Biological phosphorus removal BY Pure CULTURE of lampropedia spp.

Stante L., Cellamare C. M., Malaspina F., Bortone G. and Tilche A.

ENEA - Sezione Depurazione e Ciclo dell’Acqua - Via Martiri di Monte Sole, 4

I-40129 Bologna - Italy


Lampropedia spp. is a gram-negative, Neisser-positive coccus that was isolated from EBPR (enhanced biological phosphate removal) activated sludge laboratory plants operating on dairy and piggery wastewaters. In aerobic growth tests carried out on sodium acetate, Lampropedia spp. stored PHB up to 12% w/w. Biomass yield was estimated 0.55 gVSS·gHAc-1, and specific growth rate 0.045 h-1. The experimental maximum acetic acid removal rate resulted 71.86 mgHAc·gVSS-1·h-1 with a semisaturation constant of 71.78 mg·L-1. Batch tests were carried out to check whether Lampropedia spp. was capable of enhanced biological phosphorus removal. Under anaerobic conditions, Lampropedia spp. sequestered acetate and stored PHB with an average conversion factor of 0.33 mgPHB·mgHAc-1. The measured maximum PHB storage capacity was 31% w/w, with a maximum specific PHB accumulation rate of 17 mgPHB·gVSS-1·h-1 and a specific anaerobic acetate uptake rate of 57 mgHAc·gVSS-1·h-1. The experimental ratio between phosphorus released and acetate uptaken was low, in average 0.044 mgPO4-P·mgHAc-1, with a specific rate ranging from 1.7 to 3.6 mgPO4-P·gVSS-1·h-1 at pH 7.5. Despite of the low figure, fractionation analyses showed that in anaerobic conditions the released phosphate comes from cell polyphosphate degradation. Therefore, the whole results allow to conclude that Lampropedia spp. can be classified amongst the phosphorus accumulating bacteria.


Lampropedia spp., poly-ß-hydroxibutyrate, enhanced biological phosphorus removal, nutrient removal, polyphosphate accumulating microorganisms.


ADM     =   Chemical Defined Medium

COD      =   Chemical Oxigen Demand

EBPR     =   Enhanced Biological Phosphorus Removal

FID        =   Flame Ionization Detector

g             =   Gravity acceleration [L·T-2]

GC         =   Gas Chromatography

GTA       =   Green Top Agar

HAc       =   Acetic Acid

HPIC     =   High Performance Ion Chromatography

mod.       =   Modified

PHA       =   Polyhydroxialcanoate

PHB       =   Poly-ß-hydroxibutyrate

PHV       =   Polyhydroxivalerate

poly-P    =   Polyphosphates

rpm        =   Rounds per minute

rs            =   Specific Substrate Removal Rate [T-1]

rsac          =   Specific Acetic Acid Removal Rate [T-1]

TSS        =   Total Suspended Solids

VSS       =   Volatile Suspended Solids

µ             =   Specific Growth Rate [T-1]

µobs         =   Specific Observed Growth Rate [T-1]


In the last years, many studies on pure cultures of activated sludge polyphosphate accumulating microorganisms (PAO) have been carried out.

The common characteristics of this heterogeneous group of bacteria are their capability of synthesizing polyphosphates (poly-P) and polyhydroxyalkanoates as internal storage compounds; polyphosphates are formed during the aerobic phase, while PHA are stored during the anaerobic phase (Comeau et al, 1986; Wentzel et al., 1986; Mino et al., 1987; Mino et al., 1994).

Many different kinds of poly-P accumulating bacteria have been isolated from activated sludge. Brodisch and Joyner (1983) found that in several EBPR plants phosphorus removal was mainly carried out by bacteria belonging to the genera Aeromonas and Pseudomonas, representing up to 50% of the microbial population. The presence of those two genera has been confirmed in other studies by Florentz and Hartemann (1984) and Lötter and Murphy (1985).

Activated sludge bacteria, belonging to the genus Acinetobacter, have been isolated for the first time by Fuhs and Chen (1975), who demonstrated in batch tests that Acinetobacter was able to take up phosphate and to degrade poly-ß-hydroxibutyrate (PHB) under aerobic conditions, releasing soluble ortophosphate in anaerobic conditions. On the other hand, they did not give any figure on acetate uptake in anaerobic conditions.

Other tests have been carried out by Deinema et al. (1980) and Deinema et. al. (1985); in their experience, Acinetobacter cultures released soluble ortophosphate without synthesizing PHB under anaerobic conditions, using acetate, lactate and ethanol as carbon source.

Tandoi et al. (1987) revealed incomplete COD uptake and phosphorus release with pure cultures of Acinetobacter lwoffi, maintained in anaerobic/aerobic alternate conditions.

