Bioprocess Biosyst Eng (2015) 38:125–135 DOI 10.1007/s00449-014-1250-1
ORIGINAL PAPER
Toxic influence of silver and uranium salts on activated sludge of wastewater treatment plants and synthetic activated sludge associates modeled on its pure cultures Dmitry V. Tyupa • Sergei V. Kalenov • Dmitry A. Skladnev • Nikolay S. Khokhlachev • Marina M. Baurina • Alexander Ye. Kuznetsov
Received: 19 October 2013 / Accepted: 24 June 2014 / Published online: 16 July 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Toxic impact of silver and uranium salts on activated sludge of wastewater treatment facilities has been studied. Some dominating cultures (an active nitrogen fixer Agrobacterium tumifaciens (A.t) and micromyces such as Fusarium nivale, Fusarium oxysporum, and Penicillium glabrum) have been isolated and identified as a result of selection of the activated sludge microorganisms being steadiest under stressful conditions. For these cultures, the lethal doses of silver amounted 1, 600, 50, and 300 lg/l and the lethal doses of uranium were 120, 1,500, 1,000, and 1,000 mg/l, respectively. A.tumifaciens is shown to be more sensitive to heavy metals than micromyces. Synthetic granular activated sludge was formed on the basis of three cultures of the isolated micromyces steadiest against stress. Its granules were much more resistant to silver than the whole native activated sludge was. The concentration of silver causing 50 % inhibition of synthetic granular activated sludge growth reached 160–170 lg/l as far as for the native activated sludge it came only to 100–110 lg/l. Keywords Activated sludge Biological wastewater treatment Heavy metal bioremediation Silver toxicity Uranium toxicity Metal resistant microorganisms Synthetic sludge
D. V. Tyupa S. V. Kalenov (&) D. A. Skladnev N. S. Khokhlachev M. M. Baurina A. Ye. Kuznetsov The Department of Biotechnology, Faculty of Biotechnology and Industrial Ecology, D.I. Mendeleyev University of Chemical Technology of Russia, 9 Miusskaya Square, Moscow 125047, Russia e-mail: [email protected]; [email protected]
Abbreviations A.t Agrobacterium tumifaciens BI Bacterial isolate CFU Colony-forming units DW Oven-dry weight F.n Fusarium nivale F.o Fusarium oxysporum P.g Penicillium glabrum
Introduction The presence of salts of heavy metals such as copper, cadmium, cobalt, nickel, lead, chromium, etc. in waste waters of industrial enterprises is an essential issue at wastewater treatment because of their toxic impacts on the cultures of microorganisms which are a part of activated sludge of wastewater treatment facilities. Such powerful effects can cause death of microorganisms of those cultures which are most sensitive to metals that lead to decrease in biological activity of the whole activated sludge community of organisms and as a result, deterioration of wastewater treatment [1–7]. Not only are the processes of an exchange of nitrogen broken in the activated sludge community, but also the ability of the activated sludge to natural formation of granules decreases as well [8]. In this regard, special attention should be focused on a harmful influence of silver salts on water and soil biocenoses. Pollution of waste waters with large doses of silver salts leads to death of bacteria necessary for natural ecosystems, and also causes poisoning algae, fungi, crustaceans, fishes, and even mammals [9]. The experiments on small fish of the Danio genus showed that silver nanoparticles cause
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embryogenesis pathology. Besides, the metal accumulates in fish and men consuming the fish. It should be noted that colloidal particles of silver are capable of causing diseases of various human tissues and organs [10]. Silver inhibits the activity of nitrifying bacteria most strongly in water biocenoses that causes serious problems at wastewater treatment. There are some data on activated sludge communities of complex structure, which are capable to withstand up to 5 mg/l of Ag? due to the mechanisms of its collective resistance. But, as a rule, the majority of activated sludge cultures die already at 1 mg/l of silver ions [7, 11]. In most cases, treatment facilities cope well with the waste waters containing heavy metals, but the activated sludge so obtained is not extremely recommended to be applied as fertilizer in connection with possibility of contaminating crops with metals, and also because of impacts of metals on soil biocenoses [11]. Silver having stronger bactericidal action in comparison with heavy metals destroys soil ecosystems greatly. Nitrogen-fixing bacteria being a part of the symbiosis with bean plants, and also many cultures of chemolithotrophs necessary for soil formation processes die first. Denitrifying and ammonifying bacteria are also highly sensitive to effect of silver. The death of denitrifiers causes accumulation of excess nitrogen in the soil that leads to eutrophication of reservoirs and pollution of potable water [9]. In the light of data on negative impact of salts of heavy metals on activated sludge and natural biological communities, more detailed researches of such influence, taking specific activated sludge as a case in point, and also discussion of solutions of the problem of treatment of waste waters with a high content of metals are necessary. Some first steps taken on this way are reviewed in this paper. An initial assessment of the effect of salts of silver and uranium on one of the possible variants of activated sludge of wastewater treatment facilities both on biocenosis as a whole and on its isolated components was carried out in the work given. Some pure cultures of microorganisms resistant to stresses and suitable for modeling of synthetic activated sludge functioning stably in the presence of toxic metals were isolated from the native activated sludge.
Identification of these microorganisms and development of techniques of their cultivation and stressing with metal salts were carried out. The thresholds of toxic influence of salts of silver and uranium on the pure cultures isolated and on the activated sludge per se were determined; the steadiest cultures were revealed, and the influence of salts of silver and uranium on the activated sludge being compared. Also, synthetic activated sludge resistant to toxic metals was modeled on basis of the most viable and specially selected cultures.
Materials and methods Cultures of microorganisms The aerobic activated sludge of wastewater treatment plant (Stupino of Moscow Region, Russia) with discharge capacity of 6 9 1012 v3 of water per year was investigated. It was noted that some microorganisms of the activated sludge started dominating in the community, showing more rapid growth and more intensive accumulation of biomass in comparison with other cultures if the sludge was exposed to stressful influences such as hunger, volley dumping, an oxidative stress, and sharp changes of a number of parameters of functioning of treatment facilities. Four cultures steadiest against a stress were selected at long-term cultivation after passages of activated sludge under conditions of fractional introduction of hydrogen peroxide (at its concentration up to 2.4–3.0 g/l) and selected in the form of pure cultures. These cultures were capable to form strong granules that explains in some way their survival under conditions of a stress. Resistance of these cultures to adverse conditions made them promising for formation of the synthetic activated sludge resistant to toxic metals and other types of a stress. These objects were also suitable for researches as they had clearly defined morphological distinctions (Table 1). The cultures were defined later as follows: Agrobacterium tumifaciens—A.t, Fusarium nivale—F.n, Fusarium oxysporum—F.o, and Penicillium glabrum—P.g.
Table 1 Morphological characteristics of the cultures on the agar and liquid media Cultures of microorganisms
Morphological characteristics on the agar medium
Morphological characteristics on the liquid medium
A.t
Colorless roundish colonies with a diameter of 1.0–1.5 mm
White granules of 0.5–1.5 mm in size
F.n
Pale yellowish mycelial colonies with a diameter of 4–20 mm
Yellowish threads, agglomerates and granules of an irregular form of 2–4 mm in size
F.o
White mycelial colonies with a diameter of 4–10 mm
Strong yellowish granules of a round and wrong form of 2–4 mm in size
P.g
White mycelial colonies with a diameter of 4–10 mm, forming green spores
Very stable whitish round granules of 2–4 mm in size
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Phylogenetic identification of the microbial samples The isolates from activated sludge were tentatively identified by the 16S (or eukaryotic 18S) rRNA gene sequences. For this purpose, DNA was extracted from microbial cells using a boiling method. When using this method, the concentration of the DNA in the preparation was 30–50 lg/ ml, and RNA was present at trace quantities (\1 %). For carrying out the PCR and further sequencing of PCR fragments of 16S (or 18S) rRNA genes, a universal primer system was used [12]. The reaction mixture was of the following content: 19 buffer of Taq polymerase (17 mM of (NH4)2SO4, 67 mM of Tris–HCl, pH 8.8, 2 mM of MgCl2); 12.5 nM of each dNTP, 50 ng of microbial DNA; 5 pM corresponding primers and 3 Units of BioTaq polymerase (Dialat LTD, Russia). The PCR was carried out at corresponding temperature and time profiles. The electrophoresis of PCR products was carried out in 2 % agarose gels. All PCR products for sequencing were purified using Wizard PCR Preps DNA Purification System (Promega, Madison, WI, USA) as described by the manufacturer. Sequencing of PCR fragments of 16S and 18S rRNA genes was carried out on Senger’s method [13] with ready reaction kit Big Dye Terminator v.3.1 (Applied Biosystems, Inc., USA) on an automatic sequencer ABI PRIZM 3730 (Applied Biosystems, Inc., USA) according to instructions of the producer. Sequence similarities were identified by a BLAST search [14]. Techniques of cultivation of microorganisms Cultivation on an agar medium The culture of A.t was cultivated on a modified Ashby’s medium, a special medium for nitrogen-fixing bacteria. The composition of each medium is tabulated in Table 2, thus a classical Ashby’s medium differed from the medium given below by content of 20 g/l of sucrose and addition of solution of microelements in amount of 1 ml/l. The other cultures were incubated on a modified Gause’s No 1 medium (Table 2), and a classical Gause’s No 1 medium differed from the medium given below only by the content of starch in amount of 20 g/l. All nutrient media were autoclaved at 121 °C for 30 min. Before inoculating the cultures of F.n, F.o and P.g, their large granules from liquid media had been disintegrated sterilely in a mortar in order the biomass to be distributed uniformly in the volume of an inoculating suspension and then the suspension had been spread on agar plates. The cultivations were carried out at 30 °C during 3 days.
