ES-MS data from submerged and solid-state fermentations by paired t test analyses showed that solid-state growth significantly altered the chemical profiles of extracts from 75% of the actinomycetes evaluated. Parallel analysis of the same extracts by high-performance liquid chromatography-ES-MS-evaporative light scattering showed that the chemical differences detected by the ES-MS method were associated with growth condition-dependent changes in the yield of secondary metabolites. Our results indicate that the high-throughput ES-MS method is useful for identification of fermentation conditions that enhance expression of secondary metabolites from actinomycetes.

Identification of intermediates of in vivo trichloroethylene oxidation by the membrane-associated methane monooxygenase.

The rate and products of trichloroethylene (TCE) oxidation by Methylomicrobium album BG8 expressing membrane-associated methane monooxygenase (pMMO) were determined using 14C radiotracer techniques. [(14)C]TCE was degraded at a rate of 1.24 nmol (min mg protein)(-1) with the initial production of glyoxylate and then formate. Radiolabeled CO(2) was also found after incubating M. album BG8 for 5 h with [(14)C]TCE. Experiments with purified pMMO from Methylococcus capsulatus Bath showed that TCE could be mineralized to CO(2) by pMMO. Oxygen uptake studies verified that M. album BG8 could oxidize glyoxylate and that pMMO was responsible for the oxidation based on acetylene inactivation studies. Here we propose a pathway of TCE oxidation by pMMO-expressing cells in which TCE is first converted to TCE-epoxide. The epoxide then spontaneously undergoes HCl elimination to form glyoxylate which can be further oxidized by pMMO to formate and CO(2).

Primary structure of cytochrome c' of Methylococcus capsulatus Bath: evidence of a phylogenetic link between P460 and c'-type cytochromes.

Cytochrome c' of Methylococcus capsulatus Bath is involved in electron flow from the enzyme responsible for hydroxylamine oxidation, cytochrome P460, to cytochrome C555. This cytochrome is spectrally similar to other cytochromes c' but is larger (16,000 Da) and has a lower midpoint potential (-205 mV). By a combination of Edman degradation, mass spectroscopy, and gene sequencing, we have obtained the primary structure of cytochrome c' from M. capsulatus Bath. The cytochrome shows low sequence similarity to other cytochromes c', only residues R12, Y53, G56, and the C-terminal heme-binding region (GXXCXXCHXXXK) being conserved. In contrast, cytochrome c' from M. capsulatus Bath shows considerable sequence similarity to cytochromes P460 from M. capsulatus Bath (31% identity) and from Nitrosomonas europaea (18% identity). This suggests that P460-type cytochromes may have originated from a c'-type cytochrome which developed a covalent cross-link between a lysine residue and the c'-heme.

High-molecular-mass multi-c-heme cytochromes from Methylococcus capsulatus bath.

The polypeptide and structural gene for a high-molecular-mass c-type cytochrome, cytochrome c553O, was isolated from the methanotroph Methylococcus capsulatus Bath. Cytochrome c553O is a homodimer with a subunit molecular mass of 124,350 Da and an isoelectric point of 6. 0. The heme c concentration was estimated to be 8.2 +/- 0.4 mol of heme c per subunit. The electron paramagnetic resonance spectrum showed the presence of multiple low spin, S = 1/2, hemes. A degenerate oligonucleotide probe synthesized based on the N-terminal amino acid sequence of cytochrome c553O was used to identify a DNA fragment from M. capsulatus Bath that contains occ, the gene encoding cytochrome c553O. occ is part of a gene cluster which contains three other open reading frames (ORFs). ORF1 encodes a putative periplasmic c-type cytochrome with a molecular mass of 118, 620 Da that shows approximately 40% amino acid sequence identity with occ and contains nine c-heme-binding motifs. ORF3 encodes a putative periplasmic c-type cytochrome with a molecular mass of 94, 000 Da and contains seven c-heme-binding motifs but shows no sequence homology to occ or ORF1. ORF4 encodes a putative 11,100-Da protein. The four ORFs have no apparent similarity to any proteins in the GenBank database. The subunit molecular masses, arrangement and number of hemes, and amino acid sequences demonstrate that cytochrome c553O and the gene products of ORF1 and ORF3 constitute a new class of c-type cytochrome.

Cytochrome P460 genes from the methanotroph Methylococcus capsulatus bath.

