SEARCH

SEARCH BY CITATION

Keywords:

  • large intestine;
  • odour;
  • pig;
  • sulphate-reducing bacteria;
  • volatile sulphur compounds

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  To investigate the production of volatile sulphur compounds (VSC) in the segments of the large intestine of pigs and to assess the impact of diet on this production.

Methods and Results:  Pigs were fed two diets based on either wheat and barley (STD) or wheat and dried distillers grains with solubles (DDGS). Net production of VSC and potential sulphate reduction rate (SRR) (sulphate saturated) along the large intestine were determined by means of in vitro incubations. The net production rate of hydrogen sulphide and potential SRR increased from caecum towards distal colon and were significantly higher in the STD group. Conversely, the net methanethiol production rate was significantly higher in the DDGS group, while no difference was observed for dimethyl sulphide. The number of sulphate-reducing bacteria and total bacteria were determined by quantitative PCR and showed a significant increase along the large intestine, whereas no diet-related differences were observed.

Conclusion:  VSC net production varies widely throughout the large intestine of pigs and the microbial processes involved in this production can be affected by diet.

Significance and Impact of the Study:  This first report on intestinal production of all VSC shows both spatial and dietary effects, which are relevant to both bowel disease- and odour mitigation research.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Industrialisation of pig production has led to increased concerns regarding the environmental impact of piggeries. Of particular concern is the emission of odour, which creates a nuisance (Wing et al. 2008) and potentially poses a health threat to neighbours of the production facilities (Schiffman et al. 1995) and consequently influences decisions on positioning and expansion of piggeries in several countries. For these reasons, knowledge about the mechanisms of odorant production and odour abatement strategies are needed.

The emission of volatile sulphur compounds (VSC) from various steps in pig production and waste handling is considered a main contributor to the total odour emission from piggeries (Blanes-Vidal et al. 2009; Hansen et al. 2012). The production of VSC is initiated by the microbial metabolism of dietary and endogenously secreted sulphur compounds in the gastrointestinal tract (GI-tract) of the pig and continues in the manure. Within the GI-tract, a number of factors determine the production and concentration of these gases, such as dietary composition (Magee et al. 2000), endogenous secretions (Willis et al. 1996) and epithelial uptake (Suarez et al. 1998). Unfortunately, only little is known about the distribution and magnitude of this initial VSC production taking place inside the animal.

VSC produced in the GI-tract are excreted to the surrounding environment primarily as the constituents of the faecal material produced by the pigs, from which they are readily liberated because of their high volatility and the large surface area of the faeces. Additionally, some VSC are excreted through flatus (Suarez et al. 1997) and through respiratory gasses (Suarez et al. 1999).

In addition to its contribution to odour emission, intestinal hydrogen sulphide (H2S) has received considerable attention because of its putative role in the pathogenesis of severe inflammatory diseases (Pitcher and Cummings 1996) and colorectal cancer in humans (Attene-Ramos et al. 2007). Whether these putative harmful effects of H2S have any relevance for the well-being and growth of pigs during their short lifespan remains an open question.

The major VSC identified in the GI-tract are H2S, methanethiol (MT) and dimethyl sulphide (DMS) (Poulsen et al. 2010). Several mechanisms are involved in the production and degradation of VSC in anaerobic environments, rich in organic material comparable to the GI-tract: H2S is produced by sulphate-reducing bacteria (SRB), by bacteria that degrade cysteine and by methanogenic demethylation of MT and DMS; MT is produced by the degradation of methionine and by methylation of H2S. DMS is produced through the methylation of MT (Higgins et al. 2006).

Microbial processes in the GI-tract are strongly influenced by the dietary composition (Jensen et al. 1995). Dried distillers’ grains with solubles (DDGS) are produced from the fuel ethanol industry and as a consequence of the rapid increase in ethanol production in recent years, the quantity of DDGS used in pig feed is rising. However, feeding trials have indicated that dietary inclusion of DDGS may adversely affect VSC emissions (Li et al. 2011), a result, which warrants further investigations into the effect of DDGS-containing feed on odorant production.

The primary aim of this study was to quantify the production of VSC along the large intestine of pigs and to investigate whether this production is affected by diet, specifically, a diet consisting of typical ingredients of a Danish pig diet relative to the same diet with DDGS added at the expense of wheat and soybean meal. To do this, intestinal material was obtained from pigs fed two diets, one of which included DDGS and one which did not. In addition, we investigated the SRB in the large intestine to obtain novel insight into their distribution and population size and to correlate these parameters to the H2S production.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Diets and feeding

Two diets were formulated: a standard diet based on wheat and barley (STD) and a diet with 30% DDGS added at the expense of wheat and dehulled toasted soy bean meal (Table 1). The diets were formulated to comply with Danish recommendations for essential amino acids, calcium and phosphorous. The pigs were fed three times daily, at 0700, 1500 and 2300 h. The daily amount of feed offered to the animals was adjusted weekly to 3% of their body weight.

Table 1.   Composition of experimental diets
Item, % (as fed basis)Diet
STDDDGS
  1. DDGS, dried distillers grains with solubles; NSP, nonstarch polysaccharides.

  2. *Supplied per kilogram of diet: 4400 IU of vitamin A; 1000 IU of cholecalciferol 60 mg of alpha-tocopherol; 2·2 mg of menadione; 2·2 mg of thiamine; 4·0 mg of riboflavin, 3·3 mg of pyridoxine; 11 mg of d-pantothenic acid; 22 mg of niacin; 0·06 mg of biotin; 0·02 mg of vitamin B12; 50 mg of Fe as FeSO4·7H2O; 80 mg of Zn as ZnO; 28 mg of Mn as MnO; 20 mg of Cu as CuSO4·5H2O; 0·20 mg of I as KI; 0·30 mg of Se as Na2SeO3.