127 Table 2 Composition of modified agar media Components
Modified Ashby’s medium, (g/l)
Modified Gause’s No 1 medium, (g/l)
Sucrose
3.0
–
Starch
–
3.0
CaCO3
5.0
–
KNO3
–
1.0
K2HPO4
0.2
0.5
MgSO47H2O
0.2
0.5
NaCl K2SO4
0.2 0.1
0.5 –
FeSO47H2O
–
10-4
Agar
20
20
Cultivation in a liquid nutrient medium Liquid nutrient media of the same compositions were used for liquid-phase cultivation. At cultivation in flasks, a volume of the medium consisted of one-third of a total flask volume. Inoculants were added at the rate of 10 % of a medium volume. Large granules had been disintegrated sterilely in a mortar before inoculation. Culturing was carried out on a shake-flask propagator (at 150 rpm) at diffuse background lighting and at 27 °C during 3 days. Assessment of culture growth intensity A culture growth rate, and also quantity of an accumulated biomass of microorganisms were controlled by measuring the dry cell weight (DW). For this purpose, 10 ml of cell sample suspension was harvested by centrifugation (9,000 rpm) for 15 min, washed twice with distilled water and dried up to constant weight at 105 °C. In case of the cultures of F.o and P.g where large granules were being formed on the third day of cultivation, 30 ml of cell sample suspension was withdrawn; large granules were disintegrated in a mortar, and only then 10 ml of a test was sampled. Such an approach allowed lowering an error significantly. Determination of the DW for culture of A.t was problematic as a result of the high content of CaCO3 in the medium. Therefore, in addition, the growth rate of the culture of A.t was estimated by counting of cells in the count chamber. Besides, the amount of cells for the cultures studied in the medium was estimated by inoculation of cultural liquids diluted in series on Petri dishes with the subsequent counting of the number of colony-forming units (CFU, ml-1). Each experiment was repeated thrice, and the experimental errors were \5 %.
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Determination of lethal concentration of heavy metals Assessment of toxicity of silver and uranium salts on agar nutrient media Threshold values of toxic effect of silver and uranium salts were determined for cultures of microorganisms by adding of various amounts of water solutions of silver nitrate (AgNO3) or dihydrate of uranyl acetate (UO2(CH3COO)22H2O) onto a liquid agarized nutrient medium; after its hardening, the inoculation was carried out. The cultures’ growth inhibition rate was estimated according to reduction of a diameter of colonies and their quantities as far as the concentration of salts of heavy metals increased. The inoculums (0.1 ml of the suspension) obtained within 3 days of liquid-phase cultivation and diluted to 6 9 10-6 were spread on a sterilized agar plates that allowed not only to count the number of colony-forming units, but also to estimate the size and external characteristics of the colonies. Assessment of toxicity of silver and uranium salts in liquid nutrient media Toxic influence of salts of the metals on microorganisms in the liquid medium was determined during three-day liquidphase cultivation by the technique described in Sect. ‘‘Cultivation in a liquid nutrient medium’’, and various amounts of silver nitrate or uranyl acetate solutions had been added under sterile conditions before inoculation; the control samples were incubated in parallel. The cultures’ growth inhibition rate was estimated on decrease of biomass accumulation by DW. Synthetic activated sludge modeling Synthetic activated sludge was modeled by joint cultivating three cultures of F.n, F.o, and P.g being most resistant to silver, the cultivation being carried out in the modified Gause’s No 1 medium within 3 days. To prepare inoculums, the equal amounts of suspension of these cultures were taken as these species of microorganisms had almost identical curves of growth and accumulated approximately similar quantity of biomass for equal time. Before introduction of inoculums, the granules of these three cultures were sterilely disintegrated in a mortar and the suspensions of the cells were mixed. The synthetic activated sludge obtained showed intensive growth and biomass accumulation comparable to the corresponding characteristics of cultures of F.n, F.o, and P.g and formed the spherical white granules of 3–5 mm in a diameter, which were not similar to those types of granules that were formed by the pure cultures. The activated sludge granules formed were analyzed by a method of light microscopy. For the estimation of
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percentage species composition of the modeled activated sludge, some indicated characteristics of the cultures were used: spindle-shaped and cylindrical macroconidia mostly with three septa, without a foot-shaped basal cell, and absence of chlamydospores—for F.n; spindle-shaped and lunate macroconidia with a distinct foot-shaped basal cell or with a papilla, mainly with four or five septa and abundant chlamydospores—for F.o; and the plentiful unicellular almost round conidia formed on flask-shaped phialides—for P.g. The modeled activated sludge resistance to silver was determined by the same method as the resistance of the pure cultures was done.
Results Research of culture growth dynamics When studying the growth of microorganisms, we intended to achieve two purposes. The first one was to determine the optimum time of cultivation of the isolated activated sludge cultures for the subsequent assessment of influence of toxic metals on their growth. Secondly, it was optimization of the composition of nutrient media to decrease in amount of organic substances added into the media and capable to make a contribution to an essential toxic impact of the metals, forming evens more toxic organometallic compounds. The researches of cultivation in a liquid media showed that all four cultures of microorganisms reached a stationary growth phase synchronously on the fourth day. The Gause’s No 1 and Ashby’s media of standard composition (20 g/l of starch or sucrose, respectively) and their modified variants were used to investigate the dynamics of a culture growth on nutrient media with original and lowered contents of sources of carbon (3 g/l). The curves of growth are shown in Figs. 1, 2 and 3. Thus, it was established that cells of the cultures concerned reached the most active growth phase on the second and third days, and they had managed to accumulate the level of biomass not \90 % from the maximum by the third day. Fig. 2. A sevenfold decrease of carbon source contents did not influence a culture P.g growth in a synthetic medium, whereas accumulation of biomass for cultures of F.n and F.o decreased no more than 2.5 times in an impoverished medium reducing intensity of microbial growth slightly. These data allowed using modified nutrient medium in the subsequent experiments that decreased the level of formation of toxic organometallic compounds, thus reducing slightly the intensity of microbial growth. At the same time, the optimal duration of cultivation (3 days) for maximum biomass accumulation on these media was determined.
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129
7
F.n
6
F.o
5
Diameter of colonies, mm
DW, g/l
8
P.g
4 3 2 1 0 0
20
40
60
80
100
Cultivation time, h
F.n
DW, g/l
2.5
3
F.o P.g
2 1 0 1
10
100
1000
Fig. 4 Influence of concentration of Ag? on a diameter of colonies of four main activated sludge cultures
causing inhibition of growth of cultures, and also identification of cultures that are the steadiest ones, and also those ones which are most sensitive against impact of silver became a specific objective of the research.
