Oxygen: The tolerance of the microorganisms in relation with oxygen classifies them as aerobic (when growth requires oxygen), facultative anaerobes (when growth may occur on oxygen when available but does not require it) and anaerobes, classified further to strictly anaerobes (when oxygen is toxic) and aerotolerant anaerobes (when growth may occur in the presence of oxygen but without utilising it). Strict anaerobes include Clostridia, methanogens, sulphate reducers and homoacetogens. The sensitivity to oxygen varies widely among the strict anaerobes. All bacteria contain enzymes to react with oxygen and produce toxic free radicals that destroy vital cellular components. However, it is the presence of other enzymes that remove the toxic oxygen radicals that determine the degree of tolerance to oxygen. In anaerobic environments the traces of oxygen are rapidly consumed by the facultative anaerobes of the consortium, decreasing the redox potential to acceptable levels (-400 mV). For this reason, the facultative anaerobes are usually found in external layers in systems where spatial distribution of the various populations is possible (e. g. lagoons, heterogeneous or hybrid bioreactors).

Ammonia: It is the degradation product of nitrogenous compounds such as proteins and amino acids. Anaerobic digestion of feedstocks such as manure results in the production of high amounts of ammonia. Non-ionised ammonia is quite inhibitory to methanogens. Since the concentration of non-ionised ammonia is a function of pH, the inhibition is less at neutral pH. There are contradictory reports on the levels of tolerance to ammonia, due to differences in substrates, inocula, environmental conditions and acclimation periods. The inhibitory total ammonia concentration causing a 50% decrease in the methane production ranges from 1.7 to 14 g/L (Chen et al., 2008). Acclimation plays a significant role in making the anaerobic consortium tolerant to high levels of ammonia (Bhattacharya and Parkin, 1989; Angelidaki and Ahring, 1993).

Long chain fatty acids (LCFAs) and other organic compounds: LCFAs tend to be adsorbed on surfaces and interfere with the molecule transfer mechanisms or the protection functions of the cell wall or membrane. Moreover, flotation of biomass can occur as a result of the adsorption of LCFA (Rinzema et al., 1989). The inhibition of LCFA in thermophilic anaerobes is more severe because of the different composition of their cell membranes (Hwu and Lettinga, 1997). Biodegradation of LCFAs, although difficult, has been observed in mesophilic and thermophilic conditions.

Other organic compounds which have been found to be toxic to anaerobic digestion are: alkyl benzenes, halogenated benzenes, nitrobenzenes, phenol and alkyl phenols, halogenated phenols, nitrophenols, alkanes, halogenated aliphatics, alcohols, halogenated alcohols, aldehydes, ethers, ketones, acrylates, carboxylic acids, amines, nitriles, amides, pyridine and its derivatives (Chen et al., 2008). The extent of toxicity depends on several factors such as the toxicant concentration, microorganism concentration, toxicant exposure time, cell age, feeding pattern, acclimation and temperature (Yang and Speece, 1986).

Metals: They can be distinguished into light and heavy metals. Light metals are present in the form of cations in solution and, in the case of anaerobic digesters, they usually include sodium, potassium, calcium and magnesium. They are usually added in the form of chemicals for pH control, but they can also arise from the breakdown of biomass. They are required for microbial growth at moderate concentrations, but they can cause severe inhibition or even toxicity at high concentrations (Soto et al., 1993).

Heavy metals (e. g. chromium, iron, cobalt, copper, zinc, cadmium and nickel) can be present in significant concentrations in municipal sewage and sewage sludge as well as in industrial wastewaters. Several metals such as iron, zinc, nickel, cobalt, molybdenum and copper are constituents of vital enzymes. Due to the non biodegradability of heavy metals, they tend to biosorb and accumulate at toxic concentrations. Apart from sorption, the heavy metals may be precipitated (reacting with sulphide, carbonate or hydroxyls) or form complexes in solution with degradation compounds produced during digestion. However, only metals in soluble free ionic form exhibit toxicity (Mosey and Hughes, 1975; Oleszkiewicz and Sharma, 1990). Therefore, immobilisation of heavy metals can take place through processes such as precipitation, sorption and chelation. The relative sensitivity of acidogenesis and methanogenesis to heavy metals is Cu > Zn > Cr > Cd > Ni > Pb and Cd > Cu > Cr > Zn > Pb > Ni, respectively (Lin, 1992, 1993).

Sulphide and sulphate: The presence of sulphate in the absence of oxygen causes anoxic conditions since it can be used as an electron acceptor instead of oxygen. The sulphate reducing bacteria can utilise a number of substrates (acetate, hydrogen, propionate, butyrate) in anaerobic systems and, therefore, they compete with the groups of microorganisms that degrade the same substrates. As a result the flow of electrons is diverted mostly to sulphide instead of methane production reducing the efficiency of the anaerobic systems (in terms of biogas production).

Sulphide is toxic to methanogens but also to the sulphate reducing bacteria. There is a great discrepancy in the literature concerning the mechanism of inhibition and the toxicity levels of sulphides (Chen et al., 2008). Sulphide removal can take place through stripping, coagulation, oxidation, precipitation but also through biological processes such as oxidation to sulphur (Oude Elferink et al., 1994; Song et al., 2001). Acclimation to sulphide can also be beneficiary to methanogens, increasing their tolerance levels.