Heymann et al. (1989) emphasised the distinction between aerobic or microaerophilic growth. In the last condition, Acinetobacter lwoffi was rich either of PHB and poly-P, while the aerobic cultures had a lower poly-P content.

Bayly et al. (1990) observed phosphorus release without acetate uptake in an Acinetobacter culture that, in spite of this behaviour, showed enhanced poly-P storage capacity.

Some tests with Acinetobacter calcoaceticus failed. A significant anaerobic acetate uptake was observed without any correlation to phosphate release (Ohtake et al, 1985).

However, it is now clear that in full scale EBPR plants, many different bacterial groups are responsible for enhanced biological phosphate removal (Wagner et al., 1994; Bond et al., 1995).

Nakamura et al. (1991) worked in microaerophilic conditions with pure cultures of a bacteria thought to belong to the genus Micrococcus and then assigned to a new genus and species Microlunatus phosphorovorus (Nakamura et al., 1995). These bacteria were able phosphate uptake in oxidative conditions without organic substrate in the medium, and released phosphate when fed with glucose in anaerobic condition.

More recently, Ubukata and Takii (1994) hypotised that the enzymatic system for the assumption of phosphate could be inducted by at least two anaerobic/aerobic cycles. In that particular case, phosphorus release has been observed with a concomitant uptake of casamino acid after induction of the cell culture.

The present study regards a gram-negative coccus, Neisser-positive in aerobic conditions and Neisser negative in anaerobic conditions, that was observed from EBPR activated sludge laboratory plants operating on dairy and piggery wastewaters; it was supposed to be able of enhanced biological phosphate removal. The microorganism was morphologically identified as belonging to the genus Lampropedia.

The striking quality of these bacteria is undoubtedly the large cell sheets they use to build both in solid and liquid medium, a property which gives to Lampropedia the rare prerogative of being immediately recognized under the microscope (Murray, 1984).

In this paper, isolation procedures, growth culture conditions and alternate anaerobic/oxic batch tests for evaluating the enhanced biological phosphorus removal capacity of Lampropedia spp. are described.

Materials and methods


Bacteria were isolated from the activated sludge of a sequencing batch reactor (SBR) treating dairy and piggery wastewater as described in the isolation procedure.

The single cells have a rounded, almost cubical shape, and they are arranged in square tablets of 16-64 or more individual cells. They divide synchronously in a sheet and alternately in two planes.

Staining and microscopic observation

Gram and Sudan Black B staining were carried out as reported by Jenkins et. al. (1986); methachromatic granules were stained both with Pergola method (Pasquinelli, 1981), Albert method (Tandoi  et al., 1990) and Neisser method (Jenkins et al., 1986); Blue Nile A staining was done as reported by Ostle and Hot (1982).

Light microscopic observations were carried out using a Jenalumar A/D Contrast light microscope (1000x magnification) in bright field, phase contrast, Nomarski interferential contrast and bright field coupled to fluorescence episcopy; scanning electron microscope (SEM) observations were carried out using a Philips XL 20.


Two kinds of media were used for the tests: a modified Green Top Agar (mod.GTA) and a chemical defined medium (ADM). All media were sterilised by autoclaving at 121°C for 15 minutes.

Modified Green Top Agar (based on Green Top Agar by Pringsheim (1980) was made with: 2 g of yeast extract, 1 g of peptone, 1 g of sodium acetate, 100 mL of filtered and sterilized effluent from dairy wastewater treatment plant, 15 g of agar (only 2 g for semisolid medium), distilled water to 1 L volume and pH adjusted to 7.2. This medium was used as solid substrate during isolation and maintenance on Petri plates (100 mm) and tubes (16x200 mm).

The modified Green Top Agar without agar was used for growth, PHB storage and phosphorus uptake tests.

ADM was composed by 160 mg of NH4Cl, 64 mg of KH2PO4, 2 ml of a microelement stock solution (Puttlitz and Seeley, 1968), and distilled water to 1 L volume. After sterilisation, 1 mg of biotine, 1 µg of tiamine and a variable amount of sodium acetate (from 500 to 10000 mg, depending on test conditions) were added after filtration on 0.22 µm sterile filters. This medium was also used for growth tests. Sodium acetate in data tests was expressed as acetic acid.

The ADM medium without KH2PO4 was used in phosphorus releasing tests.