F.o
2 P.g
1.5
F.n
[Ag+], µg/l
Fig. 1 The growth of cultures on a rich Gause’s No 1 medium (20 g/l of starch)
3
A.t
4
1
Assessment of toxicity of silver salts on an agar medium
0.5 0 0
20
40
60
80
100
Cultivation time, h
CFU•10-9, ml-1
Fig. 2 The growth of cultures on a modified Gause’s No 1 medium (3.0 g/l of starch)
4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 0
A.t
F.n
F.o
P.g
20
40
60
80
100
Cultivation time, h Fig. 3 The growth of cultures on modified Gause’s No 1 medium and modified Ashby’s medium (3 g/l of starch or sucrose, respectively)
Influence of silver salts on growth of microorganisms The purpose of the experiments was to estimate a toxic influence of silver salts on the cultures of microorganisms dominating in activated sludge under stress and, hence, playing a key role in industrial wastewater treatment. Determination of a threshold concentration of silver
The quantity of silver cations added to the medium varied to 1,000 lg/l (equivalent to 9.3 lM of silver ions) that was up to such concentration of Ag? which caused absolute death of bacteria of E. coli on an agar medium [15]. When assessing an impact of silver ions on activated sludge microorganisms, not only the number of colonyforming units was compared in 3 days of growth, but also the size of the colonies. It was established that the number of colony-forming units dramatically decreased only at approaching the amount of silver in the medium to the concentration causing complete inhibition of growth of cultures, whereas the diameter of the colonies formed by them decreased gradually, allowing to assess an inhibition degree more accurately. The dependence of a diameter of colonies on the amount of silver in the medium is given in Fig. 4, allowing estimating visually the range of concentrations of the metal inhibiting growth of microorganisms, and also showing considerably higher sensitivity of bacterial cultures to silver in comparison with fungus one. Assessment of toxicity of silver salts in the liquid medium For assessment of steadiness of the cultures studied to impact of silver in liquid nutrient medium, three-day cultivation in flasks with a volume of 30 ml of medium according to the technique above mentioned was carried out. Various amounts of silver ions from 0.1 to 1,000 lg/l were added into the media before inoculation. The range of the concentration studied was expanded because toxic influence of bactericidal substances was shown, as a rule,
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more strongly in the liquid media than at surface cultivation. The growth of microorganisms was estimated by DW measurement, and also visually. The data on DW for the cultures researched are submitted in Fig. 5 and external characteristics of growth of microorganisms are tabulated in Table 3. Influence of salts of uranium on growth of four main activated sludge cultures of microorganisms The experiments with uranyl acetate were carried out to solve two following tasks. First of all, the thresholds of toxic influence of uranium were determined on four cultures studied. Secondly, comparison of toxicity of uranium and silver was carried out to find out whether resistance of
2.5 A.t
DW, g/l
2
F.n F.o
1.5
the microorganisms to silver was a reliable evidence of resistance of these cultures also to heavy metals, or an impact of the last ones on microorganisms was comparable on strength with toxicity of silver. The experiments on assessment of toxicity of uranium on agar and liquid media were carried out by the same way as the experiments with silver were, thus concentration of uranium in the form of UO2(CH3COO)22H2O varied to 1,500 mg/l (equivalent to 6.3 mM of uranium). In experiments with uranium salts, the ability of microorganisms to adsorb uranium completely from the medium without adverse effects to them was used; as evidenced by the fact that at the moment of inoculation, the cultural liquids with high content of uranyl acetate had yellow coloring, but during cultivation they became colorless, while the biomass adsorbing uranium salt gained yellow color. The results of experiments are given in Figs. 6, 7 and in Table 4. Thus, the effect of uranium salt to the main activated sludge cultures was ten thousand times less toxic than one of silver salt that testified to uniqueness of the second metal influence on the biota of the activated sludge.
P.g
Influence of silver salts on growth of activated sludge
1 0.5 0 0.1
1
10
100
1000
[Ag+], µg/l Fig. 5 Influence of concentration of Ag? on the DW of four main activated sludge cultures
The cultures of microorganisms investigated were isolated from the activated sludge containing about thirty different types of microorganisms. The activated sludge represented a stable community of microorganisms which became more viable and treated waste waters more effectively due to syntrophic interactions. It was logical to assume that resistance of microorganisms to effect of metals in the
Table 3 The visual characteristics of growth of the cultures in the liquid nutrient medium containing various concentrations of silver [Ag?], lg/l
Characteristics of growth Culture of A.t
Culture of F.n
Culture of F.o
Culture of P.g
0.1
Active growth
Active growth
Active growth
Active growth
0.3
Sluggish growth, not enough granules
1.0
No growth, no granules
10 20
Sluggish growth, not enough granules
30
No growth, but the granulated inoculant
50
Sluggish growth, not enough granules
100
No growth, but the granulated inoculant
300 600
No growth
1,000
no granules
123
No growth, no granules
Sluggish growth, not enough granules No growth, but the granulated inoculant No growth No granules
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131
2.5
A.t
2
F.n
DW, g/l
F.o 1.5
P.g
1 0.5 0 1
10
100
1000
10000
[U(VI)], mg/l Fig. 6 Influence of concentration of U(VI) on the DW of pure cultures
Diameter of colonies
4
A.t
3.5 3
F.n
2.5
F.o
2
P.g
1.5 1 0.5 0 1
10
100
1000
10000
[U(VI)], mg/l Fig. 7 Influence of concentration of U(VI) on a diameter of colonies
community similar in an activated sludge type also increased. To check this assumption, a number of experiments in which activated sludge was exposed to an effect of silver nitrate both in liquid, and on the agar media under conditions identical to conditions of researches of pure cultures were carried out. These experiments had also another purpose: to find out whether the pure cultures represented in the work were not
only dominating in the activated sludge given under stress condition, but also the cultures responsible for the activated sludge resistance to effect of silver salts. If it was not so, experiments would help to reveal the culture that was most tolerant to silver; this information would be valuable to planning of composition of the activated sludge with increased resistance to toxic metals. The activated sludge showed a weak growth in comparison with pure cultures in experiments on flasks with liquid media without Ag?; small brown granules of an irregular form being formed no more than 1 mm in size. However, the activated sludge community of microorganisms had quite a high tolerance to effect of silver, comparable to the culture of F.n, the most resistant one from the pure cultures. Slight inhibition of growth of microorganisms was observed at concentration of 100 lg/l of Ag?; only one granule was formed and the growth practically was not observed at concentration of 300 lg/l of Ag?; and a complete inhibition of the growth took place at concentration of 600 lg/l of Ag? (see Fig. 8). In experiences on agar media, the activated sludge showed greater sensitivity to silver: the mixture of many cultures with a diameter of colonies up to 3 mm was observed at the contents of 50 lg/l of silver in the agar in 3 days of cultivation. There were much less colonies and their diameter was smaller than 1.5–2.0 mm at concentration of 200 lg/l of Ag?; and colonies were absent at concentration of 500 lg/l of Ag?. And pure cultures of F.n, F.o and P.g grew in the presence of 500 lg/l of Ag? in the medium, though hereat the growth was strongly inhibited. It is interesting to note that there was a bacterial culture constituting of the activated sludge and steadiest to effect of silver, and forming white colonies of the irregular form with a diameter up to 3 mm; however, it was worse in stability than the pure cultures of F.n, F.o and P.g isolated
Table 4 The visual characteristics of growth of cultures in the liquid nutrient medium with various concentrations of uranium [U(VI)], mg/l
Characteristics of growth Culture of A.t
Culture of F.n
Culture of F.o
Culture of P.g
10
Active growth
Active growth
Active growth
Active growth
30
Slight inhibition
60 90
Sluggish growth, not enough granules
120
No growth Slight inhibition
Slight inhibition
600
300 Slight inhibition
Sluggish growth, not enough granules
Sluggish growth, not enough granules
1,000
Sluggish growth, small granules
No growth
No growth
1,500
No growth
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Bioprocess Biosyst Eng (2015) 38:125–135 2.5 1
DW, g/l
2
2 3
1.5 1 0.5 0 1
10
100
1000
[Ag+], µg/l Fig. 8 Influence of concentration of Ag? on the activated sludge (1), the pure culture of F.n (2) and the modeled activated sludge (3) by DW
from the same activated sludge. This culture was named a culture of a bacterial isolate (BI); cultured in a liquid nutrient medium and tested on resistance to silver salts. As a result, it had resistance to the metal in the liquid medium approximately the same as the culture of P.g had, and that resistance was slightly lower than the activated sludge had. It meant that symbiotic relations of activated sludge played a crucial role in increase of its resistance to metals, and communities of microorganisms were much more resistant than the individual cultures. The model of activated sludge and its resistance to effect of silver Maintaining viability of activated sludge and increasing its resistance to those harmful compounds against which it is applied is a recognized problem [1, 3]. One of the ways to overcome this problem could be formation of synthetic functional activated sludge resistant to toxicants [16, 17]. The microbial resistance of the activated sludge to effect of silver increased at growing or being in the community due to the implementation of mechanisms of collective resistance to toxic metals. The F.n, F.o and P.g isolates obtained showed an active growth and high tolerance to silver. On the basis of these facts, it was possible to assume that synthetic activated sludge formed by mixing the isolates concerned would have qualities promising for treatment of wastewaters polluted with toxic metals. The data of light microscopy testified that the ratio of F.n, F.o and P.g cultures in the activated sludge modeled was approximately 1:2:2; thick-walled unicellular conidia which are typical for P.g, macroconidia with a foot-shaped basal cell and 4–5 partitions which are a distinctive characteristic of F.o prevailed. Macroconidia without a footshaped basal cell and with three partitions which are typical for F.n were much fewer than previous cultures. During the growth of the modeled activated sludge, there was no
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elimination of any of the species that could testify to the absence of antagonistic interaction between the microorganisms studied and allowed assuming possibility of symbiosis between them resulted in increase of the modeled activated sludge resistance to toxic metals. The modeled activated sludge continued to grow intensively even at concentration of 100 lg/l of Ag? that testified to resistance of the synthetic associate to effect of silver, surpassing the resistance of any of the pure cultures, and also the initial ordinary activated sludge. Strong inhibition of the modeled activated sludge development was observed only at concentration of 300 lg/l of Ag? (only 1–2 granules were formed in 30 ml of the medium), and the growth stopped absolutely at concentration of 600 lg/l of Ag?. The influence of silver on the activated sludge, the modeled activated sludge and the most resistant culture of F.n is compared in Fig. 8. The experiments on agar media gave the similar results. Pure cultures of F.n, F.o and P.g showed very weak growth at concentration of 600 lg/l of Ag?, and they did not grow at concentration of 1,000 lg/l of Ag? at all. The modeled activated sludge grew on the medium containing 1,000 lg/l of silver though the growth was thus hardly showed up: after 3 days of cultivation, a diameter of colonies was 0.3 mm. One of the possible reasons of increase of microbial resistance to silver at formation of microbial community is an ordered structure of granules of modeled activated sludge. At concentration of 100 lg/l of Ag? the culture of F.n, most resistance to silver prevailed in a cover of an activated sludge granule, and the core of the granule consisted mainly of the culture of F.o which was least resistant to silver; whereas the culture of P.g was localized on the average granule layer. Such spatial distribution of the microbial cultures in the activated sludge modeled resulted from natural selection of the microorganisms more tolerant to silver in the cover of the granule and also provided the survival of the cultures less resistant to silver in its internal layers.
