M.L. Siebert

Determination of protein content of anaerobic digesting sludge

Abstract Methods to determine the protein content (acid hydrolysis) as well as the protein-nitrogen (alkali extraction) content of anaerobic sludges are presented. Recovery tests revealed that alkali extracted about 93 per cent of the protein-nitrogen present in the sludge. A correction factor was therefore used to calculate the protein-nitrogen content of a sludge from the analytically determined value after alkali extraction and a factor of 5–15 used to calculate the protein content of the sludge from the protein-nitrogen content.

The proteolytic bacteria present in the anaerobic digestion of raw sewage sludge

Abstract The occurrence and identity of proteolytic bacteria in the anaerobic digestion of raw sewage sludge were investigated. Proteolytic bacteria occurred in numbers of 65 × 106/ml. A high proportion of the isolates (65 per cent) was sporeforming bacteria, and included seven Clostridium species. Cocci comprised 21 per cent of the isolates and consisted mainly of Peptococcus anaerobius and one isolate of Staphylococcus aureus. The remaining isolates were non-sporeforming rod-shaped bacteria and included bifid-like bacteria, Bacteroides species and Eubacterium species.

A biological and chemical study of several anaerobic digesters

Abstract Biological and chemical analyses were carried out on several digesters operated on laboratory, pilot and full-scale. These digesters received a variety of substrates which included raw sewage sludge, industrial effluents and a synthetic substrate. Hexose monophosphate shunt enzymes could only be demonstrated in very active digesters receiving substrates high in carbohydrate material. The glycolytic pathway, glyoxylic acid and citric acid cycles were found in all the anaerobic digesters analysed. Enzyme activity tests revealed different stages of adaptation of a raw sewage digester adapted to a synthetic substrate. Digesters receiving different substrates showed characteristic enzyme activity patterns, but chemical analysis such as pH, alkalinity and volatile fatty acid content did not demonstrate any obvious differences between these digesters.

Estimation of methane-producing bacterial numbers by the most probable number (MPN) technique

Abstract Heukelekian and Heinemann proposed a method in 1939 of enumerating the methane-producing bacteria that occur in anaerobic digesters. A modification of their method is presented here which is simpler to use and yields more consistent results. This method makes use of a mineral medium supplemented with vitamins and uses sodium formate as the sole source of carbon. The bacterial numbers are determined by the most probable number technique.

The bacterial nature of the acid-forming phase of anaerobic digestion

Abstract During bacteriological studies on several laboratory-scale digesters aerobic and facultative anaerobic bacteria occurred in all digesters at all stages, but numerically these bacteria were found to comprise less than 1 per cent of the total acid-forming bacterial population. Obligate anaerobic acid-forming bacteria were also found in all digesters at all stages, usually in numbers 100–200 times greater than the aerobic and facultative anaerobic bacteria present. The numbers of the obligate anaerobic acid-forming bacteria were highly significantly correlated with the deoxyribonucleic acid (DNA) and volatile suspended solids (VSS) contents of the anaerobic digesters. The same was not true for the aerobic and facultative anaerobic bacteria. A highly significant correlation between DNA and VSS was also observed. It was concluded that the concept that facultative anaerobic bacteria are the most important acid producers in anaerobic digestion is incorrect, and that obligate anaerobic bacteria are the important group in acid production.

Biological changes during the adaptation of an anaerobic digester to a synthetic substrate

Abstract A study of the biological changes taking place in the heterogenous microbial population of an anaerobic digester during the period of adaptation to a synthetic substrate and the procedure used to adapt the population to the new substrate is described. Adaptation from raw sewage sludge to the new substrate was followed by chemical, enzymic and bacteriological analyses of the liquid and the solid phases in the digester. Results indicate that although the chemical composition of the effluent did not show any significant changes over the period of adaptation, the chemical, enzymic and bacteriological properties of the sludge phase changed markedly. Results also indicate that adaptation to the new substrate was not complete after 60 days operation since the microbial population was still changing as indicated by intermediate enzyme activities and the number of micro-organisms present per unit volume.

A method for the enumeration and cultivation of anaerobic “acid-forming” bacteria present in digesting sludge

Abstract A method is described for the enumeration and cultivation of anaerobic “acid-forming” bacteria from digesting sludge. In this method use is made of pre-reduced media in roll tubes containing oxygen-free gas atmospheres. Digester supernatant liquor is included in the culture media to provide the growth factors normally present in anaerobic digesters.

Interrelations between biological and chemical characteristics in anaerobic digestion

Abstract A thorough study of the ecosystem of anaerobic digestion should include determinations of both biotic and environmental characteristics and the interrelations between these characteristics. This paper describes such a study executed on three experimental laboratory-scale digesters receiving limiting amounts of nitrogen in their substrate. The characteristics of these three digesters were followed daily until failure of digestion occurred as a result of a deficiency in nitrogen. The daily observations included gas production data, chemical quality determinations, counts of different groups of bacteria, and the determination of a wide range of intermediary metabolic enzymes. The changes taking place in these characteristics until failure occurred are discussed. Linear correlation coefficients were determined between all the different characteristics. A large number of significant correlations was found between the different characteristics of the ecosystem. The implications of some of these correlations are discussed, as well as the advantages of such an approach to the study of ecosystems in general.

Enumeration studies on methanogenic bacteria

Abstract Various media for the enumeration of methanogenic bacteria were compared statistically. It is suggested that a reduced medium containing mineral salts, vitamins, small amounts of fatty acids, carbon dioxide (20 per cent) and hydrogen (80 per cent) should be used for the enumeration of methanogenic bacteria from natural habitats. The addition of rumen fluid and digester fluids to media seemed to have stimulated the growth of more colonies, some of which may have been non-methanogenic in origin. Repressive effects of formate and acetate on colony counts in certain circumstances were observed.

Factor analysis as an aid in an ecological study of anaerobic digestion

Abstract The interrelations between the chemical and biological characteristics of an anaerobic digesting ecosystem were investigated by means of principal component analysis of the linear correlation coefficient matrix of these characteristics. Principal components (factors) were obtained which presented various related characteristics of the ecosystem. The behaviour of the factors during a stress period was graphically demonstrated in relation to time. A three-dimensional model yielded visual representation of the behaviour of three arbitrary chosen factors in relation to each other. The importance of these findings was discussed in relation to the process of anaerobic digestion, and in relation to ecological studies on ecosystems in general.