Instruments and analytical methods

Analyses of TSS and VSS were performed on the residue retained by GF filter following Standard Methods (1989). The different forms of phosphorus were measured according to the same manual. Phosphorus fractionation was carried out as reported by De Haas (1989). Nitrate, ortophosphate and ammonia were determined using a HPIC (Dionex 4000i). Acetic acid was determined gaschromatographically, using a DANI 8510 GC equipped with a 25 m 0.53 mm 1.2mm capillary wide bore column (Alltech SO FA bound FSOT) and a FID detector; hydrogen was used as carrier gas and 2,2-dimethylbutyric acid as internal standard. GC working conditions were the following: oven temperature from 107°C to 140°C with an increasing rate of 6 °C·min-1, injector temperature of 220°C and FID port temperature of 230°C.

PHB was determined by GC with a modified method as proposed by Braunegg et al. (1978) and Gerhardt et al. (1994). 10 mL of culture media were centrifuged at 4000 g x 15 min at 4°C. After centrifugation, the supernatant was discharged and replaced with an equal volume of a solution of sodium hypochlorite; after incubation at 37°C for 1 hour, the sample was centrifuged at 9000 g x 15 min at 4°C. The solid fraction was washed with water and centrifuged at 9000 g x 15 min at 4°C. The solid fraction was then mixed with 2 ml of acidified methanol (3% H2SO4) and 1 ml of chloroform and then heated for 3.5 h at 100°C.

After the digestion/methylation, 1 ml of water and 25 µL of internal standard (4000 mg·L-1 2,2-dimethylbutyric acid solution) were added to the sample; after 10 min. of vigorous shaking, phase separation follows. 0.1 µL of the chloroform fraction is injected in the GC, equipped with the same column and detector described above; hydrogen was used as carrier gas. GC working condition were: oven temperature from 60°C to 120°C with an increasing rate of 4.7 °C·min-1, injector temperature of 220°C and FID port temperature of 230°C. Pure PHB (Sigma) was used for GC calibration.

Test methods

The growth tests were carried out under aerobic conditions in a 2 L well mixed glass bottle (magnetic stirrer at 60 rpm) with 0.5 L of media (modified GTA or ADM) covered with a cotton lid. The inoculum was taken from a stock plate colony. Temperature was kept at 25°C and the initial pH was 7.5.

Cells for phosphorus release tests were cultured batchwise under aerobic condition in modified GTA medium.

Phosphorus release and PHB storage tests were carried out on centrifuged cells (4000 g x 15 min.) washed with phosphorus free media. The washed cells were resuspended in 0.3-0.5 L of ADM without phosphate in a 2 L glass bottle. Anaerobic conditions were obtained by bubbling sterilised (by filtration on 0.22 µm sterile filter) nitrogen gas.

Temperature was kept at 20°C and, during the tests, pH reached 8.3-8.5.

Results and discussion

Isolation and identification

After Neisser staining, these bacteria showed the presence of metachromatic granules; besides, they were Sudan Black positive (Murray, 1963) and the isolate was also positive to Albert staining (Tandoi et al, 1990) that, together with Neisser staining, is recommended for evidencing the presence of volutine granules (Jenkins et al, 1986). On the basis of these characteristics, it was supposed that Lampropedia might belong to the functional group of poly-P accumulating microorganisms.

Activated sludge was sampled from two SBR reactors operating on dairy and piggery wasterwater and observed by a phase contrast light microscope. Some particular shaped bacteria belonging to the genus Lampropedia were observed. Other authors before (Standridge, 1981) have identified Lampropedia spp. in an activated sludge-trickling filter plant treating cheese factory wastes.

Lampropedia. hyalina  was isolated for the first time by Schroeter (1886) and more in detail by Pringsheim (1955). Lampropedia cells are enclosed in a Gram-negative type of cell wall; no flagella occur, they are strictly aerobic, chemoorganotrophic and usually include PHB granules, as described in Bergey’s manual. Lampropedia was first observed in muddy water and probably its name has been originated because they appear like tablets “glistening”. They have been isolated in organic matter rich environment, although a definite ecological niche is not clearly definable.

Growth occurs as a thin, hydrophobic and extended cell pellicle spreading on the solid surface or liquid media (Murray, 1963). Each cell is usually linked to the near cells by a remarkable proteic surface structure or S layer (Murray, 1963 ; Austin and Murray, 1990). Murray (1984) and Khun and Starr (1965) reported the presence of refractile PHB inclusions when cells grow in presence of sodium acetate.

Several isolation techniques have been tested; among them, the most reliable was the direct spreading of sludge samples onto agar plates. Sequential steps onto agar plates allowed culture isolation.

The solid medium was modified GTA, kept at 25 °C in aerobic conditions. Once Lampropedia colonies were isolated from the rest of the sludge population, several cultural enrichments on a liquid medium containing 2 g·L-1 agar were tested. In this liquid medium, Lampropedia cells are mainly growing on the liquid surface.