Discussion Comparison of toxicity of salts of uranium and silver Among noble metals, silver has the greatest bactericidal action and its wide application for disinfection in medicine is based on this. Therefore, when metals get into waste waters of industrial enterprises, silver causes the greatest damage to natural ecosystems. There are some data on toxic influence of silver, first of all, on nitrogen-fixing and denitrifying bacteria [9, 11]. As there are a very limited number of the microorganisms fixing nitrogen in nature, their inhibition slows down growth of the majority of types
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of soil and activated sludge communities, and the death of denitrifiers, on the contrary, leads to an eutrophication of reservoirs. It is possible to assume that presence of silver in waste waters of enterprises also breaks a nitrogen exchange in microbial community of activated sludge or wastewater treatment plants that leads if not to death of all community but, at least, to deterioration of water treatment quality and decrease in its rate. At solid-phase cultivation, an oppressing impact of silver on the culture of A.t arose already at concentration of 3 lg/l of metal, and almost complete inhibition of growth was observed at concentration of 50 lg/l of silver (Fig. 4). The other three cultures showed much higher level of resistance to silver salts than nitrogen-fixing ones did. The inhibiting effect started being shown at concentration of 50 lg/l of metal, and almost complete inhibition of growth took place at concentration of 600 lg/l of silver (equivalent to 5.6 lM). Thus, the results of the experiments confirmed the data on hypersensibility of nitrogen-fixing bacteria A.t to effect of silver: for the activated sludge studied critical lethal concentration of Ag? is about 10 times lower for nitrogen-fixing A.t than for the other three dominating cultures [9, 11]. By results of cultivation in the liquid medium, (Fig. 5, Table 3) levels of stability of the cultures studied to silver could be rowed as follows: A.t F.o \ P.g \ F.n, unlike the experiments on the agar medium in which cultures of F.n, F.o and P.g showed approximately an identical resistance to silver. Such difference could be connected with the fact that an aeration level increased greatly at liquid-phase cultivation, so did the total metabolic rate, therefore the extent of resistance to silver could decrease, and so microbial sensitivity to metal in the liquid medium increased in comparison with the agar medium. So, for example, tolerance of the cultures of F.n and P.g to silver decreased three times at submerged cultivation in comparison with surface cultivation, and tolerance of the cultures of A.t and F.o decreased ten times. In these experiments, another interesting regularity was found: at growth in the medium with high concentration of silver (causing almost complete inhibition of growth of the cultures), the cells of the inoculating material formed large agglomerates and they were granulated; no increase of a biomass was practically observed. When the concentration of complete inhibition of growth was reached, granulation of microorganisms did not occur that allowed establishing a lethality threshold quite accurately even visually. On this basis, it was suggested that formation of a usual type of granules required rather a high level of metabolism uninhibited by silver ions. Therefore, silver, breaking granulation of activated sludge, could provoke its inflation and reduce its resistance to other toxicants and so the efficiency of wastewater treatment.
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It is possible to mention the following most significant results of the research carried out. First, thresholds of toxic influence of silver on the activated sludge cultures which were the most significant under stress were established that gave direct information on viability of the activated sludge studied in the waste waters containing silver ions. Secondly, the experiment confirmed that the culture of A.t, i.e., nitrogen-fixing bacteria, was least steady among the main cultures of the activated sludge under consideration. And if sensitivity of nitrogen fixers to silver ions was 10 times more on the agar nutrient medium than one of the other cultures, this difference increased hundred times in the liquid medium. It is possible to make a conclusion that the culture of A.t which was the strongest nitrogen fixer and most essential for the activated sludge community would die one of the first at polluting of waste waters with silver salts. In that case just this culture would need to be brought additionally periodically into treatment facilities to maintain the microbial community integrity. Soluble salts of U(VI) also made negative impact both on activated sludge, and on microbial communities of soils polluted with this metal. Poisoning effect of U(VI) on the microorganisms started being shown at its concentration more than 25 mg/l (more than 60 mg/l for especially resistant bacteria). Therefore, in the present paper, the influence of salts of uranium on the activated sludge was investigated in detail on the example of two-water uranyl acetate, a reactant often used at the industrial enterprises [18–24]. In the experiments with uranium, the threshold values of toxic effect of uranyl acetate were determined on four pure cultures of microorganisms (Figs. 6, 7; Table 4), and it was also established that nitrogen fixers showed low tolerance concerning other cultures to effect of not only silver but also other metals. The cultures of F.n, F.o and P.g grew actively at concentration up to 300 mg/l of uranium, completely stopping their growth only at 1,500 mg/l (equivalent to 6.3 mM) of uranium, whereas a delay of growth of the culture of A.t began at concentration of 30 mg/l of uranium, and its growth stopping completely at 120 mg/l of uranium. The established thresholds of influence of uranium on the activated sludge cultures allowed predicting approximately the extent of influence of this toxic metal on the activated sludge in case of its getting into sewage, for example, in the form of such a reactant as uranyl acetate often used in chemical laboratories and at the industrial enterprises. As a whole, the experiments carried out on cultivation of four main cultures of the activated sludge showed that toxicity of silver cations was ten thousand times higher than toxicity of the uranium salt. It confirmed the assumption that the microorganisms which were rather
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steady against effect of silver would show their resistance also to heavy metals even at higher concentrations. Resistance of microorganisms to silver can be taken for a universal characteristic of resistance to metals. It is also possible to make a conclusion that as far as silver surpasses uranium so strongly in toxicity, it is silver salts that are the main aspects of the problem connected with polluting waste waters with salts of metals on a number of occasions, and it is expedient to use activated sludge resistant to silver for treatment of waste waters with high content of other toxic metals. Influence of salts of silver on activated sludge and model activated sludge Initial activated sludge surpassed all the pure cultures in resistance to silver that could testify to either symbiotic interactions in the community of microorganisms or existence of one or several cultures especially resistant to effect of silver as a part of the activated sludge and which had not been isolated in a pure form yet. However, the especially resistant isolates were not revealed in the experiments on agar media that testified for existence of symbiotic mechanisms of activated sludge resistance to Ag?. As for the culture of microorganisms being most resistant to effect of silver as a part of the activated sludge and named BI, it showed lower tolerance to metal in its pure form than the pure culture of F.n. Besides, it showed rather slow growth and low accumulation of biomass in comparison with pure cultures that means it is not promising enough for creation of activated sludge effective at treatment of waste waters with toxic metals. Further researches in overcoming the problem of activated sludge resistance to salts of heavy metals were continued by formation of an artificial consortium of activated sludge on the basis of the isolated microorganisms. The prepared mix (denoted as synthetic activated sludge) of the pure cultures of F.n, F.o b P.g, the most tolerant to silver surpassed separate pure cultures in resistance to silver that confirmed the influence of symbiotic relationships of microorganisms on tolerance of the community to effect of metals (Fig. 8). Moreover, such synthetic activated sludge excelled considerably in silver salt resistance and also in biomass accumulation intensity. At formation of a community, one of the possible mechanisms explaining increase of stability of microorganisms to metals is connected with formation of multilayered activated sludge granules with the structure ordered. The most tolerant to silver culture of F.n which was localized in a cover of synthetic activated sludge granule reduced ions of Ag? to elementary silver that led to its precipitation on the surface of a granule and prevented
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penetration of the metal into its core. It increased chances of a survival of less resistant to silver culture of F.o which was a constituent of the center of an activated sludge granule. While the culture of F.n carried out mainly a protective function in the microbial community, the cultures of F.o and P.g could make the contribution to collective resistance of the modeled activated sludge, for example, secreting the enzymes which were responsible for reduction of ions of Ag? to atomic silver. The mechanisms of collective resistance of the modeled activated sludge demand further studying as the data of experiments testify to prospects of purposeful modeling of special activated sludge intended for treatment of sewage with high content of metals by mixing microbial cultures which were most resistant to effect of metals and most actively growing.