Dried distillers’ grains w. solubles (wheat)30·0
Barley26·026·0
Wheat66·741·4
Dehulled toasted soybean meal4·0
l-Lysine HCl, 78%0·40·3
dl-Methionine, 40%0·1
L-Threonine, 50%0·20·1
Calcium carbonate0·60·7
Dicalcium phosphate1·50·9
Sodium chloride0·40·4
Vitamin and mineral premix*0·20·2
Analysed composition (DM basis)
 Crude protein (N × 6·25) (%)14·1119·70
 Lignin (%)1·92·8
 NSP (%)12·617·7
 Sulphur (g kg−1)1·412·19
 Cysteine (g kg−1)2·684·10
 Methionine (g kg−1)2·632·85
 Lysine (g kg−1)8·097·96
 Threonine (g kg−1)6·176·41

Animals, housing and sampling procedure

The experiment complied with the guidelines of the Danish Ministry of Justice with respect to animal experimentation and care of animals under study. A total of eight crossbred (Danish Landrace × Yorkshire × Duroc) gilts from Aarhus University Swine Herd, Foulum, Denmark were used. The animals were housed in individual pens (1·65 × 1·50 m, of which 0·80 × 1·50 m were slatted) while fed the experimental diets for a minimum of 2 weeks. At a body weight of 71·9 ± 1·5 kg, the animals were killed with a bolt pistol 3 h after the morning meal, exsanguinated, the GI-tract removed and the large intestine divided into four segments: caecum and three equally long segments of the colon including the rectum. The digesta of each segment was retrieved, and samples for in vitro incubations, DNA extraction and measurements of short chain fatty acid (SCFA) and sulphate were obtained. The digesta for in vitro incubations was processed within 0·5 h and the remaining samples were stored at −20°C until further processing.

In vitro incubations

In order to determine the production rate of VSC in digesta, slurry incubations of digesta were set up. The in vitro incubations [modified from Jensen and Jorgensen (1994) and Jensen et al. (1995)] were carried out in serum bottles (330 ml) containing 10 g of digesta and 40 ml deoxygenated phosphate buffer (NaH2PO4·H2O) with a pH of 6·0, characteristic of digesta from the large intestine. Digesta incubations from each segment were run in triplicates. Phosphate buffer was included to keep the pH stable during the incubations and to ensure a relatively thin mixture, which could be thoroughly mixed during incubation. Preliminary incubations showed only minor changes in pH during the first hours (up to 6 h) of incubation and around half a pH unit after 24 h of incubation, thus proving an efficient buffering (data not shown). Digesta was added to the serum bottles under a constant flow of N2. The bottles were capped with butyl rubber stoppers and repeatedly (three times) evacuated and filled with N2 before incubation on a shaker in a water bath at 37°C. After 1, 2 and 3 h of incubation, gas samples (3 ml in total) for VSC and methane analysis were withdrawn using polypropylene syringes.

Analytical methods

VSC were analysed by injecting the gas samples directly into the sample inlet of a Clarus 5000 gas chromatograph equipped with an amperometric sulphur detector (Perkin-Elmer-Arnel, Waltham, MA) and a capillary column (30 m × 0·32 mm, 4 μm polydimethylsiloxane film). The detector is specific for sulphur-containing gasses and has an equimolar sulphur response. Helium was used as carrier gas with a column head pressure of 26 psi (8·2 ml min−1 at 40°C). To measure in the detector’s dynamic range, some samples were diluted in a N2 filled 100-ml polypropylene syringe prior to injection. For calibration, a permeation chamber (Dynacal; Valco Instruments, Houston, TX) containing DMS (73 ng min−1 at 50°C) was used. The limit of detection was c. 13 ng S l−1.

For methane measurements, the samples were analysed in a gas chromatograph (Mikrolab, Aarhus, Denmark) equipped with a thermal conductivity detector and a Hayesep Q column (80/100 mesh; 1·5 m ×¼ inches) Argon was used as carrier gas at a flow rate of c. 58 ml min−1, and the oven, injection port and detector temperature were 50, 50 and 70°C, respectively.

Total nitrogen in feed was determined by the Dumas method using an elemental analyser, model CNS-2000 (Leco, St. Joseph, MI, USA), as described by Hansen (1989). Amino acid analyses were carried out according to Mason et al. (1980) and Commission Directive 2000/45/EC. Nonstarch polysaccharides (NSP) were determined by a modification of the Uppsala procedure and that of Englyst et al. (1982) as described by Knudsen (1997). The concentration of SCFA in digesta was determined as described by Canibe et al. (2007). The sulphur content of diets was detected by spectrophotometry after wet ashing with magnesium nitrate and perchloric acid and the addition of barium chloride (Nes 1979). Digesta samples for the measurements of sulphate concentration were prepared by the method of Florin et al. (1991) and measured using a BioLC ion chromatograph (Dionex, Sunnyvale, CA, USA) equipped with a 4 × 250 mm IonPac AS18 column and an ED50 electrochemical detector.

