Microthrix parvicella...

Microthrix parvicella is the most controversial filamentous microorganism of Eikelboom's system, a real enfant terrible. The problems start with its taxonomic position, which is not clear and firm. The physiology and etiology of Microthrix parvicella exhibit many features similar to nocardioform actinomycetes. Thus, some authors agree that Microthrix parvicella could be an actinomycete while others consider this possibility unlikely because Microthrix parvicella filaments have not been shown to branch. However, the diversity in metabolic abilities, the specialization in hydrophobic substrates and the extremely high hydrophobicity of trichomes confirm that Microthrix parvicella is not an ordinary filamentous bacterium.

Thus, although Microthrix parvicella was given a Latin name, it should be treated as an Eikelboom type and not as a taxonomic species. The identification of the type based on a microscopic observation of morphology and staining reactions, and on a subsequent comparison to the description of Microthrix parvicella in the literature is usually unambiguous, although some doubts may arise.

In native samples, Microthrix parvicella can be observed as a thin, coiled long filament (100 - 400 micro - m) and sometimes even longer) which grows both inside the flocs and in the bulk liquid where it forms loose patches.

Under low magnifications, the filaments of Microthrix parvicella may appear morphologically similar to Nostocoida limicola from which, however, Microthrix parvicella differs clearly in the reactions to the Neisser stain. Another filamentous microorganism, Type 0581, also appears similar to Microthrix parvicella, but again it differs in staining reactions. In addition, Type 0581 is not reported very frequently in activated sludges. Another less - frequently observed filamentous microorganism, which can be confused with Microthrix parvicella, seems to be Type 0803. A quite famous story in the literature on Microthrix parvicella is a description of a filamentous form of Escherichia coli by a researcher. The filamentous form of E. coli was in later Dutch papers redescribed as Microthrix parvicella. However, the filamentous microorganism reported by him exhibited some properties different from those of Microthrix parvicella described below.

The manuals and papers on Microthrix parvicella describe this filamentous microorganism as unsheathed. As mentioned above, the presence or absence of a sheath is one of the most controversial identification features of filamentous microorganisms. For Microthrix parvicella some authors observed in phase contrast and at magnification higher than 5OO x a kind of membrane enclosing clear areas in the trichome. These clear areas were fragile and represented potential breaking points of the trichomes.

Until recently, growth in the form of long coiled filaments, positive reaction to the Gram stain and the presence of Neisser positive granules were considered key identification features of Microthrix parvicella. However, research at the University of Birmingham in cooperation with Wessex Water showed that the morphology and the reaction to the Gram stain of Microthrix parvicella can be variable. Depending on the rate of substrate supply, Microthrix parvicella can grow in long coiled filaments (substrate deficiency) or as individual cells or short filaments when substrate is present in excess (organic loading about 2 g / g . day; COD, biomass as MLSS). The short form of Microthrix parvicella was Gram negative, quite contrary to the typical reactions described in the manuals. The authors explain the Gram stain variability by changing the cell surface properties under substrate deficiency and substrate abundance (accumulation of lipids and PHB) conditions. Fortunately, one identification feature remains the same for both growth forms, namely the presence of Neisser positive granules.

From the practical point of view, the other finding of the Birmingham Group is even more interesting - the morphological change from long coiled filaments to the short form of Microthrix parvicella was accompanied by a decrease in hydrophobicity. As it is the hydrophobicity of cell surfaces that supports the stability of biological foams, it can be concluded that the short form of Microthrix parvicella is less prone to foaming problems than the conventional long form. On the other hand, the presence of the nonfilamentous or short form of Microthrix parvicella can easily be overlooked in activated sludge samples when the examiner is not aware of these unusual growth forms. This may represent a particular threat for an activated sludge plant, as the regrowth of the conventional filamentous form can occur rapidly. The results of the Birmingham experiments can explain many cases of a sudden outburst of Microthrix parvicella bulking or foaming problems.