Conclusion The promising approach for formation of the activated sludge resistant to effect of silver and uranium salts consisting in selection of the cultures dominating under the conditions of an oxidative stress has been offered. Four cultures of microorganisms (Agrobacterium tumifaciens, Fusarium nivale, Fusarium oxysporum, and Penicillium glabrum) isolated from the activated sludge of treatment facilities and the steadiest against an oxidative stress were investigated on the subject of silver and uranium salts resistance. The lethal doses of silver for these microorganisms were 1 and 600, 50 and 300 lg/l and the lethal doses of uranium were 120, 1,500; 1,000, and 1,000 mg/l, respectively. It was established that nitrogen-fixing bacteria (Agrobacterium tumifaciens) were more sensitive to heavy metals in comparison with micromyces of the activated sludge. The toxic impact of uranium and silver on the activated sludge was compared: the toxicity of the later was ten thousand times higher, silver made a strong impact on the activated sludge already at concentration of 1 lg/l. It was suggested that at selection of activated sludge or its components for treatment of metallic waste waters, it is possible to estimate the resistance of microorganisms to all other metals on their resistance to silver. On the basis of three activated sludge cultures most resistant to silver, the synthetic activated sludge shows resistance to silver, surpassing the stability of any of the pure cultures and native activated sludge. The concentration at which 50 % inhibition of growth of biomass came amounted to 100–110 lg/l for the native activated sludge and came up to 160–170 lg/l for the synthetic modeled activated sludge.
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References 1. Principia P, Villab F, Bernasconib M, Zanardini E (2006) Metal toxicity in municipal wastewater activated sludge investigated by multivariate analysis and in situ hybridization. Water Res 40:99–106 2. Laurent J, Casellas M, Dagot C (2009) Heavy metals uptake by sonicated activated sludge: relation with floc surface properties. J Hazard Mater 162:652–660 3. Gikas P (2008) Single and combined effects of nickel (Ni(II)) and cobalt (Co(II)) ions on activated sludge and on other aerobic microorganisms. J Hazard Mater 159:187–203 4. Weishuang Z, Yingheng F, Yi H (2009) Soluble protein and acid phosphatase exuded by ectomycorrhizal fungi and seedlings in response to excessive Cu and Cd. J Environ Sci 21:1667–1672 5. Gikas P, Romanos P (2006) Effects of tri-valent (Cr(III)) and hexa-valent (Cr(VI)) chromium on the growth of activated sludge. J Hazard Mater B133:212–217 6. Hammaini A, Gonzalez F, Ballester A, Blazquez ML, Munoz JA (2007) Biosorption of heavy metals by activated sludge and their desorption characteristics. J Environ Manage 84(4): 419–426 7. Yuncu B, Sanin FD, Yetis U (2006) An investigation of heavy metal biosorption in relation to C/N ratio of activated sludge. J Hazard Mater B137:990–997 8. Nguyen TP, Hankinsb NP, Hilal N (2007) A comparative study of the flocculation behaviour and final properties of synthetic and activated sludge in wastewater treatment. Desalination 204:277–295 9. Panyala NR, Pen˜a-Me´ndez EM, Havel J (2008) Silver or silver nanoparticles: a hazardous threat to the environment and human health? J Appl Biomed 6:117–129 10. Seaton A, Tran L, Aitken R, Donaldson K (2009) Nanoparticles, human health hazard and regulation. J R Soc Interface 11. Sharma VK, Yngard RA, Lin Y (2009) Silver nanoparticles: green synthesis and their antimicrobial activities. Adv Colloid Interface Sci 145(1–2):83–96 12. Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic Acids Techniques in Bacterial Systematics. John Wiley & Sons, Chichester, pp 115–147 13. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci 84:5463–5467
135 14. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST? : architecture and applications. BMC Bioinform Dec 15:10:421 15. Le AT, Huy PT, Tam PD, Huy TQ, Cam PD, Kudrinskiy AA, Krutyakov YA (2010) Green synthesis of finely-dispersed highly bactericidal silver nanoparticles via modified Tollens technique. Current Appl Phys 10:910–916 16. Sirotkin A (2003) Technological and ecological bases of biosorption processes of sewage treatment. Doctoral Dissertation. Kazan National Research Technological University, Kazan 17. Sirotkin A, Shaginurova G, Ippolitov K (2007) Aggregation of microorganisms: flocculi, biofilms, microbic granules. Publishing house ‘‘Fan’’ of Academy of Sciences of the Republic of Tatarstan, Kazan 18. Veeramani H, Alessi DS, Suvorova EI, Lezama-Pacheco JS, Stubbs JE, Sharp JO, Dippon U, Kappler A, Bargar JR, Bernier-Latmani R (2011) Products of abiotic U(VI) reduction by biogenic magnetite and vivianite. Geochim Cosmochim Acta 75:2512–2528 19. Marshall MJ, Beliaev AS, Dohnalkova AC, Kennedy DW, Shi L (2006) c-Type cytochrome-dependent formation of U(IV) nanoparticles by Shewanella oneidensis. PLoS Biol 4(8):1324–1333 20. Bernier-Latmani R, Veeramani H, Vecchia ED, Junier P, Lezama-Pacheco JS, Suvorova EI, Sharp JO, Wigginton NS, Bargar JR (2010) Non-uraninite Products of Microbial U(VI) Reduction. Environ Sci Technol 44:9456–9462 21. Vecchia ED, Veeramani H, Suvorova EI, Wigginton NS, Bargar JR, Bernier-Latmani R (2010) U(VI) reduction by spores of Clostridium acetobutylicum. Res Microbiol 161:765–771 22. Sharp JO, Schofield EJ, Veeramani H, Suvorova EI, Kennedy DW, Marshall MJ, Mehta A, Bargar JR, Bernier-Latmani R (2009) Structural similarities between biogenic uraninites produced by phylogenetically and metabolically diverse bacteria. Environ Sci Technol 43:8295–8301 23. Schofield EJ, Veeramani H, Sharp JO, Suvorova EI, Marshall MJ, Mehta A, Stahlaman J, Webb SM, Clark DL, Conradson SD, Ilton ES, Bargar JR (2008) Structure of biogenic uraninite produced by Shewanella oneidensis strain MR-1. Environ Sci Technol 42:7898–7904 24. Veeramani H, Schofield EJ, Sharp JO, Suvorova EI, Kai-Uwe U, Mehta A, Giammar DE, Bargar JR, Bernier-Latmani R (2009) Effect of Mn(II) on the structure and reactivity of biogenic uraninite. Environ Sci Technol 43:6541–6547
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ORIGINAL PAPER
Toxic influence of silver and uranium salts on activated sludge of wastewater treatment plants and synthetic activated sludge associates modeled on its pure cultures Dmitry V. Tyupa • Sergei V. Kalenov • Dmitry A. Skladnev • Nikolay S. Khokhlachev • Marina M. Baurina • Alexander Ye. Kuznetsov
Received: 19 October 2013 / Accepted: 24 June 2014 / Published online: 16 July 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Toxic impact of silver and uranium salts on activated sludge of wastewater treatment facilities has been studied. Some dominating cultures (an active nitrogen fixer Agrobacterium tumifaciens (A.t) and micromyces such as Fusarium nivale, Fusarium oxysporum, and Penicillium glabrum) have been isolated and identified as a result of selection of the activated sludge microorganisms being steadiest under stressful conditions. For these cultures, the lethal doses of silver amounted 1, 600, 50, and 300 lg/l and the lethal doses of uranium were 120, 1,500, 1,000, and 1,000 mg/l, respectively. A.tumifaciens is shown to be more sensitive to heavy metals than micromyces. Synthetic granular activated sludge was formed on the basis of three cultures of the isolated micromyces steadiest against stress. Its granules were much more resistant to silver than the whole native activated sludge was. The concentration of silver causing 50 % inhibition of synthetic granular activated sludge growth reached 160–170 lg/l as far as for the native activated sludge it came only to 100–110 lg/l. Keywords Activated sludge Biological wastewater treatment Heavy metal bioremediation Silver toxicity Uranium toxicity Metal resistant microorganisms Synthetic sludge
D. V. Tyupa S. V. Kalenov (&) D. A. Skladnev N. S. Khokhlachev M. M. Baurina A. Ye. Kuznetsov The Department of Biotechnology, Faculty of Biotechnology and Industrial Ecology, D.I. Mendeleyev University of Chemical Technology of Russia, 9 Miusskaya Square, Moscow 125047, Russia e-mail: [email protected]; [email protected]
Abbreviations A.t Agrobacterium tumifaciens BI Bacterial isolate CFU Colony-forming units DW Oven-dry weight F.n Fusarium nivale F.o Fusarium oxysporum P.g Penicillium glabrum