Potential sulphate reduction rates

Subsamples of the digesta used in the in vitro incubations were used to determine the potential sulphate reduction rate (SRR). A slurry of digesta and deoxygenated phosphate buffer (NaH2PO4·H2O, pH 6·0) was mixed (1 : 9) and transferred under constant flow of N2 to 12-ml gas-tight glass containers (exetainers) containing 0·4 ml of a Na2SO4 stock solution (100 mmol l−1), yielding an initial concentration of sulphate of minimum 4 mmol l−1. The addition of inline image ensured a sulphate-saturated sulphate reduction throughout the incubation (Ingvorsen and Jorgensen 1984), and hence the term: potential SRR. Five μl of 35S-inline image (20 kbq μl−1) was injected and the exetainers were incubated in a shaker for 1·5 h at 37°C. At the end of the incubation, 1 ml of a 20% zinc acetate solution was injected and the exetainers were stored at −20°C until distillation. To determine the SRR during the incubations, 35S-labelled H2S was separated from the 35S-labelled sulphate by acid chromium distillation as described by Fossing and Jorgensen (1989). After separation, the activity of the sulphide and the sulphate pool was determined using a scintillation counter and the SRR per gram of digesta was calculated by the following equation (Fossing and Jorgensen 1989): SRR = (a (A + a)−1 × [inline image] × 1·06) (h × g)−1, in which a is the radioactivity of the volatile sulphide, A the radioactivity of inline image after the incubation, [inline image] the concentration of sulphate, 1·06 is a constant that accounts for the percentage of fractionation between 35S and 32S by the SRB, h is the incubation time and g is the gram of digesta per litre of the digesta slurry used in the incubations.

Quantification of SRB and total bacteria by quantitative PCR (qPCR)

DNA extraction from digesta was performed as described by Jozefiak et al. (2010) with one modification: 2 min of bead beating was replaced by 2 min of vortex mixing. The quantification of SRB and total bacteria in digesta was performed using qPCR assays employing the primer pair DSR1F+ and DSR-R (Kondo et al. 2004), which targeted the dsrA gene of SRB and the primer pair 1055F and 1392R (Harms et al. 2003), which targeted the 16S rRNA gene for the enumeration of total bacteria. The assays were performed on a Rotorgene 6000 (Corbett Robotics Inc., San Francisco, CA, USA) using the Quantitect SYBR Green PCR kit (Qiagen, Valencia, CA, USA) as described by Spence et al. (2008), except for the annealing temperature in the dsrA assay, which was 62°C in the present study. A final melt curve analysis was performed to determine the presence or absence of nonspecific amplification products. All samples were run in triplicate and dH2O replaced the template in control reactions. The procedure for the determination of dsrA and bacterial 16S rRNA gene copy numbers was as described in Spence et al. (2008). To estimate the number of SRB and the total bacterial number per gram of digesta from the measured numbers of dsrA and 16S rRNA gene copies, it was assumed that the bacterial genome contains on average 3·6 rRNA operons (Klappenbach et al. 2001) and the SRB genome a single dsrA copy (Klein et al. 2001).

Statistical methods

The statistical model used to estimate the effect of diet and segment along the GI-tract on various response variables in digesta was a mixed model, which included diet, segment of the GI-tract and diet × segment as fixed effects. To capture the correlation between measurements in different segments of the GI-tract on each pig, the random errors were allowed to be correlated (the statement ‘repeated’ in SAS). The analyses were performed with SAS for Windows ver. 8.2 (SAS Institute, Cary, NC, USA). When there was an overall effect of diet at an alpha of  0·05, differences between means were compared pairwise using an t-test.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Diet composition

The DDGS diet had higher concentration of crude protein, sulphur-containing amino acids, NSP and total sulphur compared to the STD diet (Table 1).

Net production rates of VSC production in digesta from the large intestine

Given that the three measured VSC are both produced and consumed in the digesta during our incubations, the term ‘net production rate’ will be used here when describing the rates determined during the in vitro incubations. In the in vitro incubations, an initial burst of VSC into the gas phase was observed during the first hour, after which the production was constant for the next 2 h (Fig. 1). Therefore, the VSC net production rate was calculated from the slope between the concentrations measured at 1 and 3 h. In incubations showing a negative net production rate, no or very low concentrations of VSC were measured in the gas phase at the end of incubation.

image

Figure 1.  Time course of gas-phase concentration of hydrogen sulphide, methanethiol and dimethyl sulphide in the incubation of digesta from proximal colon of one pig fed the STD diet. The broken line illustrates the expected development in gas concentration during the first hour of incubation and the intercept with y-axis illustrates the burst.

Download figure to PowerPoint

The net production rate of H2S (Fig. 2) was significantly higher in the digesta of the STD-fed pigs compared to the DDGS-fed pigs [P (diet) = 0·04]. In the middle and distal colon, the mean H2S net production rate of the STD group was approximately double the rate of the DDGS group. Along the large intestine, the H2S net production rate increased significantly from the caecum (−2·4 vs−4·4 nmol g−1 h−1 in STD vs DDGS group) to reach the highest rates in the distal colon (147·3 vs 71·1 nmol g−1 h−1, STD vs DDGS). A significant diet-related difference was also observed for MT net production rates (Fig. 2), which were higher in the DDGS group compared to the STD group [P (diet) < 0·001]. In addition, a different, although not statistically significant [P (diet × seg) = 0·17], pattern of net production rates was observed between the two groups along the large intestine: in the STD group, the highest net production rate was observed in the caecum (16·2 nmol g−1 h−1), followed by the proximal colon (5·3 nmol g−1 h−1) and consumption in both the middle (−2·8 nmol g−1 h−1) and the distal segment of the colon (−1·7 nmol g−1 h−1). In contrast, in the DDGS group, an almost constant and relatively high net production rate (ranging from 26·6 to 33·5 nmol g−1 h−1) was measured throughout all segments. For both diets, the highest DMS net production rates (Fig. 2) were measured in the caecum and in the proximal colon. No diet effect was observed on the rates of DMS net production. Large differences between individual pigs of both dietary groups were observed for all VSC. The total VSC net production rate showed a significant increase from caecum to distal colon, but no diet-related difference was measured.

image

Figure 2.  Mean volatile sulphur compound net production rate in digesta from various segments determined by in vitro incubations of digesta. Error bars represent standard error of the means (n = 4). Segments without a common letter are significantly different ( 0·05). (inline image) STD diet, (□) dried distillers grains with solubles diet.