P460 cytochromes catalyze the oxidation of hydroxylamine to nitrite. They have been isolated from the ammonia-oxidizing bacterium Nitrosomonas europaea (R. H. Erickson and A. B. Hooper, Biochim. Biophys. Acta 275:231-244, 1972) and the methane-oxidizing bacterium Methylococcus capsulatus Bath (J. A. Zahn et al., J. Bacteriol. 176:5879-5887, 1994). A degenerate oligonucleotide probe was synthesized based on the N-terminal amino acid sequence of cytochrome P460 and used to identify a DNA fragment from M. capsulatus Bath that contains cyp, the gene encoding cytochrome P460. cyp is part of a gene cluster that contains three open reading frames (ORFs), the first predicted to encode a 59,000-Da membrane-bound polypeptide, the second predicted to encode a 12, 000-Da periplasmic protein, and the third (cyp) encoding cytochrome P460. The products of the first two ORFs have no apparent similarity to any proteins in the GenBank database. The overall sequence similarity of the P460 cytochromes from M. capsulatus Bath and N. europaea was low (24.3% of residues identical), although short regions of conserved residues are present in the two proteins. Both cytochromes have a C-terminal, c-heme binding motif (CXXCH) and a conserved lysine residue (K61) that may provide an additional covalent cross-link to the heme (D. M. Arciero and A. B. Hooper, FEBS Lett. 410:457-460, 1997). Gene probing using cyp indicated that a cytochrome P460 similar to that from M. capsulatus Bath may be present in the type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP but not in the type I methanotrophs Methylobacter marinus A45, Methylomicrobium albus BG8, and Methylomonas sp. strains MN and MM2. Immunoblot analysis with antibodies against cytochrome P460 from M. capsulatus Bath indicated that the expression level of cytochrome P460 was not affected either by expression of the two different methane monooxygenases or by addition of ammonia to the culture medium.

Copper-binding compounds from Methylosinus trichosporium OB3b.

Two copper-binding compounds/cofactors (CBCs) were isolated from the spent media of both the wild type and a constitutive soluble methane monooxygenase (sMMOC) mutant, PP319 (P. A. Phelps et al., Appl. Environ. Microbiol. 58:3701-3708, 1992), of Methylosinus trichosporium OB3b. Both CBCs are small polypeptides with molecular masses of 1,218 and 779 Da for CBC-L1 and CBC-L2, respectively. The amino acid sequence of CBC-L1 is S?MYPGS?M, and that of CBC-L2 is SPMP?S. Copper-free CBCs showed absorption maxima at 204, 275, 333, and 356 with shoulders at 222 and 400 nm. Copper-containing CBCs showed a broad absorption maximum at 245 nm. The low-temperature electron paramagnetic resonance (EPR) spectra of copper-containing CBC-L1 showed the presence of a copper center with an EPR splitting constant between those of type 1 and type 2 copper centers (g = 2.087, g = 2.42 G, A = 128 G). The EPR spectrum of CBC-L2 was more complex and showed two spectrally distinct copper centers. One signal can be attributed to a type 2 Cu2+ center (g = 2.073, g = 2.324 G, A = 144 G) which could be saturated at higher powers, while the second shows a broad, nearly isotropic signal near g = 2.063. In wild-type strains, the concentrations of CBCs in the spent media were highest in cells expressing the pMMO and stressed for copper. In contrast to wild-type strains, high concentrations of CBCs were observed in the extracellular fraction of the sMMOC mutants PP319 and PP359 regardless of the copper concentration in the culture medium.

Cytochrome c peroxidase from Methylococcus capsulatus Bath.

A bacterial cytochrome c peroxidase was purified from the obligate methanotroph Methylococcus capsulatus Bath in either the fully oxidized or the half reduced form depending on the purification procedure. The cytochrome was a homo-dimer with a subunit mol mass of 35.8 kDa and an isoelectric point of 4.5. At physiological temperatures, the enzyme contained one high-spin, low-potential (Em7 = -254 mV) and one low-spin, high-potential (Em7 = +432 mM ) heme. The low-potential heme center exhibited a spin-state transition from the penta-coordinated, high-spin configuration to a low-spin configuration upon cooling the enzyme to cryogenic temperatures. Using M. capsulatus Bath ferrocytochrome c555 as the electron donor, the KM and Vmax for peroxide reduction were 510 +/- 100 nM and 425 +/- 22 mol ferrocytochrome c555 oxidized min-1 (mole cytochrome c peroxidase)-1, respectively.

Evidence for an iron center in the ammonia monooxygenase from Nitrosomonas europaea.

Binding of the ligand, nitric oxide, in the presence of reductant was used to identify a ferrous S = 3/2 signal, characteristic of a ferrous nitrosyl complex, and a g= 2.03 copper or iron signal in membranes of the ammonia-oxidizing bacterium, Nitrosomonas europaea. The same ferrous S = 3/2 signal is thought to be a component of the membrane-associated methane monooxygenase (pMMO) of Methylococcus capsulatus Bath, since it is seen in the membrane fraction of cells expressing pMMO and in the purified enzyme, but not in the membrane fraction of cells expressing the soluble MMO [Zahn, J.A. and DiSpirito, A.A. (1996) J. Bacteriol. 178, 1018-1029]. Treatment of resting membranes or cells of N. europaea with nitrapyrin, 2-chloro,6-trichloromethylpyridine, resulted in the increase in magnitude of a g = 6, high-spin ferric iron signal. In the presence of NO and reductant, nitrapyrin prevented the formation of the S = 3/2 nitrosyl-iron complex while increasing the intensity of the g = 6 signal. Nitrapyrin is a specific inhibitor of, and is reduced by, the ammonia monoxygenase (AMO) [Bédard, C. and Knowles, R. (1989) Microbiol. Rev. 53, 68-83]. Taken together the data suggest that iron capable of forming the S = 3/2 complex is a catalytic component of AMO of N. europaea, possibly a part of the oxygen-activating center. Inactivation of the membrane-associated AMO with acetylene did not diminish the S = 3/2 nitrosyl-iron signal, the g = 6 signal, or the g = 6 signal.

