The OLR, HRT, and SRT are other essential characteristics of ADP. The rapid increase of OLR, especially of readily digestible substrate, causes fast acid forma­tion, leads to alkalinity depletion and a drop in pH. The HRT determines the volume and capital cost for an AD system. The SRT influences the volatile solids (VS) reduction and, thus the methane yield from biomass. Significant fluctuations in OLR, HRT, and SRT lead to upset of the ADP and inhibition of the methane production.

1.1.2 Toxicants

Hydrolytic, fermenting, and acidifying organisms are tolerant to the presence of oxygen but methanogens are strict anaerobes. Oxygen concentration as low as 0.1 mg/L starts to inhibit the production of methane.

High salts concentration (e. g., NaCl) can affect methane production when marine algae are used for anaerobic digestion. Nevertheless, the methane yield from green macroalgae diluted with seawater was comparable to the methane yield from a sam­ple diluted by fresh water [77] . A shock increase in salt concentration in a fixed bacteria reactor caused inhibition only at 35 g/L. When the NaCl concentration was increased gradually, methanogens adapted to concentrations up to 65 g/L [ 78]. Moreover, desalination of macroalgae by heat and pressure resulted in less methane yield compared to untreated algae likely because of the loss of easily digestible organic matter [77, 79].

Heavy metals, such as lead, cadmium, copper, zinc, nickel, and chromium, are well-known toxicants for bacteria. Some algae accumulate heavy metals but their negative effect can be decreased by precipitation with sulfide compounds.

By-products, including ammonia and hydrogen sulfide, at high concentrations can be toxic for methanogenic microorganisms [62]. Generally, the main source of nitrogen and sulfur in AD is proteins, but some seaweeds have a high amount of sulfated carbohydrates. The toxicity of ammonia and sulfide is related to the pres­ence of metals, temperature, and pH in digesters since neutral forms of ammonia and hydrogen sulfide are more toxic, possibly because they can more rapidly pene­trate the cell membrane [65, 80-82]. On the other hand, other authors have reported increasing sulfide toxicity with increasing pH [83]. This discrepancy is possibly due to different mechanisms of sulfide toxicity on different species. The mechanism of sulfide toxicity is usually associated with the following factors: sulfate reducing bacteria that are able to outcompete methanogens for hydrogen and acetate [84]; denaturation of native proteins through the formation of sulfide and disulfide cross­linkage between polypeptide chains [85]; interference with the assimilatory metabolism of sulfur [86]; and the ability to remove essential metals (nickel, iron, cobalt) from the solution.

The mechanisms of ammonia toxicity are possibly associated with disruption of intracellular pH, potassium deficiency, and inhibition of a specific enzymatic reac­tion [87,88]. Several studies showed that ammonia is toxic for methanogenic micro­organisms at concentration 1.5-1.7 g N/L at pH 7.4 and above [89, 90]. Whereas methanogens tolerate ammonia concentration up to 3-4 g N/L at lower pH [90-92]. Moreover, microorganisms are able to acclimate to high ammonia concentration, and ADP can be stable at nitrogen concentrations as high as 5-7 g/L [93-96] .

Organic acids are common intermediate products of AD but accumulation of them, especially in nonionic form, inhibits the overall process. Decline of hydrogen utilization causes accumulation of propionate, leading to failure of the acetoclastic methanogenesis, and therefore causing acetate accumulation and a drop in pH [97]. The mechanism of inhibition by organic acids is probably the denaturation of cell proteins.