Download figure to PowerPoint

Methane production rates in in vitro incubations

No significant diet-related difference was observed concerning the measured methane production rates (Fig. 3). The production of methane was highly variable among the different colon segments of individual pigs. In the caecum, the sample of only one pig showed methane production, while it was observed in the proximal colon of five pigs, in the middle colon of six pigs and in the distal colon of seven pigs. One of the DDGS-fed pigs did not show methane production in any segment.

image

Figure 3.  Mean methane production rate in digesta from various segments determined by in vitro incubations of digesta. Error bars represent standard errors of the means (n = 4). The caecum did only show methane production in one pig and is consequently not included in the statistical model. (inline image) STD diet and (□) dried distillers grains with solubles diet.

Download figure to PowerPoint

Potential SRRs

Digesta from the STD group showed a significantly higher [P (diet) = 0·003] potential SRR along the large intestine compared to the DDGS group (Fig. 4). The pattern of potential SRR showed a significant increase from the caecum (22·4 vs 6·9 nmol g−1 h−1 in STD vs DDGS group) towards the middle colon (99·9 vs 59·8 nmol g−1 h−1 in STD vs DDGS group), after which the rate increase levelled off in the distal colon segment.

image

Figure 4.  Potential sulphate reduction rate in digesta from various segments determined by 35S-tracer method. Error bars represent standard error of the means (n = 4). Segments without a common letter are significantly different ( 0·05). (inline image) STD diet and (□) dried distillers grains with solubles diet.

Download figure to PowerPoint

Sulphate concentration in digesta

The concentration of free sulphate in digesta showed a tendency towards a higher concentration in the DDGS-fed pigs [P (diet) = 0·09] (Table 2). No significant difference between segments was found, but a slight numerical increase from caecum to distal colon was observed in both groups.

Table 2.   Free sulphate concentration* in digesta of pigs fed the experimental diets
SegmentSulphate concentration (μmol kg−1 digesta)
STDDDGSSEM
  1. DDGS, dried distillers grains with solubles.

  2. *Values are least squares means and standard error of the means (SEM) (n = 4).

Caecum32944566·09
Proximal colon33548766·09
Middle colon38047266·09
Distal colon51854066·09
P-value
 Diet  0·09
 Segment  0·19
 Diet × segment  0·80

SCFA concentration in digesta

The total concentration of straight-chain SCFA (Table 3) showed no diet-related difference, whereas the concentration of branched-chain SCFA (iso-valeric and iso-butyric acid) was significantly higher [P (diet) = 0·02] in the STD group compared to the DDGS group. Additionally, the concentration of branched-chain SCFA showed a significant increase along the large intestine.

Table 3.   Concentration* of short chain fatty acids (SCFA) in digesta of pigs fed the experimental diets
SegmentStraight-chain SCFA† (mmol kg−1 digesta)Branched-chain SCFA‡ (mmol kg−1 digesta)
STDDDGSSEM§STDDDGSSEM§
  1. DDGS, dried distillers grains with solubles.

  2. *Values are least squares means and standard error of the means (SEM) (n = 4).

  3. †Acetic, propionic, butyric and valeric acid.

  4. ‡Iso-butyric and iso-valeric acid.

  5. §Segments without a common letter are significantly different ( 0·05).

Caecum136·1125·56·58a0·40·10·26a
Proximal colon143·1133·36·58a0·90·40·26b
Middle colon108·4108·36·58b2·11·40·26c
Distal colon93·794·06·58c3·22·10·26d
P-value
 Diet  0·32  0·02
 Segment  <0·001  <0·001
 Diet × segment  0·74  0·16

Enumeration of SRB and total bacteria by qPCR

The conditions reported by Kondo et al. (2004) for the DSR1F+/DSR-R primer pair resulted in the formation of amplification products of the desired length (182 bp) as well as shorter products, which was evident by gel visualization. Higher annealing temperatures were tested to eliminate the nontarget products, and it was found that at 62°C, only fragments of the right size were formed and accordingly, this temperature was used for the qPCR assay. Cloning and sequencing of the PCR products yielded only dsrA-related sequences, thus suggesting no co-amplification of nontarget sequences of similar length.

The mean number of SRB per gram of digesta was significantly higher in samples from the distal colon (5·2 vs 5·0 log10 SRB per g in STD vs DDGS group) compared to the caecum samples (4·6 vs 4·2 log10 SRB per g in STD vs DDGS group) in both diet groups (Table 4). No diet-related difference was found.

Table 4.   Numbers* of sulphate-reducing bacteria (SRB) and total bacteria determined by qPCR
Segmentlog10 (SRB g−1 digesta)†log10 (total bacteria g−1 digesta)‡
STDDDGSSEM§STDDDGSSEM§
  1. DDGS, dried distillers grains with solubles; qPCR, quantitative PCR

  2. *Values are least squares means and standard error of the means (SEM) (n = 4).

  3. †Assuming one dsrA gene copy per SRB genome.

  4. ‡Assuming 3·6 rRNA operons per bacterial genome.

  5. §Segments without a common letter are significantly different ( 0·05).

Caecum4·64·20·14a10·09·80·10a
Proximal colon4·64·70·14b10·110·10·10ab
Middle colon4·94·90·14bc10·210·30·10bc
Distal colon5·25·00·14c10·210·40·10c
P-value
 Diet  0·22  0·72
 Segment  <0·001  0·007
 Diet × segment  0·52  0·32

The total number of bacteria in the digesta showed no diet-related difference, but a significant increase from the caecum to the distal colon was found.