Cytochrome c' of Methylococcus capsulatus Bath.

Cytochrome c' was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The native and subunit molecular masses of the cytochrome were 34.9 kDa and 16.2 kDa, respectively, with an isoelectric pH of 7.0. The amino acid composition and N-terminal amino acid sequence were consistent with identification of the protein as a cytochrome c'. The electron paramagnetic resonance spectrum of the monoheme cytochrome indicated the presence of a high spin, S = 5/2, heme center that is diagnostic of cytochromes c'. The optical absorption spectra of ferric or ferrous cytochrome c' were also characteristic of cytochromes c'. The ferrocytochrome bound carbon monoxide and nitric oxide, but not isocyanide, cyanide, or azide. Changes in physical properties due to binding of CO or NO to some other c'-type cytochromes have been interpreted as an indication of dimer dissociation. In the case of cytochrome c' from M. capsulatus Bath, analytical ultracentrifugation of the ferricytochrome, the ferrocytochrome, and the ferrocytochrome-CO complex indicate that the changes induced by binding of CO are conformational and are not consistent with dimer dissociation. EPR spectra show that cytochrome c' was reduced in the presence of hydroxylamine only when in a complex with cytochrome P-460. The value of the midpoint potential, Em 7.0, was -250 mV for cytochrome c' from M. capsulatus Bath, which is well below the range of values reported for other cytochromes c'. The values of midpoint potentials for cytochrome P-460 (Em 7.0 = -300 mV to -380 mV) and cytochrome C555 (Em 7.0 = +175 mV to +195 mV) are less than and greater than, respectively, the value for cytochrome c' and suggest the possibility that the latter may function as an electron shuttle between cytochrome P-460 and cytochrome C555.

Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath).

An active preparation of the membrane-associated methane monooxygenase (pMMO) from Methylococcus capsulatus Bath was isolated by ion-exchange and hydrophobic interaction chromatography using dodecyl beta-D-maltoside as the detergent. The active preparation consisted of three major polypeptides with molecular masses of 47,000, 27,000, and 25,000 Da. Two of the three polypeptides (those with molecular masses of 47,000 and 27,000 Da) were identified as the polypeptides induced when cells expressing the soluble MMO are switched to culture medium in which the pMMO is expressed. The 27,000-Da polypeptide was identified as the acetylene-binding protein. The active enzyme complex contained 2.5 iron atoms and 14.5 copper atoms per 99,000 Da. The electron paramagnetic resonance spectrum of the enzyme showed evidence for a type 2 copper center (g perpendicular = 2.057, g parallel = 2.24, and magnitude of A parallel = 172 G), a weak high-spin iron signal (g = 6.0), and a broad low-field (g = 12.5) signal. Treatment of the pMMO with nitric oxide produced the ferrous-nitric oxide derivative observed in the membrane fraction of cells expressing the pMMO. When duroquinol was used as a reductant, the specific activity of the purified enzyme was 11.1 nmol of propylene oxidized.min-1.mg of protein-1, which accounted for approximately 30% of the cell-free propylene oxidation activity. The activity was stimulated by ferric and cupric metal ions in addition to the cytochrome b-specific inhibitors myxothiazol and 2-heptyl-4-hydroxyquinoline-N-oxide.

Oxidation of hydroxylamine by cytochrome P-460 of the obligate methylotroph Methylococcus capsulatus Bath.

An enzyme capable of the oxidation of hydroxylamine to nitrite was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The absorption spectra in cell extracts, electron paramagnetic resonance spectra, molecular weight, covalent attachment of heme group to polypeptide, and enzymatic activities suggest that the enzyme is similar to cytochrome P-460, a novel iron-containing protein previously observed only in Nitrosomonas europaea. The native and subunit molecular masses of the M. capsulatus Bath protein were 38,900 and 16,390 Da, respectively; the isoelectric point was 6.98. The enzyme has approximately one iron and one copper atom per subunit. The electron paramagnetic resonance spectrum of the protein showed evidence for a high-spin ferric heme. In contrast to the enzyme from N. europaea, a 13-nm blue shift in the soret band of the ferrocytochrome (463 nm in cell extracts to 450 nm in the final sample) occurred during purification. The amino acid composition and N-terminal amino acid sequence of the enzyme from M. capsulatus Bath was similar but not identical to those of cytochrome P-460 of N. europaea. In cell extracts, the identity of the biological electron acceptor is as yet unestablished. Cytochrome c-555 is able to accept electrons from cytochrome P-460, although the purified enzyme required phenazine methosulfate for maximum hydroxylamine oxidation activity (specific activity, 366 mol of O2 per s per mol of enzyme). Hydroxylamine oxidation rates were stimulated approximately 2-fold by 1 mM cyanide and 1.5-fold by 0.1 mM 8-hydroxyquinoline.