The physiology of Microthrix parvicella seems to be quite strange. The substrate and nutrient requirements were studied on Microthrix parvicella isolates from activated sludges by two researchers in the Netherlands in the 1980s, and their conclusions are repeated in the introductory parts of most papers on Microthrix parvicella. The following main features can be concluded from the Dutch studies in axenic cultures:
  • Microthrix parvicella cannot use common readily biodegradable substrates such as glucose, fructose, succinic, citric and other simple organic acids and protein hydrolysates.
  • The preferable substrates are long chain acids in esterified forms. Unsaturated oleic acid can be utilized as oleate directly up to 150 mg / L.
  • Short - chain fatty acids and glycerol are used only together with a soluble ester of oleic acid.
  • Microthrix parvicella is able to store oleic acid in esterified form inside the cells. The stored material can subsequently be metabolized for energy generation or protein production, which would represent a strong selective advantage for this microorganism in competition with other filaments or flocformers. The content of lipoid components may reach up to 35 % of the dry weight of Microthrix parvicella.
  • For synthesis purposes, Microthrix parvicella requires reduced forms of nitrogen and sulphur, i.e., assimilative reduction of nitrate or sulphate by Microthrix parvicella is not possible.
  • Microthrix parvicella is more psychrophilic than mesophilic, as its growth occurs down to 8 deg C but ceases at 35 deg C.
  • The optimum observed pH range is 7.7 - 8.0. No growth occurs at pH below 7.1, which is a pH value quite normal in activated sludges.
  • The growth of Microthrix parvicella is retarded under continuous oxygen supply to the cultivation medium, while an intermittent aeration supports the growth.
  • The maximum growth rate of Microthrix parvicella for oleic acid was 1.4 1 / day, which proves that Microthrix parvicella is a slow - growing microorganism. On the other hand, the growth rate is not so low that Microthrix parvicella can be washed out from the mixed culture of activated sludge, especially at higher retention times (sludge ages).

The substrate and nutrient requirements of Microthrix parvicella found by two researchers are unusual for a filamentous microorganism, and do not correspond very well with the frequent occurrence of this filament in activated sludge systems worldwide. Esters of long chain fatty acids are not a common component of municipal wastewaters. In well - controlled laboratory experiments, Microthrix parvicella proliferated in a system fed with synthetic wastewater based on glucose and ethanol.

The inability of Microthrix parvicella to use other than reduced forms of nitrogen and sulphur corresponds to the fact that Microthrix parvicella did not proliferate in a well - aerated environment in an author's experiments. The research on the low F : M filament bulking in South Africa came to a similar conclusion - that the exposition of activated sludge to purely oxic conditions (continuous aeration, DO above 2 - 3 mg / L) reduced the presence of Microthrix parvicella and other low F : M filaments. Also, two researchers found in an activated sludge system with alternately aerated and nonaerated compartmentalized contact zones prior to the main part that the growth of Microthrix parvicella was suppressed only when return activated sludge was mixed with coming wastewater under oxic conditions.

The observation made by an author that Microthrix parvicella grows better at lower temperatures is in good agreement with practical experience. In many foaming activated sludge plants, the foams are dominated by nocardioforms during the summer but Microthrix parvicella in the winter. In Czech Republic, a researcher observed filamentous bulkig by Microthrix parvicella in an activated sludge system with compartmentalized predenitrification zone only in cold periods when the temperature of mixed liquor dropped below 12 - 15 deg C for a long period of time. In the wastewater treatment plant Johannesburg Northern Works Microthrix parvicella is the dominant filamentous microorganism during winters, while during summers Type 0092 dominates the biocenosis. The relationship between Microthrix parvicella proliferation and lower temperatures needs more data to be explained correctly. An author suggests that the growth of Microthrix parvicella at lower temperatures is favored due to reduced solubility of fat. This material is then concentrated at activated sludge surfaces and becomes more available to Microthrix parvicella.

The high occurrcnce of Microthrix parvicella in nutrient removal systems in which the most substantial part of (readily) biodegradable substrates is removed under anoxic and / or anaerobic conditions indicates that this microorganism should be equipped with the enzymatic apparatus for anoxic and anaerobic substrate uptake. Most filamentous microorganisms cannot use nitrate nitrogen as a terminal electron acceptor, or can perform only the first step of denitrification, i.e., the reduction of nitrate to nitrite. Some researchers used this postulate as the basis for their hypothesis on the cause of bulking in nutrient removal activated sludge systems. However, for Microthrix parvicella this presupposition still waits for experimental verification in pure cultures. On the contrary, the data from mixed cultures of activated sludges do not confirm that the increased presence of Microthrix parvicella results in an elevated concentration of nitrite as a product of anoxic respiration. In one of some researchers' experiments with activated sludge systems with a predenitrification zone, Microthrix parvicella became the dominant filamentous microorganism in spite of the fact that all available substrate (glucose and ethanol) was removed under anoxic conditions. In fact, after 40 days of operation, the microscopic picture of the activated sludge resembled a technical culture of Microthrix parvicella contaminated by some residual flocs. The rates of denitrification, rXD, and substrate uptake (as COD), rXCOD, measured in batch tests were unexpectedly high for a bulking sludge as can be seen from table given below.