Estimation of number of SRB based on potential SRRs

The SRB numbers calculated based on the potential SRR ranged from 7·0 to 7·7 log10 SRB per g and 6·4 to 7·4 log10 SRB per g for STD and DDGS group, respectively (Table 5). As expected, these numbers, deriving from potential SRR, also showed a clear effect of diet and an increase from caecum to middle colon.

Table 5.   Numbers* of sulphate-reducing bacteria (SRB) calculated from the potential sulphate reduction rate (SRR)
Segmentlog10 (SRB g−1 digesta)†
STDDDGSSEM‡
  1. DDGS, dried distillers grains with solubles.

  2. *Values are least squares means and standard error of the means (SEM) (n = 4).

  3. †Assuming a cell-specific SRR of 48 fmol per cell per day.

  4. ‡Segments without a common letter are significant different ( 0·05).

Caecum7·06·40·13a
Proximal colon7·57·10·13b
Middle colon7·77·40·13c
Distal colon7·77·40·13c
P-value
 Diet  0·005
 Segment  <0·001
 Diet × segment  0·38

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The two experimental diets were formulated with the objective of manipulating the microbial ecosystem to a great extent to study its impact on the production of VSC. Addition of DDGS at the expense of wheat and soybean meal was expected to add protein and NSP to the diet (Stein et al. 2006) and accordingly the DDGS diet had a considerably higher content of NSP, crude protein, cysteine, methionine and total sulphur.

We hypothesize that the observed initial burst of VSC in the in vitro incubations derived from an entrapment of VSC in the digesta, which were not released and eliminated from the bottles during the repeated evacuations. Some samples showed a negative net production rate, however, as the initial amount of VSC available for consumption (deriving from the burst) was depleted during incubation, the negative rates are only indicative of consumption exceeding production and their magnitude cannot be interpreted.

The measured net production rates of H2S from the colonic segments (41·7–145·6 nmol g−1 h−1) are within the range of SRR reported for the large intestine of a human sudden death victim (c. 240 nmol g−1 dry weight per h) (Macfarlane et al. 1992) and human faecal samples (c. 2·8–51·7 nmol g−1 h−1) (Christl et al. 1992; Lewis and Cochrane 2007) and so there seems to be conformity among intestinal environments of humans and pigs, whereas somewhat lower rates (c. 1·8 nmol g−1 h−1) (Deplancke et al. 2003) have been found in digesta from the colon of mice. It should be noted, though, that the H2S net production rate measured by the in vitro incubations of the present study are the collective outcome of all H2S producing and consuming processes, whereas the SRR from the literature, like the potential SRR of the present study, only accounts for H2S produced by SRB.

The increase in H2S net production rates observed along the sequence of segments implies that the producing processes, primarily sulphate reduction and cysteine degradation, rise distally in the large intestine. Amino acid fermentation in the hindgut of pigs (Jensen et al. 1995) and humans (Macfarlane et al. 1992) has been reported to increase distally in the large intestine, which is supported by the increased concentration of branched-chain SCFA [exclusively produced by amino acid degradation (Allison 1978)] along the large intestine observed in the present study. Similarly, the potential SRR and the number of SRB (qPCR) increased from the caecum towards the distal colon segment, and as free sulphate was found in all segments, it seems plausible that an increasing SRR from caecum to distal colon contributes further to the increasing H2S net production rate along the hindgut.

From the potential SRR and the measured free sulphate concentration in digesta, it follows that the free sulphate found in the caecum is insufficient to maintain sulphate reduction in the digesta as it moves through the colon, and thus would have to be replenished continuously, which is supported by the uniform free sulphate concentration measured in all segments. At the measured potential SRR, the pool of free sulphate in each colon segment would be consumed relatively fast (c. 4–5 h and c. 8–18 h in STD and DDGS group, respectively) compared to the residence time of digesta in the colon (c. 48 h). In the colon, sulphate may be liberated from dietary components or from sulphated mucin secreted in the intestine (Gibson et al. 1988), whereas in the in vitro incubations, only the dietary sulphate source is present. The free sulphate found in the digesta would result in initial free sulphate concentrations of only c. 65–110 μmol l−1 in the in vitro incubations, concentrations which approach half-saturation constants (Km) of Desulfovibrio for sulphate (Ingvorsen and Jorgensen 1984), and so the early sulphate concentration of the incubations would probably only just be sufficient to saturate the initial sulphate-reducing activity of the SRB. A comparison of the potential SRR (not sulphate limited) and the total VSC net production rate (sulphate reduction, cysteine and methionine degradation collectively) of the STD group showed a numerically higher potential SRR in the caecum, proximal and middle colon segment, thus indicating that the SRB in these segments were, to some extent, limited by a lack of oxidized sulphur.

The higher H2S net production rate in the STD group could be a result of diet-related differences in both sulphate reduction and S-amino acid degradation. As no difference in the concentration of free sulphate along the hindgut was measured, higher SRR of the STD (indicated by higher potential SRR) is likely to contribute to the higher H2S net production rate of this group. Additionally, the significantly higher concentration of branched-chain SCFA in the STD group indicates higher amino acid fermentation rates in this group, adding to the higher H2S net production rate. The NSP content of the DDGS diet is likely to have effected an increased incorporation of amino acids into bacterial protein in the large intestine by supplying a substrate for fermentation and consequent increased bacterial growth (Jensen et al. 1995).