Introduction The presence of salts of heavy metals such as copper, cadmium, cobalt, nickel, lead, chromium, etc. in waste waters of industrial enterprises is an essential issue at wastewater treatment because of their toxic impacts on the cultures of microorganisms which are a part of activated sludge of wastewater treatment facilities. Such powerful effects can cause death of microorganisms of those cultures which are most sensitive to metals that lead to decrease in biological activity of the whole activated sludge community of organisms and as a result, deterioration of wastewater treatment [1–7]. Not only are the processes of an exchange of nitrogen broken in the activated sludge community, but also the ability of the activated sludge to natural formation of granules decreases as well [8]. In this regard, special attention should be focused on a harmful influence of silver salts on water and soil biocenoses. Pollution of waste waters with large doses of silver salts leads to death of bacteria necessary for natural ecosystems, and also causes poisoning algae, fungi, crustaceans, fishes, and even mammals [9]. The experiments on small fish of the Danio genus showed that silver nanoparticles cause
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embryogenesis pathology. Besides, the metal accumulates in fish and men consuming the fish. It should be noted that colloidal particles of silver are capable of causing diseases of various human tissues and organs [10]. Silver inhibits the activity of nitrifying bacteria most strongly in water biocenoses that causes serious problems at wastewater treatment. There are some data on activated sludge communities of complex structure, which are capable to withstand up to 5 mg/l of Ag? due to the mechanisms of its collective resistance. But, as a rule, the majority of activated sludge cultures die already at 1 mg/l of silver ions [7, 11]. In most cases, treatment facilities cope well with the waste waters containing heavy metals, but the activated sludge so obtained is not extremely recommended to be applied as fertilizer in connection with possibility of contaminating crops with metals, and also because of impacts of metals on soil biocenoses [11]. Silver having stronger bactericidal action in comparison with heavy metals destroys soil ecosystems greatly. Nitrogen-fixing bacteria being a part of the symbiosis with bean plants, and also many cultures of chemolithotrophs necessary for soil formation processes die first. Denitrifying and ammonifying bacteria are also highly sensitive to effect of silver. The death of denitrifiers causes accumulation of excess nitrogen in the soil that leads to eutrophication of reservoirs and pollution of potable water [9]. In the light of data on negative impact of salts of heavy metals on activated sludge and natural biological communities, more detailed researches of such influence, taking specific activated sludge as a case in point, and also discussion of solutions of the problem of treatment of waste waters with a high content of metals are necessary. Some first steps taken on this way are reviewed in this paper. An initial assessment of the effect of salts of silver and uranium on one of the possible variants of activated sludge of wastewater treatment facilities both on biocenosis as a whole and on its isolated components was carried out in the work given. Some pure cultures of microorganisms resistant to stresses and suitable for modeling of synthetic activated sludge functioning stably in the presence of toxic metals were isolated from the native activated sludge.
Identification of these microorganisms and development of techniques of their cultivation and stressing with metal salts were carried out. The thresholds of toxic influence of salts of silver and uranium on the pure cultures isolated and on the activated sludge per se were determined; the steadiest cultures were revealed, and the influence of salts of silver and uranium on the activated sludge being compared. Also, synthetic activated sludge resistant to toxic metals was modeled on basis of the most viable and specially selected cultures.
Materials and methods Cultures of microorganisms The aerobic activated sludge of wastewater treatment plant (Stupino of Moscow Region, Russia) with discharge capacity of 6 9 1012 v3 of water per year was investigated. It was noted that some microorganisms of the activated sludge started dominating in the community, showing more rapid growth and more intensive accumulation of biomass in comparison with other cultures if the sludge was exposed to stressful influences such as hunger, volley dumping, an oxidative stress, and sharp changes of a number of parameters of functioning of treatment facilities. Four cultures steadiest against a stress were selected at long-term cultivation after passages of activated sludge under conditions of fractional introduction of hydrogen peroxide (at its concentration up to 2.4–3.0 g/l) and selected in the form of pure cultures. These cultures were capable to form strong granules that explains in some way their survival under conditions of a stress. Resistance of these cultures to adverse conditions made them promising for formation of the synthetic activated sludge resistant to toxic metals and other types of a stress. These objects were also suitable for researches as they had clearly defined morphological distinctions (Table 1). The cultures were defined later as follows: Agrobacterium tumifaciens—A.t, Fusarium nivale—F.n, Fusarium oxysporum—F.o, and Penicillium glabrum—P.g.
Table 1 Morphological characteristics of the cultures on the agar and liquid media Cultures of microorganisms
Morphological characteristics on the agar medium
Morphological characteristics on the liquid medium
A.t
Colorless roundish colonies with a diameter of 1.0–1.5 mm
White granules of 0.5–1.5 mm in size
F.n
Pale yellowish mycelial colonies with a diameter of 4–20 mm
Yellowish threads, agglomerates and granules of an irregular form of 2–4 mm in size
F.o
White mycelial colonies with a diameter of 4–10 mm
Strong yellowish granules of a round and wrong form of 2–4 mm in size
P.g
White mycelial colonies with a diameter of 4–10 mm, forming green spores
Very stable whitish round granules of 2–4 mm in size
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Phylogenetic identification of the microbial samples The isolates from activated sludge were tentatively identified by the 16S (or eukaryotic 18S) rRNA gene sequences. For this purpose, DNA was extracted from microbial cells using a boiling method. When using this method, the concentration of the DNA in the preparation was 30–50 lg/ ml, and RNA was present at trace quantities (\1 %). For carrying out the PCR and further sequencing of PCR fragments of 16S (or 18S) rRNA genes, a universal primer system was used [12]. The reaction mixture was of the following content: 19 buffer of Taq polymerase (17 mM of (NH4)2SO4, 67 mM of Tris–HCl, pH 8.8, 2 mM of MgCl2); 12.5 nM of each dNTP, 50 ng of microbial DNA; 5 pM corresponding primers and 3 Units of BioTaq polymerase (Dialat LTD, Russia). The PCR was carried out at corresponding temperature and time profiles. The electrophoresis of PCR products was carried out in 2 % agarose gels. All PCR products for sequencing were purified using Wizard PCR Preps DNA Purification System (Promega, Madison, WI, USA) as described by the manufacturer. Sequencing of PCR fragments of 16S and 18S rRNA genes was carried out on Senger’s method [13] with ready reaction kit Big Dye Terminator v.3.1 (Applied Biosystems, Inc., USA) on an automatic sequencer ABI PRIZM 3730 (Applied Biosystems, Inc., USA) according to instructions of the producer. Sequence similarities were identified by a BLAST search [14]. Techniques of cultivation of microorganisms Cultivation on an agar medium The culture of A.t was cultivated on a modified Ashby’s medium, a special medium for nitrogen-fixing bacteria. The composition of each medium is tabulated in Table 2, thus a classical Ashby’s medium differed from the medium given below by content of 20 g/l of sucrose and addition of solution of microelements in amount of 1 ml/l. The other cultures were incubated on a modified Gause’s No 1 medium (Table 2), and a classical Gause’s No 1 medium differed from the medium given below only by the content of starch in amount of 20 g/l. All nutrient media were autoclaved at 121 °C for 30 min. Before inoculating the cultures of F.n, F.o and P.g, their large granules from liquid media had been disintegrated sterilely in a mortar in order the biomass to be distributed uniformly in the volume of an inoculating suspension and then the suspension had been spread on agar plates. The cultivations were carried out at 30 °C during 3 days.