Day of experiment Substrate rXD (mg / g . h) rXCOD (mg / g . h)
57 Ethanol 11.5 64.0
57 Glucose 9.8 186.0
87 Ethanol 16.2 80.7
87 Glucose 11.3 191.0

No significant nitrite production was observed during the batch tests. Another interesting result from these batch tests is the rate of COD removal for glucose. The rate for glucose is more than double the rate for ethanol. The amount of glucose removed during the denitrification tests is also much higher than that which would correspond to the stoichiometry of denitrification. This means that glucose was accumulated in the cells without its simultaneous oxidation under anoxic conditions. The phenomenon of accumulation of saccharidic substrates is well - known for flocforming microbes. Because of the high abundance of Microthrix parvicella in the tested activated sludge, one can expect that Microthrix parvicella participated significantly in the observed accumulation of glucose under anoxic conditions.

Microthrix parvicella is able to synthesize intracellular storage products like PHB and polyphosphate. This ability is the first condition for Microthrix parvicella to utilize substrate and obtain energy under alternating anaerobic and oxic cultivation conditions. There is some question as to whether or not Microthrix parvicella can do it at a rate comparable to that of polyphosphate - accumulating bacteria like Acinetobacter spp. The probable answer is yes. If the answer is not yes, it would be very difficult to explain so frequent and so significant an occurrence of Microthrix parvicella in activated sludge systems with anaerobic zones.

For instance, two researchers described a case of Microthrix parvicella bulking at the Renkum wastewater treatment plant in the Netherlands, which had been retrofitted for enhanced biological phosphorus removal. After the conversion of one of three parallel conventional lanes to an anaerobic / oxic system, Microthrix parvicella developed in this lane and increased the SVI - values to more than 200 mL / g. The presence of Microthrix parvicella in neighboring conventional, fully aerobic lanes was not recorded. The presence of Microthrix parvicella in anaerobic / oxic activated sludge systems may lead to a complete failure of the enhanced biological phosphorus mechanism. Microthrix parvicella sequestered readily biodegradable substrates under anaerobic conditions, thus driving Acinetobacter from the system. The compartmentalization of the anaerobic / oxic system (five compartments anaerobic, five compartments oxic) did not prevent Microthrix parvicella from excessive growth.

The persistence of problems caused by Microthrix parvicella can be illustrated by the example of the Johannesburg Northern Works wastewater treatment plant. The head end of a corridor of a five - zone PHOREDOX activated sludge system was divided into five compartments. The first compartment was mostly anoxic (nitrate in the return activated s1udge), he remaining four compartments were anaerobic. The conversion of the Johannesburg Northern Works PHOREDOX module is schematically depicted in figure shown below. Although the readily biodegradable substrate was removed in this compartmentalized anoxic / anaerobic part of the system, Microthrix parvicella was not eliminated from the biocenosis.

"Definition Sketch for Operational Modes"...

The indications from wastewater treatment practice stress the necessity of further research into Microthrix parvicella. The pure culture studies should confirm the presumptions on the metabolic diversity of Microthrix parvicella. For practical purposes, the rates of substrate utilization under anoxic and anaerobic conditions by the organism have to be determined.

The description of Microthrix parvicella can be finished with a conclusion that the literature on the physiology and metabolic abilities of this filamentous microorganism contain many contradictions. This inconsistency prompted some researchers to ask whether the Microthrix parvicella filaments observed in Australia, France, South Africa and other countries were physiologically the same organisms as the original isolates described by Slijkhuis in the Netherlands, especially when they had little success in growing Microthrix parvicella on Slijkhuis's medium.