Finally, it should be pointed out that the lower H2S net production rate in the hindgut of the DDGS group might be the result of a higher net production of MT by methylation of H2S in these incubations. If the produced MT had been degraded to H2S, there would be no significant difference in H2S net production, as seen for the total VSC net production rate.

The distribution of MT and DMS net production rates along the large intestine of the STD-fed pigs could be inferred as a result of the distribution of methane production: MT and DMS accumulate in the caecum because of no or little methanogenic degradation, followed by an emerging methanogenic degradation in the proximal colon, and finally a balanced production and degradation in the middle and distal segments of the colon, where methane production is (numerically) highest. Such a balanced production and degradation of MT and DMS have also been observed for MT and DMS production in other environments (Visscher et al. 1995; Lomans et al. 1997). However, the pattern of high and relatively even MT net production in all segments of the DDGS-fed pigs does not fit the distribution of methane production and most likely derives from a combination of higher production (methionine degradation and methylation of H2S) and lower degradation (methanogenic degradation). The lower concentration of branched-chain SCFA in digesta from the DDGS-fed pigs indicates lower amino acid fermentation and consequently a lower MT production from methionine degradation in this group, despite the slightly higher methionine concentration in the DDGS diet. The other MT-producing process is methylation of H2S. The source of methyl groups in this process is methoxylated aromatic compounds (Finster et al. 1990) that are primarily found in the NSP fraction of the diet (Besle et al. 1995; Bravo 1998). As the DDGS diet contained more NSP compared to the STD diet, this might be the reason for the observed difference. Finally, the higher MT net production rate in the colon of the DDGS group may be a result of no or very limited methanogenic degradation of MT. The reason for such low or absent MT degradation could be speculated to be the result of a diet-related shift in the community of methanogens, because not all methanogens are capable of MT degradation (Boone et al. 1993; Lomans et al. 2002).

Based exclusively on odour threshold values (1·1 vs 17·8 ppb for MT and H2S, respectively (Devos et al. 1990)) the potential of MT to affect odour emission clearly outweighs that of H2S. Consequently, the markedly higher MT net production throughout the large intestine of the DDGS-fed pigs is likely to result in an increased odour production in this group compared to the STD. Additionally, inclusion of DDGS in pig diets has been found to result in increased slurry production (Jarret et al. 2011), which would further enhance the odorous impact of the higher MT net production. However, no significant effect of diet was found on the amount of digesta in each segment in present study (data not shown).

The reports on numbers of SRB in the hindgut of pigs obtained by both culture dependent (Butine and Leedle 1989) and culture-independent methods (Kerr et al. 2011) are only few and the results vary greatly. In the light of the significant diet effect on potential SRR, it was surprising not to find a parallel difference in SRB numbers estimated by qPCR. A calculation of cell-specific rates, based on the qPCR-determined SRB numbers and the potential SRR, yielded cell-specific rates far beyond previously reported rates (Detmers et al. 2001) for both dietary groups, and on this basis, we suspect the qPCR assay to have underestimated the number of SRB in the digesta – most pronounced in the STD group. The reasons for such an underestimation may be poor DNA extraction efficiency and incomplete SRB diversity coverage by the primers, which would have been exacerbated by the necessary use of a raised annealing temperature.

To provide an alternative estimate of the number of SRB in the digesta, cell numbers were calculated based on the measured potential SRR and a cell-specific SRR of 48 fmol per cell per day (Detmers et al. 2001). The obtained numbers of SRB (Table 5) were in good agreement with the values reported by Butine and Leedle (1989) for caecum and colon contents of pigs and human faecal samples (Lewis et al. 2005) and were approximately two orders of magnitude higher than those obtained by qPCR.

The number of SRB from caecum towards distal colon increased in both dietary groups, as indicated by both qPCR and potential SRR. This result is supported by Kerr et al. (2011) and Butine and Leedle (1989), who both reported increasing numbers of SRB distally in the large intestine of pigs. The pattern of increasing numbers of SRB might be ascribed to a mere upgrowth of SRB in the digesta as it moves through the colon, but factors such as the distribution of sulphated mucin-excreting cells (available sulphate) (Deplancke et al. 2003) and physicochemical conditions (Macfarlane et al. 1992) are likely to play a role, too. In support of this, pronounced variation in the relative numbers of SRB genera have been found between different sites in the colon of both pigs (Kerr et al. 2011) and humans (Croix et al. 2011), indicating that the SRB community changes in both numbers and composition along the colon.