The pure culture of Lampropedia were almost daily renewed in new agar plates or in test tubes; the latter was more stable than plates, since this kind of environment is slightly damp, as Lampropedia requires (Murray, 1963). The properties of Lampropedia hyalina as originaly described by Puttlitz and Seeley (1968) correspond to the used strain.

Characterisation tests, a group of those enumerated in Bergey’s manual (Tab.1), were carried out in order to confirm the visual identification of Lampropedia;. According to test results, the used strain corresponds to Lampropedia. The species attribution, however, is not clearly defined.


Tab. 1. Identification tests for Lampropedia spp.


reaction or result obtained

reaction or result for Lampropedia spp. (Bergey’s)

Gram staining

Neisser staining


not reported

Growth under anaerobic conditions

Intracellular PHB formed



Catalase test



Nitrate reduction


Shape characteristic

cocci in sheet

cocci in sheet

Cell arranged in square tablets of 16-64 cells



Final pH culture media




To check the presence of PHB granules in the cells (Kuhn and Starr, 1965), Sudan Black B  and the more specific Blue Nile A stains were used for microscopic observations. PHB was also measured by GC analysis.

Fig. 1. Lampropedia spp. in pure culture (Sudan Black stain, white bar is 10 µm)

Fig. 2. Lampropedia spp. (scanning electron microscope, bar corresponds to 2 µm)



Growth tests have been carried out in aerobic conditions on enriched (mod. GTA) and minimal media (ADM), to calculate stoichiometric and kinetic parameters.

Trends of substrate (sodium acetate) and biomass (calculated from VSS, not including the weight of PHB) during 9 growth tests showed a decrease of substrate concentration with correspondent biomass growth. In some tests, a slight biomass growth after substrate consumption, probably due to degradation of stored PHB, was observed. The same trend was observed with different initial substrate concentration; however, growth rates varied depending on substrate/biomass ratios.

PHB was stored also during aerobic cell growth, particularly with high substrate/biomass ratios (Fig. 3). This phenomenon has been already described for other microorganisms in unbalanced growth conditions (Dawes & Senior 1973).

PHB ranged from 3% to 12% w/w, with initial acetic acid concentrations ranging from 450 to 3500 mg·L-1 and initial acetic acid/VSS ratios of 3.5 and of 8.8 gHAc·gVSS-1 respectively. PHB/Acetate conversion factors were 6.5% and 16%  at the acetic acid concentrations reported above.

Fig. 4 shows the VSS increase as a function of the removed substrate. The curve slope represents the average biomass yield (0.55 gVSS·gHAc-1). The observed COD:Nitrogen ratio consumed for growth was in average 100:2.

Fig. 3. Trend of PHB/VSS and acetic acid during a growth test in aerobic conditions.

Fig. 4. VSS trend as a function of acetic acid depletion (curve slope represents the average yield).





Fig. 5. Acetic acid specific removal rate during growth as a function of acetic acid concentration (averaged values of three parallel tests).


On enriched medium, the specific growth rate (µobs) was quite stable in the substrate concentration range from 100 to 550 mg·L-1 of acetic acid, with an average value of 0.045 h-1. Similar results were obtained on minimal medium.

The specific substrate removal rate (rsac) followed a Michaelis-Menten curve, as reported in Fig. 5. From data interpolation, the estimated maximum rate was 71.9 mgHAc·gVSS-1·h-1 and the semisaturation constant 71.8 mgHAc·L-1.

Tests were carried out to evaluate the capability of Lampropedia spp to grow with nitrate as the final electron acceptor instead of oxygen: no growth was detected.

Substrate uptake and PHB storage under anaerobic conditions

To evaluate stoichiometric and kinetic parameters in anaerobic conditions, tests without any inorganic electron acceptor and with sodium acetate as the sole substrate were carried out.

From Fig. 6 it can be seen that acetate uptake is strictly related to PHB storage. The average PHB yield, calculated as the slope of the curve in Fig. 7, was about 0.33 mgPHB·mgHAc-1; this value is lower than the one reported by Comeau (1986) and by Smolders et al. (1994) for mixed cultures, but it is quite similar to those reported by Fukase et al. (1982) and by Arun et al. (1988) in pure culture. This discrepancy can partially depend on uncomplete extraction of PHB and on the fact that the analytical method allowed only the estimation of PHB and not of other PHA. The chromatograms showed qualitatively the presence of other methyl-volatile fatty acids.

PHB was stored into the cells in considerable amounts, about 31% of VSS dry weight.