127 Table 2 Composition of modified agar media Components
Modified Ashby’s medium, (g/l)
Modified Gause’s No 1 medium, (g/l)
Sucrose
3.0
–
Starch
–
3.0
CaCO3
5.0
–
KNO3
–
1.0
K2HPO4
0.2
0.5
MgSO47H2O
0.2
0.5
NaCl K2SO4
0.2 0.1
0.5 –
FeSO47H2O
–
10-4
Agar
20
20
Cultivation in a liquid nutrient medium Liquid nutrient media of the same compositions were used for liquid-phase cultivation. At cultivation in flasks, a volume of the medium consisted of one-third of a total flask volume. Inoculants were added at the rate of 10 % of a medium volume. Large granules had been disintegrated sterilely in a mortar before inoculation. Culturing was carried out on a shake-flask propagator (at 150 rpm) at diffuse background lighting and at 27 °C during 3 days. Assessment of culture growth intensity A culture growth rate, and also quantity of an accumulated biomass of microorganisms were controlled by measuring the dry cell weight (DW). For this purpose, 10 ml of cell sample suspension was harvested by centrifugation (9,000 rpm) for 15 min, washed twice with distilled water and dried up to constant weight at 105 °C. In case of the cultures of F.o and P.g where large granules were being formed on the third day of cultivation, 30 ml of cell sample suspension was withdrawn; large granules were disintegrated in a mortar, and only then 10 ml of a test was sampled. Such an approach allowed lowering an error significantly. Determination of the DW for culture of A.t was problematic as a result of the high content of CaCO3 in the medium. Therefore, in addition, the growth rate of the culture of A.t was estimated by counting of cells in the count chamber. Besides, the amount of cells for the cultures studied in the medium was estimated by inoculation of cultural liquids diluted in series on Petri dishes with the subsequent counting of the number of colony-forming units (CFU, ml-1). Each experiment was repeated thrice, and the experimental errors were \5 %.
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Determination of lethal concentration of heavy metals Assessment of toxicity of silver and uranium salts on agar nutrient media Threshold values of toxic effect of silver and uranium salts were determined for cultures of microorganisms by adding of various amounts of water solutions of silver nitrate (AgNO3) or dihydrate of uranyl acetate (UO2(CH3COO)22H2O) onto a liquid agarized nutrient medium; after its hardening, the inoculation was carried out. The cultures’ growth inhibition rate was estimated according to reduction of a diameter of colonies and their quantities as far as the concentration of salts of heavy metals increased. The inoculums (0.1 ml of the suspension) obtained within 3 days of liquid-phase cultivation and diluted to 6 9 10-6 were spread on a sterilized agar plates that allowed not only to count the number of colony-forming units, but also to estimate the size and external characteristics of the colonies. Assessment of toxicity of silver and uranium salts in liquid nutrient media Toxic influence of salts of the metals on microorganisms in the liquid medium was determined during three-day liquidphase cultivation by the technique described in Sect. ‘‘Cultivation in a liquid nutrient medium’’, and various amounts of silver nitrate or uranyl acetate solutions had been added under sterile conditions before inoculation; the control samples were incubated in parallel. The cultures’ growth inhibition rate was estimated on decrease of biomass accumulation by DW. Synthetic activated sludge modeling Synthetic activated sludge was modeled by joint cultivating three cultures of F.n, F.o, and P.g being most resistant to silver, the cultivation being carried out in the modified Gause’s No 1 medium within 3 days. To prepare inoculums, the equal amounts of suspension of these cultures were taken as these species of microorganisms had almost identical curves of growth and accumulated approximately similar quantity of biomass for equal time. Before introduction of inoculums, the granules of these three cultures were sterilely disintegrated in a mortar and the suspensions of the cells were mixed. The synthetic activated sludge obtained showed intensive growth and biomass accumulation comparable to the corresponding characteristics of cultures of F.n, F.o, and P.g and formed the spherical white granules of 3–5 mm in a diameter, which were not similar to those types of granules that were formed by the pure cultures. The activated sludge granules formed were analyzed by a method of light microscopy. For the estimation of
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percentage species composition of the modeled activated sludge, some indicated characteristics of the cultures were used: spindle-shaped and cylindrical macroconidia mostly with three septa, without a foot-shaped basal cell, and absence of chlamydospores—for F.n; spindle-shaped and lunate macroconidia with a distinct foot-shaped basal cell or with a papilla, mainly with four or five septa and abundant chlamydospores—for F.o; and the plentiful unicellular almost round conidia formed on flask-shaped phialides—for P.g. The modeled activated sludge resistance to silver was determined by the same method as the resistance of the pure cultures was done.
Results Research of culture growth dynamics When studying the growth of microorganisms, we intended to achieve two purposes. The first one was to determine the optimum time of cultivation of the isolated activated sludge cultures for the subsequent assessment of influence of toxic metals on their growth. Secondly, it was optimization of the composition of nutrient media to decrease in amount of organic substances added into the media and capable to make a contribution to an essential toxic impact of the metals, forming evens more toxic organometallic compounds. The researches of cultivation in a liquid media showed that all four cultures of microorganisms reached a stationary growth phase synchronously on the fourth day. The Gause’s No 1 and Ashby’s media of standard composition (20 g/l of starch or sucrose, respectively) and their modified variants were used to investigate the dynamics of a culture growth on nutrient media with original and lowered contents of sources of carbon (3 g/l). The curves of growth are shown in Figs. 1, 2 and 3. Thus, it was established that cells of the cultures concerned reached the most active growth phase on the second and third days, and they had managed to accumulate the level of biomass not \90 % from the maximum by the third day. Fig. 2. A sevenfold decrease of carbon source contents did not influence a culture P.g growth in a synthetic medium, whereas accumulation of biomass for cultures of F.n and F.o decreased no more than 2.5 times in an impoverished medium reducing intensity of microbial growth slightly. These data allowed using modified nutrient medium in the subsequent experiments that decreased the level of formation of toxic organometallic compounds, thus reducing slightly the intensity of microbial growth. At the same time, the optimal duration of cultivation (3 days) for maximum biomass accumulation on these media was determined.
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129
7
F.n
6
F.o
5
Diameter of colonies, mm
DW, g/l
8
P.g
4 3 2 1 0 0
20
40
60
80
100
Cultivation time, h
F.n
DW, g/l
2.5
3
F.o P.g
2 1 0 1
10
100
1000
Fig. 4 Influence of concentration of Ag? on a diameter of colonies of four main activated sludge cultures
causing inhibition of growth of cultures, and also identification of cultures that are the steadiest ones, and also those ones which are most sensitive against impact of silver became a specific objective of the research.
F.o
2 P.g
1.5
F.n
[Ag+], µg/l
Fig. 1 The growth of cultures on a rich Gause’s No 1 medium (20 g/l of starch)
3
A.t
4
1
Assessment of toxicity of silver salts on an agar medium
0.5 0 0
20
40
60
80
100
Cultivation time, h
CFU•10-9, ml-1
Fig. 2 The growth of cultures on a modified Gause’s No 1 medium (3.0 g/l of starch)
4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 0
A.t
F.n
F.o
P.g
20
40
60
80
100
Cultivation time, h Fig. 3 The growth of cultures on modified Gause’s No 1 medium and modified Ashby’s medium (3 g/l of starch or sucrose, respectively)
Influence of silver salts on growth of microorganisms The purpose of the experiments was to estimate a toxic influence of silver salts on the cultures of microorganisms dominating in activated sludge under stress and, hence, playing a key role in industrial wastewater treatment. Determination of a threshold concentration of silver
The quantity of silver cations added to the medium varied to 1,000 lg/l (equivalent to 9.3 lM of silver ions) that was up to such concentration of Ag? which caused absolute death of bacteria of E. coli on an agar medium [15]. When assessing an impact of silver ions on activated sludge microorganisms, not only the number of colonyforming units was compared in 3 days of growth, but also the size of the colonies. It was established that the number of colony-forming units dramatically decreased only at approaching the amount of silver in the medium to the concentration causing complete inhibition of growth of cultures, whereas the diameter of the colonies formed by them decreased gradually, allowing to assess an inhibition degree more accurately. The dependence of a diameter of colonies on the amount of silver in the medium is given in Fig. 4, allowing estimating visually the range of concentrations of the metal inhibiting growth of microorganisms, and also showing considerably higher sensitivity of bacterial cultures to silver in comparison with fungus one. Assessment of toxicity of silver salts in the liquid medium For assessment of steadiness of the cultures studied to impact of silver in liquid nutrient medium, three-day cultivation in flasks with a volume of 30 ml of medium according to the technique above mentioned was carried out. Various amounts of silver ions from 0.1 to 1,000 lg/l were added into the media before inoculation. The range of the concentration studied was expanded because toxic influence of bactericidal substances was shown, as a rule,
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more strongly in the liquid media than at surface cultivation. The growth of microorganisms was estimated by DW measurement, and also visually. The data on DW for the cultures researched are submitted in Fig. 5 and external characteristics of growth of microorganisms are tabulated in Table 3. Influence of salts of uranium on growth of four main activated sludge cultures of microorganisms The experiments with uranyl acetate were carried out to solve two following tasks. First of all, the thresholds of toxic influence of uranium were determined on four cultures studied. Secondly, comparison of toxicity of uranium and silver was carried out to find out whether resistance of
2.5 A.t
DW, g/l
2
F.n F.o
1.5
the microorganisms to silver was a reliable evidence of resistance of these cultures also to heavy metals, or an impact of the last ones on microorganisms was comparable on strength with toxicity of silver. The experiments on assessment of toxicity of uranium on agar and liquid media were carried out by the same way as the experiments with silver were, thus concentration of uranium in the form of UO2(CH3COO)22H2O varied to 1,500 mg/l (equivalent to 6.3 mM of uranium). In experiments with uranium salts, the ability of microorganisms to adsorb uranium completely from the medium without adverse effects to them was used; as evidenced by the fact that at the moment of inoculation, the cultural liquids with high content of uranyl acetate had yellow coloring, but during cultivation they became colorless, while the biomass adsorbing uranium salt gained yellow color. The results of experiments are given in Figs. 6, 7 and in Table 4. Thus, the effect of uranium salt to the main activated sludge cultures was ten thousand times less toxic than one of silver salt that testified to uniqueness of the second metal influence on the biota of the activated sludge.