In conclusion, the results demonstrate that VSC are produced throughout the large intestine of pigs at rates dependent on both diet composition and location in the intestine. Feeding the DDGS-containing diet resulted in a significantly higher net production rate of MT, yet a lower H2S net production rate and potential SRR. In relation to odour, this increased MT net production overshadows that of H2S, and consequently, feeding the DDGS diet may lead to increased odour emission compared to the STD diet, although this needs to be verified through studies including actual olfactometry measurements.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Morten Poulsen, Thomas Rebsdorf and Tove Wiegers for excellent technical assistance. The study was financially supported by the Directorate for Food, Fisheries and Agri Business, Copenhagen, Denmark (Strategies for odour reduction from pig production units and slurry application) and the Faculty of Science and Technology, Aarhus University, Denmark.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Allison, M.J. (1978) Production of branched-chain volatile fatty-acids by certain anaerobic bacteria. Appl Environ Microbiol 35, 872877.
  • Attene-Ramos, M.S., Wagner, E.D., Gaskins, H.R. and Plewa, M.J. (2007) Hydrogen sulfide induces direct radical-associated DNA damage. Mol Cancer Res 5, 455459.
  • Besle, J.M., Jouany, J.P. and Cornu, A. (1995) Transformations of structural phenylpropanoids during cell-wall digestion. FEMS Microbiol Rev 16, 3352.
  • Blanes-Vidal, V., Hansen, M.N., Adamsen, A.P.S., Feilberg, A., Petersen, S.O. and Jensen, B.B. (2009) Characterization of odor released during handling of swine slurry: part I. Relationship between odorants and perceived odor concentrations. Atmos Environ 43, 29973005.
  • Boone, D.R., Whitman, W.B. and Rouviére, P. (1993) Diversity and taxonomy of methanogens. In Methanogenesis ed. Ferry, J.G. pp. 3580 New York: Chapman & Hall.
  • Bravo, L. (1998) Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr Rev 56, 317333.
  • Butine, T.J. and Leedle, J.A.Z. (1989) Enumeration of selected anaerobic bacterial groups in cecal and colonic contents of growing-finishing pigs. Appl Environ Microbiol 55, 11121116.
  • Canibe, N., Hojberg, O., Badsberg, J.H. and Jensen, B.B. (2007) Effect of feeding fermented liquid feed and fermented grain on gastrointestinal ecology and growth performance in piglets. J Anim Sci 85, 29592971.
  • Christl, S.U., Gibson, G.R. and Cummings, J.H. (1992) Role of dietary sulfate in the regulation of methanogenesis in the human large-intestine. Gut 33, 12341238.
  • Croix, J.A., Carbonero, F., Nava, G.M., Russell, M., Greenberg, E. and Gaskins, H.R. (2011) On the relationship between sialomucin and sulfomucin expression and hydrogenotrophic microbes in the human colonic mucosa. PLoS ONE 6, e24447.
  • Deplancke, B., Finster, K., Graham, W.V., Collier, C.T., Thurmond, J.E. and Gaskins, H.R. (2003) Gastrointestinal and microbial responses to sulfide-supplemented drinking water in mice. Exp Biol Med 228, 424433.
  • Detmers, J., Bruchert, V., Habicht, K.S. and Kuever, J. (2001) Diversity of sulfur isotope fractionations by sulphate-reducing prokaryotes. Appl Environ Microbiol 67, 888894.
  • Devos, M., Patte, F., Rouault, J., Faffort, P. and Van Gemert, L. (1990) Standardized Human Olfactory Thresholds. New York: IRI Press at Oxford University Press.
  • Englyst, H., Wiggins, H.S. and Cummings, J.H. (1982) Determination of the non-starch polysaccharides in plant foods by gas-liquid-chromatography of constituent sugars as alditol acetates. Analyst 107, 307318.
  • Finster, K., King, G.M. and Bak, F. (1990) Formation of methylmercaptan and dimethylsulfide from methoxylated aromatic-compounds in anoxic marine and fresh-water sediments. FEMS Microbiol Ecol 74, 295301.
  • Florin, T., Neale, G., Gibson, G.R., Christl, S.U. and Cummings, J.H. (1991) Metabolism of dietary sulfate – absorption and excretion in humans. Gut 32, 766773.
  • Fossing, H. and Jorgensen, B.B. (1989) Measurement of bacterial sulfate reduction in sediments – evaluation of a single-step chromium reduction method. Biogeochemistry 8, 205222.
  • Gibson, G.R., Cummings, J.H. and Macfarlane, G.T. (1988) Use of a 3-Stage continuous culture system to study the effect of mucin on dissimilatory sulfate reduction and methanogenesis by mixed populations of human gut bacteria. Appl Environ Microbiol 54, 27502755.
  • Hansen, B. (1989) Determination of nitrogen as elementary-N, an alternative to Kjeldahl. Acta Agric Scand 39, 113118.
  • Hansen, M.J., Adamsen, A.P.S., Feilberg, A. and Pedersen, P. (2012) Prediction of odor from pig production based on chemical odorants. J Environ Qual 41, 436443.
  • Harms, G., Layton, A.C., Dionisi, H.M., Gregory, I.R., Garrett, V.M., Hawkins, S.A., Robinson, K.G. and Sayler, G.S. (2003) Real-time PCR quantification of nitrifying bacteria in a municipal wastewater treatment plant. Environ Sci Technol 37, 343351.
  • Higgins, M.J., Chen, Y.C., Yarosz, D.P., Murthy, S.N., Maas, N.A., Glindemann, D. and Novak, J.T. (2006) Cycling of volatile organic sulfur compounds in anaerobically digested biosolids and its implications for odors. Water Environ Res 78, 243252.
  • Ingvorsen, K. and Jorgensen, B.B. (1984) Kinetics of sulfate uptake by fresh-water and marine species of Desulfovibrio. Arch Microbiol 139, 6166.
  • Jarret, G., Cozannet, P., Martinez, J. and Dourmad, J.Y. (2011) Effect of different quality wheat dried distiller’s grain solubles (DDGS) in pig diets on composition of excreta and methane production from faeces and slurry. Livest Sci 140, 275282.
