Type 021N and Thiothrix spp...

Eikelboom's Type 021N and Thiothrix sp. are usually considered together because of their similar morphology and many common kinetic and metabolic features. The taxonomic position of Type 021N within the current bacteriological system is still unresolved. In the early 1970s, some Czech authors described a filamentous microorganism morphologically resembling the later Type 021N. They put this microorganism in the genus Leucothrix (Leucothrix cohaerens, Leucothrix mucor). A Leucothrix - type filamentous bacterium which caused bulking in an activated sludge system treating petrochemical wastewaters was later described also by another author. However, Eikelboom did not recognize the positioning of this microorganism within the genus Leucothrix and claimed that Type 021N is identical neither with Leucothrix nor with Thiothrix. The primary reason that Leucothrix spp. were not included in the system of filamentous microorganisms occurring in activated sludges seemed to be that these organisms are halophilic and of marine origin.

The filamentous organisms described as Type 021N, Thiothrix and Leucothrix have been intensively studied. Thanks to these exhaustive studies we can conclude that Eikelboom's supposition was right. Type 021N can be placed within the family Leucotrichaceae but it appears to occupy an intermediate position between genera Leucothrix and Thiothrix. A final decision will only be possible when the genetic relatedness studies of these three microorganisms are available. Because of a great similarity in morphology, nutritional requirements and growth habits they cannot be distinguished unanimously by means of conventional microbiological methods.

These studies can also be used to determine the characteristic features of both filaments. Type 021N exhibits a nutritional versatility and is able to utilize a great variety of readily biodegradable substrates and carbon sources, such as simple sugars, alcohols and organic acids. Its affinity to these substrates under low concentrations is high because of low value of the half - saturation constant, Ks. Its value was found to be less than 1 mg/L for glucose and 0.07 mg/L for acetic acid. In addition, Type 021N can successfully compete for energy and carbon sources during unbalanced growth under nutrient limiting conditions, especially as far as ammonia nitrogen is concerned. Some authors explained this selective advantage over floc - forming microorganisms by a rapid assimilation and storage of ammonium under these conditions in comparison with floc - formers. This is why Type 021N is often classified as the nutrient deficiency bulking filamentous microorganism. The remediation of bulking problems caused by Type 021N by adding nutrients in a proper balance to organic loading was also verified in full - scale plants outside the US.

Type 021N is reportedly not able to denitrify. The trichomes react negatively to the Gram stain, but PHB and volutine granules are regularly observed. However, the formation and utilization of volutine granules, which can be detected by the Neisser stain, is extremely slow. Thus Type 021N is not able to compete for substrates with true poly - P bacteria.

Sulphur granules can also be observed in cells of Type 021N, after the S-test with thiosulphate, although not very often in situ. This fact indicates that Type 021N is connected with sulphur metabolism. The ability to metabolize reduced sulphur compounds is one of the most distinguishing features between Type 021N and Leucothrix spp. Type 021N exhibits mixotrophic characteristics when it gains energy from the oxidation of both reduced sulphur compounds and organic substrates. Nevertheless, Type 021N is probably not able to oxidize reduced sulphur compounds in the absence of organic carbon compounds, i.e., it is not chemolithotrophic. The preferred sulphur compound for Type 021N seems to be thiosulphate. Fortunately, the supportive biochemical mechanism of mixotrophic utilization of thiosulphate is probably not efficient enough to lead to bulking problems caused by Type 021N.

Thiothrix can be found under conditions which also favor the growth of Type 021N, because these two types are metabolically rather close. Thiothrix utilizes many readily biodegradable compounds, especially organic acids but the importance of sulphur metabolism is more pronounced than for Type 021N. The sulphur granules are frequently present in Thiothrix trichomes in situ while in Type 021N, filaments can be observed mostly after the S-test.

Thiothrix seems to be an obligate mixotroph requiring reduced sulphur compounds (sulphide, thiosulphate) in combination with readily biodegradable organic substrates. Reduced sulphur and organic compounds can be utilized by Thiothrix either simultaneously or in two phases. For instance, Some authors were observed for one Thiothrix strain a biphasic growth scheme when thiosulphate was nearly completely oxidized without any significant consumption of acetate. When thiosulphate was exhausted, acetate consumption and oxidation of cell-stored sulphur followed the first phase.

The nutritional requirements of Thiothrix predict its preferred environment with low fatty acids and reduced sulphur compounds. Such conditions can be found in EBPR activated sludge systems when the utilization of fermentation products in the anaerobic zone is incomplete. In addition, there is a question still unresolved about whether Thiothrix can also contribute to the reduction of sulphur compounds in anaerobic conditions (dissimilatory sulphate reduction), or whether it only utilizes in oxic conditions the reduced sulphur compounds generated by other than Thiothrix sulphate-reducing microbes.

Morphologically, the trichomes of Type 021N and Thiothrix resemble the filaments of colorless Cyanophyceae. The trichomes of Type 021N are straight or curved. They are robust and their length can reach several hundred micrometers. The individual filaments often form "ropes" of a macroscopic length.

The cells in the trichomes are easy to observe, as they are clearly distinguished by septa. The shape of individual cells is rather variable from rectangular to discoid, which is most typical. The filaments of Type 021N cause the interfloc bridging kind of bulking. Because of the length of robustness of trichomes, Type 021N usually causes severe bulking problems with "extremely" high SVI values. From our experience, the onset of bulking troubles caused by Type 021N is very rapid.

According to the manuals, Type 021N filaments are not en-sheathed. However, some outer slime coat was found by some authors for Type 021N isolates. Also an author observed a sheath-like structure surrounding the filament and asked the question about the difference between a true sheath and false sheath.

Contrary to Type 021N, Thiothrix is reported in the manuals to have a sheath which is prominent for Type I and thin and difficult to see for Type II. Both Type 021N and especially Thiothrix filaments form rosettes. An increased number of rosettes may indicate nutrient defiency conditions in the activated sludge system. Thiothrix filaments are distinguished into two types (I and II) according to the filament length and width. The more common Type I has robust filaments of up to 2.5 micro-m in diameter and 500 micro-m in length while Type II filaments are more subtle. Thiothrix Type II looks similar to another sulphur-containing Eikelboom type-Type 0914. However, the sulphur granules observed in Type 0914 are more rectangular than spherical as in Thiothrix. In addition, Type 0914 filaments do not form rosettes. The bulking problems caused by Thiothrix (especially Type I) are of the same nature and extent as in the case of Type 021N.

Under normal conditions, attached growth is not typical either of Type 021N or of Thiothrix. However, some authors were observed heavy attached growth by unicellular epiphytic bacteria on Type 021N and Thiothrix trichomes from an activated sludge system which was operated at about pH 5.5 to control bulking. Their explanation for the appearance of attached growth was that the modification of the trichome surface, including cell walls, by the action of low pH allowed the epiphytic population to colonize the trichome surface. This finding is in good agreement with the above statement that the presence or absence of attached growth is not a very characteristic identification feature, and that the presence of attached growth rather reflects the unhealthy conditions of a given trichome.