Nitrification - inhibitors of ammonia oxidation

Nitrification, the conversion of ammonia into nitrate, is a key metabolic process in many different wastewater treatment systems. This post follows up on one about inhibitors of nitrite oxidation. We will update this post as we find more compounds to add in.

The first step, the conversion of ammonia to nitrite, is carried out by ammonia-oxidizing bacteria (AOBs), which in most systems are generally members of the family Nitrosomonadaceae or the genus Nitrosomonas. The second step, the conversion of nitrite to nitrate, is carried out by nitrite-oxidizing bacteria (NOBs), with Nitrospira the most common genus. Although Nitrobacter has been well studied in the lab, in most systems it is a minor contributor to NOB activity.

Ammonia in the effluent of a WWTP is never a good thing. It can be caused by different circumstances - see Erik Rumbaugh's recent blog post on potential causes and possible responses. There are many chemicals that can cause inhibition, acute or chronic toxicity that can reduce or eliminate the AOB population. Again, we're just trying to gather this information into one place, along with links to references. This information is divided into two different tables: metals and organic chemicals/other.

You may notice some wide ranges on inhibitory concentrations of different compounds. The suspected reason? The sludges have different strains of bacteria in them, with different susceptibilities. Also, researchers use different techniques to measure these values. In some cases, they test pure cultures or nitrifier enrichments for either ammonia or oxygen uptake rates. In other cases, they test activated sludge.

If you find other compounds, please let me know - I'll add them to the tables.

METALS

A good review is available, by Li et al. (2015). Three additional references include:

• Hu et al. (2003)

• Juliastuti et al. (2003)

• Hu et al. (2004)

It's worth noting that the extracellular polymeric substances (EPS) - the stuff that helps floc stick together - is capable of binding to and accumulating metals over time. So, while a short, moderate dose of a metal may have little or no impact on nitrification, it's possible that metals can accumulate in floc over time, leading to chronic inhibition of nitrification. This is particularly important for systems with long SRTs.

The following table summarizes the 2015 review.

Metal	Inhibitory Concentration (mg/L)
Cadmium [Cd(II)]	0.2 - 14
Chromium [Cr(III) or Cr(IV)]*	38 - 90
Copper [Cu(II)]	​3.7 - 61
Mercury [Hg(II)]	8 - 10
Nickel [Ni(II)	3 - 30
Silver [Ag(I)]	​< 1
Zinc [Zn(II)]	0.1 - 10

* It seems to make little difference if it is Cr(III) or Cr(IV).

ORGANIC CHEMICALS/OTHER

Some inhibitors of ammonia oxidation are well known: sulfide (7 - 14 mg/L) and allyl thiourea (1 mg/L), for example. Many funky triazines are used in agricultural applications as inhibitors to prevent the loss of nitrogen from soil. But, there are also a lot of industrial chemicals that can end up in wastewater, too.

I really recommend one specific paper that is almost 40 years old: "Inhibition of nitrification - effects of selected organic compounds" by Hockenbury and Grady. The paper provides good insight into what is, or is not, an inhibitor. High levels of some solvents are not a problem (acetone, ethanol, butanol, propanol), while low levels of some nitrogen-containing compounds are very inhibitory (aniline, dodecylamine, allylthiourea, allyl isothiocyanate, and even the amino acids L-histidine and L-valine). The authors note that many tested compounds inhibit all bacteria, not simply nitrifiers, and are therefore not listed. They also point out that sulfur-containing compounds, and in particular chelators of metals, can be potent inhibitors. This is particularly important for ammonia oxidation as the key enzyme complex, ammonia monooxygenase, uses a copper ion for its catalytic activity (so, yes, copper is toxic at some level, but it is also a required micronutrient).

The following tables are based on the Hockenbury and Grady reference, above, and a second by Tomlinson, Boon and Trotman. I really recommend reading both papers, along with keeping in mind the differences between testing on (relatively)pure cultures or activated sludge.

Inhibition of pure cultures of Nitrosomonas

Compound	Inhibitory Concentration (mg/L, 50% inhibition)
Acetone	8,100
Allyl thiourea	1.2
Aminoethanol	12,200
Aminoguanidine	74
3-Aminotriazole	70
L-Arginine	1.7
N-Butanol	8,200
2-Chloro-6-trichloromethyl pyridine	11
Dichlorophenolindophenol	250
Dicyclohexylcarbodiimide	10
Diethyldithiocarbamate	10
2,4-Dinitrophenol	37
Diphenylthiocarbazone	7.5
Dipyridyl	16
Ethanol	4,100
Ethyl acetate	18,000
Ethyl xanthate	12
L-Histidine	0.5
Hydrazide	300
L-Lysine	4
Methanol	160
L-Methionine	9
Methylamine	310
o-Phenanthroline	9
Phenazine methosulfate	10
N-Propanol	20,000
8-Quinolinol	1.5
Tetramethylammonium chloride	2,200
Thiosemicarbazide	0.9
L-Threonine	3.6
Trimethylamine	590
L-Valine	1.8

Inhibition of nitrification in activated sludge

Compound	Inhibitory Concentration (mg/L, 75% inhibition)
Acetone	2,000
Allyl alcohol	19.5
Allyl chloride	180
Allyl isothiocyanate	1.9
Aniline	7.7
Benzothiazole disulfide	38
Carbon disulfide	35
Chloroform	18
Cresol (o-, p-, or m-)	11.4 - 16.8
Diallyl ether	100
Dicyandiamide	250
Diguanide	50
2,4-Dinitrophenol	460
Dithiooxamide	1.1
Ethanol	2,400
Guanidine carbonate	16.5
Hydrazine	58
8-Hydroxyquinoline	72.5
Mercaptobenzothiazole	3
Methylamine hydrochloride	1,550
Methyl isothiocyanate	0.8
S-Methylisothiourea hemisulfate	6.5
Phenol	5.6
Potassium thiocyanate	300
Skatol	7
Sodium dimethyl dithiocarbamate	0.9
Tetramethylthiuram disulfide	30
Thioacetamide	0.53
Thiosemicarbazide	0.18
Thiourea	0.076
Trimethylamine	118