  • Jensen, B.B. and Jorgensen, H. (1994) Effect of dietary fiber on microbial activity and microbial gas-production in various regions of the gastrointestinal-tract of pigs. Appl Environ Microbiol 60, 18971904.
  • Jensen, M.T., Cox, R.P. and Jensen, B.B. (1995) Microbial-production of skatole in the hind gut of pigs given different diets and its relation to skatole deposition in backfat. Anim Sci 61, 293304.
  • Jozefiak, D., Rutkowski, A., Kaczmarek, S., Jensen, B.B., Engberg, R.M. and Hojberg, O. (2010) Effect of -glucanase and xylanase supplementation of barley- and rye-based diets on caecal microbiota of broiler chickens. Br Poult Sci 51, 546557.
  • Kerr, B.J., Weber, T.E., Ziemer, C.J., Spence, C., Cotta, M.A. and Whitehead, T.R. (2011) Effect of dietary inorganic sulfur level on growth performance, fecal composition, and measures of inflammation and sulfate-reducing bacteria in the intestine of growing pigs. J Anim Sci 89, 426437.
  • Klappenbach, J.A., Saxman, P.R., Cole, J.R. and Schmidt, T.M. (2001) rrndb: the ribosomal RNA operon copy number database. Nucleic Acids Res 29, 181184.
  • Klein, M., Friedrich, M., Roger, A.J., Hugenholtz, P., Fishbain, S., Abicht, H., Blackall, L.L., Stahl, D.A. et al. (2001) Multiple lateral transfers of dissimilatory sulfite reductase genes between major lineages of sulfate-reducing prokaryotes. J Bacteriol 183, 60286035.
  • Knudsen, K.E.B. (1997) Carbohydrate and lignin contents of plant materials used in animal feeding. Anim Feed Sci Tech 67, 319338.
  • Kondo, R., Nedwell, D.B., Purdy, K.J. and de Queiroz Silva, S. (2004) Detection and enumeration of sulphate-reducing bacteria in estuarine sediments by competitive PCR. Geomicrobiol J 21, 145157.
  • Lewis, S. and Cochrane, S. (2007) Alteration of sulphate and hydrogen metabolism in the human colon by changing intestinal transit rate. Am J Gastroenterol 102, 624633.
  • Lewis, S., Brazier, J., Beard, D., Nazem, N. and Proctor, D. (2005) Effects of metronidazole and oligofructose on faecal concentrations of sulphate-reducing bacteria and their activity in human volunteers. Scand J Gastroenterol 40, 12961303.
  • Li, W., Powers, W. and Hill, G.M. (2011) Feeding distillers dried grains with solubles and organic trace mineral sources to swine and the resulting effect on gaseous emissions. J Anim Sci 89, 32863299.
  • Lomans, B.P., Smolders, A.J.P., Intven, L.M., Pol, A., denCamp, H.J.M.O. and vanderDrift, C. (1997) Formation of dimethyl sulfide and methanethiol in anoxic freshwater sediments. Appl Environ Microbiol 63, 47414747.
  • Lomans, B.P., van der Drift, C., Pol, A. and Op den Camp, H.J.M. (2002) Microbial cycling of volatile organic sulfur compounds. Cell Mol Life Sci 59, 575588.
  • Macfarlane, G.T., Gibson, G.R. and Cummings, J.H. (1992) Comparison of fermentation reactions in different regions of the human colon. J Appl Bacteriol 72, 5764.
  • Magee, E.A., Richardson, C.J., Hughes, R. and Cummings, J.H. (2000) Contribution of dietary protein to sulfide production in the large intestine: an in vitro and a controlled feeding study in humans. Am J Clin Nutr 72, 14881494.
  • Mason, V.C., Bechandersen, S. and Rudemo, M. (1980) Hydrolysate preparation for amino-acid determinations in feed constituents. 8. Studies of oxidation conditions for streamlined procedures. Z Tierphysiol Tierernahr Futtermittelkd 43, 146164.
  • Nes, P. (1979) Routine measurement of total sulfur in biological-material. NZ J Sci 22, 269272.
  • Pitcher, M.C.L. and Cummings, J.H. (1996) Hydrogen sulphide: a bacterial toxin in ulcerative colitis? Gut 39, 14.
  • Poulsen, H.V., Canibe, N., Finster, K. and Jensen, B.B. (2010) Concentration of volatile sulphur-containing compounds along the gastrointestinal tract of pigs fed a high-sulphur or a low-sulphur diet. Livest Sci 133, 128131.
  • Schiffman, S.S., Miller, E.A.S., Suggs, M.S. and Graham, B.G. (1995) The effect of environmental odors emanating from commercial swine operations on the mood of nearby residents. Brain Res Bull 37, 369375.
  • Spence, C., Whitehead, T.R. and Cotta, M.A. (2008) Development and comparison of SYBR Green quantitative real-time PCR assays for detection and enumeration of sulfate-reducing bacteria in stored swine manure. J Appl Microbiol 105, 21432152.
  • Stein, H.H., Gibson, M.L., Pedersen, C. and Boersma, M.G. (2006) Amino acid and energy digestibility in ten samples of distillers dried grain with solubles fed to growing pigs. J Anim Sci 84, 853860.
  • Suarez, F., Furne, J., Springfield, J. and Levitt, M. (1997) Insights into human colonic physiology obtained from the study of flatus composition. Am J Physiol 35, G1028G1033.
  • Suarez, F., Furne, J., Springfield, J. and Levitt, M. (1998) Production and elimination of sulfur-containing gases in the rat colon. Am J Physiol 37, G727G733.
  • Suarez, F., Springfield, J., Furne, J. and Levitt, M. (1999) Differentiation of mouth versus gut as site of origin of odoriferous breath gases after garlic ingestion. Am J Physiol 276, G425G430.
  • Visscher, P.T., Taylor, B.F. and Kiene, R.P. (1995) Microbial consumption of dimethyl sulfide and methanethiol in coastal marine-sediments. FEMS Microbiol Ecol 18, 145153.
  • Willis, C.L., Cummings, J.H., Neale, G. and Gibson, G.R. (1996) In vitro effects of mucin fermentation on the growth of human colonic sulphate-reducing bacteria. Anaerobe 2, 117122.
  • Wing, S., Horton, R.A., Marshall, S.W., Thu, K., Taiik, M., Schinasi, L. and Schiffman, S.S. (2008) Air pollution and odor in communities near industrial swine operations. Environ Health Perspect 116, 13621368.