The specific PHB accumulation rate was strongly dependent on initial acetic acid concentration, with higher values obtained at higher substrate concentration. The maximum rate obtained was 17 mgPHB·gVSS-1·h-1. In accordance to this, specific acetic acid uptake rate (31-57 mgHAc·gVSS-1·h-1) grew as a function of initial acetic acid concentration.

Fig. 6. Trends of acetic acid and PHB during a PHB storage test.

Fig. 7. PHB vs acetic acid concentration (the slope represents the average yield).


Phosphorus release and uptake

Tests on phosphorus release under anaerobic conditions were carried out using acetic acid as substrate. The rate of phosphorus release and acetic acid uptake was always higher at the beginning of the test (Fig. 8). Fig. 9 shows the relationship between released orthophosphate and acetic acid uptaken in one representative test; the slope represents the average stoichiometric ratio obtained. The ratios between released orthophosphate and acetic acid removed ranged in the various tests from 0.02 to 0.11 mgPrel·mgHAc-1rem, with biomass concentration ranging from 0.2 to 0.64 gVSS·L-1. The average value was 0.044 mgPrel·mgHAcrem-1. These low values are comparable to those reported for Acinetobacter calcoaceticus by Ohtake et al. (1985) and for an isolated Gram + coccus by Ubukata and Takii (1994), but lower than data obtained from poly-P activated sludge mixed cultures. In average, the amount of P-PO4 released per gram of biomass during the anaerobic phase was around 4 mg, in accordance to values of 2.5-4 mg reported for Acinetobacter calcoaceticus by Ohtake et al. (1985).

Biomass phosphorus fractionation puts in evidence the presence of a significant amount of polyphosphates. The cell culture showed a relevant difference in polyphosphate content (5 mgP·gVSS-1) between the end of the anaerobic phase and the end of the aerobic one (Tab. 2). It is possible to observe that poly-P content drastically increases during the anaerobic/aerobic tests. These figures confirm the previous values on phosphorus release reported above, and are also similar to the values reported by Ohtake et al. (1985).


Tab. 2. Phosphorus fractionation in Lampropedia cells exposed to alternate aerobic/anaerobic conditions, compared to cells before alternate exposure.


polyphosphate phosphorus (mgP·gVSS-1)

other cellular phosphorus (mgP·gVSS-1)

end of anaerobic



end of aerobic






not exposed cells




In anaerobic conditions, the specific PO4-P release rate ranged from 1.7 to 3.6 mgP-PO4·gVSS-1·h-1, with a pH near neutrality (pH = 7.5). After about 5 hours, with only slight differences among different tests, the rate slows down to values below 0.1 mgPO4-P·gVSS-1·h-1. During all tests, the specific rate of PO4-P release did not show statistically significant correlation with acetic acid and phosphorus concentrations in the media, even if the highest values were obtained with the highest substrate concentration and the lowest PO4-P concentration at the beginning of each test.

On the contrary, the specific acetic acid removal rate showed a significant correlation to substrate concentration. The highest value obtained was 70 mgHAc·gVSS-1·h-1.

Phosphorus uptake under aerobic condition showed a specific rate from 0.25 to 0.54 mgPO4-P·gVSS-1·h-1. This leads to an accumulation of phosphorus in the biomass up to 3.5 % of VSS.

Fig. 8. Trend of acetic acid and phosphorus during one of the P-release tests.

Fig. 9. Phosphorus release vs acetic acid uptake during one of the P-release tests.


Enhanced biological phosphate removal is extensively reported in literature on mixed culture, but very few works have been successfully performed in pure culture, and most of them do not report complete data on the whole uptake/release mechanism of acetate and phosphorus.

In the present work, a bacterium belonging to the genus Lampropedia isolated from EBPR activated sludge has been studied for its capacity of performing enhanced biological phosphate removal. Acetate uptake, PHB formation and phosphate release in anaerobic conditions as well as phosphate uptake and PHB consumption in aerobic conditions have been recorded. Stoichiometric and kinetic values were usually lower than those obtainable in mixed culture, but cell phosphorus fractionation analyses demonstrated a cyclic increase and decrease of the poly-P fraction from aerobic to anaerobic conditions. All these evidences allow to conclude that Lampropedia spp. can be classified as a poly-P accumulating microorganism.

The easiness of its visual recognition and its isolation in pure culture make Lampropedia spp. a candidate bacterium for future pure culture experiments. Its study might contribute to a better comprehension of the biochemical mechanism involved in enhanced biological phosphate removal process.


Authors wish to thank B. Biavati for their precious suggestions, A. Tarozzi, S. Gemelli and the personnel and the director of Montecatini Environmental Research Center for their collaboration to this research.


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