Microbial aspects of the formation of malodorous compounds in anaerobically stored piggery wastes

In modern intensive animal farming labour-saving waste handling systems are applied. As such, slatted floors have found wide application. In this system large amounts of malodorous wastes are stored in pits under the slatted floors, resulting in the emission of malodours with the ventilation air. Complaints about odour nuisance caused by piggeries do frequently occur.

In this thesis the results are reported of investigations on the microbiology of anaerobically stored piggery wastes and on the formation in such wastes of some malodorous components.

Chapter 1.

In general, odour can be measured only by sensorial methods and this applies exclusively to the odour intensity. Odour quality can be approached only by comparing it with other odours as present in an odour classification system. The lack of instrumental methods for measuring odour can partially be overcome by estimating concentrations of components considered to be characteristic of odorous mixtures.

About 150 volatile compounds have been identified in piggery wastes by different workers. Based on literature data the possible origin of some groups of volatile compounds in piggery wastes has been indicated in this chapter. In addition the composition of urine, faeces and mixed wastes has been given.

Chapter 2.

Anaerobically stored piggery wastes as found in farm pits contain large amounts of volatile fatty acids. The amounts measured in samples from different farms ranged from 4-27 g/l. About 20% of these acids were already present in the freshly voided faeces. During anaerobic storage large amounts of volatile fatty acids are formed by degradation of macromolecular constituents of the faeces. In an experiment with a mixture of faeces and urine 43% of the raw protein and 24% of the fibre were degraded during 70 days storage. Acetic, propionic, and butyric acids and small amounts of other lower fatty acids and carbon dioxide were the main products formed; in addition some methane was produced.

Chapter 3.

Colony counts of piggery wastes on an average numbered 5.10 9on media containing 80-100% farm slurry supernatant which were prepared and inoculated according to the strictly anaerobic methods developed by Hungate. These colony counts amounted to about 20% of the cell numbers found by direct microscopic observation. Pure cultures were obtained from only 20% of the colonies picked for isolation. The isolated bacteria apparently belonged to the genera Ruminococcus, Peptococcus, Peptostreptococcus and Bacteroides. The bacteria belonging to these genera are usually isolated from the intestinal tract of man and mammals; however, many of the isolates from piggery wastes were unable to grow at 37°C.

Addition of different carbohydrates to media with suboptimal concentrations of farm slurry supernatant revealed that the colony counts could be increased by incorporating hemicellulose preparations in the media. From these results it was concluded that the degradation of hemicellulose is an important process in piggery wastes.

Chapter 4.

Piggery wastes always contain simple phenols. Notably p -cresol was usually present in considerable amounts (up to 350 mg/l). Phenol and 4-ethylphenol were present in concentrations from 10 to 50 mg/l). Phenol and p -cresol originate from the anaerobic degradation of tyrosine, a product of protein break-down. These compounds are in the pig formed by the intestinal microflora as well as during anaerobic storage of the slurry. The simple phenols formed in the gut are partially absorbed by the animal and detoxicated by conjugation with glucuronic acid. The phenylglucuronides are excreted into the urine. The phenols, and also other compounds excreted as glucuronides, can be liberated by the high activity of the enzyme beta-glucuronidase which has been found in the faeces of the pig as well as in farm slurry. The phenols bound to glucuronic acid and excreted into the urine were liberated nearly instantaneously upon contact with slurry. The production of phenols from tyrosine during waste storage proceeded more slowly and was strongly influenced by storage temperature.

The concentrations of indole and skatole in farm slurry were considerably lower than those of p -cresol. Indole was found in amounts of 0-15 mg/l). and skatole of 10-50 mg/l). Indole and skatole are produced in the intestinal tract where tryptophan is their precursor. Urine contained constituent(s) which were readily converted to indole upon contact with faeces. Such constituents were not found in the case of skatole. During anaerobic storage of farm slurry indole and skatole were produced. However, on long-term storage the concentrations of these compounds diminished.

Chapter 5.

The experiments described in this chapter showed that the pathway of tyrosine degradation in faeces differs from that in farm slurry. In faeces, tyrosine is converted to about equal quantities of p -cresol and phenylpropionic acid. p -Cresol is formed from tyrosine according to the scheme tyrosine ->(hydroxyphenyl)pyruvic acid ->(hydroxyphenyl)acetic acid ->(hydroxyphenyl)pyruvic acid ->(hydroxyphenyl)acetic acid ->(hydroxyphenyl)acetic acid ->p -cresol. The pathway leading to phenylpropionic acid can be summarized as follows: tyrosine ->(hydroxyphenyl)pyruvic acid ->(hydroxyphenyl)lactic acid ->(hydroxyphenyl)pyruvic acid ->(hydroxyphenyl)lactic acid ->(hydroxyphenyl)lactic acid ->p -coumaric acid ->(hydroxyphenyl)propionic acid ->phenylpropionic acid.

In farm slurry the same pathways were found to exist, however, here only about 1% of the tyrosine was converted to phenylpropionic acid, the main products of tyrosine degradation being p -cresol and phenol. The formation of the latter product is catalysed by the enzyme tyrosine phenol lyase. An explanation of the observed differences in tyrosine decomposition between faeces and farm slurry can possibly be found in the higher degree of decomposition of farm slurry compared with faeces. Therefore, in faeces tyrosine functions as H-acceptor (formation of phenylpropionic acid) and after decay of easily degradable carbohydrates as is the case in farm slurry, it is used as energy and carbon source (formation of phenol). A further difference between faeces and farm slurry. was observed concerning the metabolism of p -coumaric acid. In faeces this compound was decarboxylated to 4-hydroxystyrene and this product was subsequently reduced to 4-ethylphenol. These reactions did not occur in farm slurry.

For the experiments concerning the degradation of tyrosine described in Chapter 5, 14C-labeled intermediates were used. Some of these labeled intermediates were prepared microbiologically by incubating clostridial strains with 14C-tyrosine and isolating the products formed.

Chapter 6.

The tyrosine metabolism of two Clostridium strains was described. Clostridium sporogenes NCIB 10696 accumulated the following degradation products of tyrosine in its culture media: (hydroxyphenyl)acetic acid, Chydroxyphenyl)lactic acid, (hydroxyphenyl)-propionic acid and tyrosol. p -Coumaric acid was demonstrated to be an intermediate product in tyrosine degradation, probably in the conversion of (hydroxyphenyl)lactic acid to (hydroxyphenyl)propionic acid. p -Coumaric acid accumulated in small quantities when washed cells were incubated with tyrosine and alpha-ketoglutaric acid.

Cl. ghoni accumulated the same products in its culture medium as Cl. sporogenes did, except that (hydroxyphenyl)propionic acid was not formed. Cl. ghoni decarboxylated p -coumaric acid, when added to nutrient media and subsequently hydrogenated the product 4-hydroxystyrene to 4-ethylphenol. The combination of these two clostridial strains suggests a possible pathway for the conversion of tyrosine to 4-ethylphenol.

Chapter 7.

In this chapter overall aspects of microbial conversions in piggery wastes are discussed. The accumulation of volatiles in farm slurry is attributed to the unbalance between the acid stage fermentation and methane fermentation which is nearly absent in the slurry. The methane fermentation in piggery wastes is presumably inhibited by the relatively low storage temperature and high concentrations of ammonia, H 2 S, and cations. The influences of these factors were not investigated. It is concluded that the accumulation of volatile products of the acid stage fermentation (as fatty acids and phenols) is the cause of the stench of piggeries.

In addition it is stated that concentrations of p -cresol and volatile fatty acids can give a good indication concerning changes in odour levels in relation to the development and abatement of odour.