P.g
Influence of silver salts on growth of activated sludge
1 0.5 0 0.1
1
10
100
1000
[Ag+], µg/l Fig. 5 Influence of concentration of Ag? on the DW of four main activated sludge cultures
The cultures of microorganisms investigated were isolated from the activated sludge containing about thirty different types of microorganisms. The activated sludge represented a stable community of microorganisms which became more viable and treated waste waters more effectively due to syntrophic interactions. It was logical to assume that resistance of microorganisms to effect of metals in the
Table 3 The visual characteristics of growth of the cultures in the liquid nutrient medium containing various concentrations of silver [Ag?], lg/l
Characteristics of growth Culture of A.t
Culture of F.n
Culture of F.o
Culture of P.g
0.1
Active growth
Active growth
Active growth
Active growth
0.3
Sluggish growth, not enough granules
1.0
No growth, no granules
10 20
Sluggish growth, not enough granules
30
No growth, but the granulated inoculant
50
Sluggish growth, not enough granules
100
No growth, but the granulated inoculant
300 600
No growth
1,000
no granules
123
No growth, no granules
Sluggish growth, not enough granules No growth, but the granulated inoculant No growth No granules
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131
2.5
A.t
2
F.n
DW, g/l
F.o 1.5
P.g
1 0.5 0 1
10
100
1000
10000
[U(VI)], mg/l Fig. 6 Influence of concentration of U(VI) on the DW of pure cultures
Diameter of colonies
4
A.t
3.5 3
F.n
2.5
F.o
2
P.g
1.5 1 0.5 0 1
10
100
1000
10000
[U(VI)], mg/l Fig. 7 Influence of concentration of U(VI) on a diameter of colonies
community similar in an activated sludge type also increased. To check this assumption, a number of experiments in which activated sludge was exposed to an effect of silver nitrate both in liquid, and on the agar media under conditions identical to conditions of researches of pure cultures were carried out. These experiments had also another purpose: to find out whether the pure cultures represented in the work were not
only dominating in the activated sludge given under stress condition, but also the cultures responsible for the activated sludge resistance to effect of silver salts. If it was not so, experiments would help to reveal the culture that was most tolerant to silver; this information would be valuable to planning of composition of the activated sludge with increased resistance to toxic metals. The activated sludge showed a weak growth in comparison with pure cultures in experiments on flasks with liquid media without Ag?; small brown granules of an irregular form being formed no more than 1 mm in size. However, the activated sludge community of microorganisms had quite a high tolerance to effect of silver, comparable to the culture of F.n, the most resistant one from the pure cultures. Slight inhibition of growth of microorganisms was observed at concentration of 100 lg/l of Ag?; only one granule was formed and the growth practically was not observed at concentration of 300 lg/l of Ag?; and a complete inhibition of the growth took place at concentration of 600 lg/l of Ag? (see Fig. 8). In experiences on agar media, the activated sludge showed greater sensitivity to silver: the mixture of many cultures with a diameter of colonies up to 3 mm was observed at the contents of 50 lg/l of silver in the agar in 3 days of cultivation. There were much less colonies and their diameter was smaller than 1.5–2.0 mm at concentration of 200 lg/l of Ag?; and colonies were absent at concentration of 500 lg/l of Ag?. And pure cultures of F.n, F.o and P.g grew in the presence of 500 lg/l of Ag? in the medium, though hereat the growth was strongly inhibited. It is interesting to note that there was a bacterial culture constituting of the activated sludge and steadiest to effect of silver, and forming white colonies of the irregular form with a diameter up to 3 mm; however, it was worse in stability than the pure cultures of F.n, F.o and P.g isolated
Table 4 The visual characteristics of growth of cultures in the liquid nutrient medium with various concentrations of uranium [U(VI)], mg/l
Characteristics of growth Culture of A.t
Culture of F.n
Culture of F.o
Culture of P.g
10
Active growth
Active growth
Active growth
Active growth
30
Slight inhibition
60 90
Sluggish growth, not enough granules
120
No growth Slight inhibition
Slight inhibition
600
300 Slight inhibition
Sluggish growth, not enough granules
Sluggish growth, not enough granules
1,000
Sluggish growth, small granules
No growth
No growth
1,500
No growth
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Bioprocess Biosyst Eng (2015) 38:125–135 2.5 1
DW, g/l
2
2 3
1.5 1 0.5 0 1
10
100
1000
[Ag+], µg/l Fig. 8 Influence of concentration of Ag? on the activated sludge (1), the pure culture of F.n (2) and the modeled activated sludge (3) by DW
from the same activated sludge. This culture was named a culture of a bacterial isolate (BI); cultured in a liquid nutrient medium and tested on resistance to silver salts. As a result, it had resistance to the metal in the liquid medium approximately the same as the culture of P.g had, and that resistance was slightly lower than the activated sludge had. It meant that symbiotic relations of activated sludge played a crucial role in increase of its resistance to metals, and communities of microorganisms were much more resistant than the individual cultures. The model of activated sludge and its resistance to effect of silver Maintaining viability of activated sludge and increasing its resistance to those harmful compounds against which it is applied is a recognized problem [1, 3]. One of the ways to overcome this problem could be formation of synthetic functional activated sludge resistant to toxicants [16, 17]. The microbial resistance of the activated sludge to effect of silver increased at growing or being in the community due to the implementation of mechanisms of collective resistance to toxic metals. The F.n, F.o and P.g isolates obtained showed an active growth and high tolerance to silver. On the basis of these facts, it was possible to assume that synthetic activated sludge formed by mixing the isolates concerned would have qualities promising for treatment of wastewaters polluted with toxic metals. The data of light microscopy testified that the ratio of F.n, F.o and P.g cultures in the activated sludge modeled was approximately 1:2:2; thick-walled unicellular conidia which are typical for P.g, macroconidia with a foot-shaped basal cell and 4–5 partitions which are a distinctive characteristic of F.o prevailed. Macroconidia without a footshaped basal cell and with three partitions which are typical for F.n were much fewer than previous cultures. During the growth of the modeled activated sludge, there was no
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elimination of any of the species that could testify to the absence of antagonistic interaction between the microorganisms studied and allowed assuming possibility of symbiosis between them resulted in increase of the modeled activated sludge resistance to toxic metals. The modeled activated sludge continued to grow intensively even at concentration of 100 lg/l of Ag? that testified to resistance of the synthetic associate to effect of silver, surpassing the resistance of any of the pure cultures, and also the initial ordinary activated sludge. Strong inhibition of the modeled activated sludge development was observed only at concentration of 300 lg/l of Ag? (only 1–2 granules were formed in 30 ml of the medium), and the growth stopped absolutely at concentration of 600 lg/l of Ag?. The influence of silver on the activated sludge, the modeled activated sludge and the most resistant culture of F.n is compared in Fig. 8. The experiments on agar media gave the similar results. Pure cultures of F.n, F.o and P.g showed very weak growth at concentration of 600 lg/l of Ag?, and they did not grow at concentration of 1,000 lg/l of Ag? at all. The modeled activated sludge grew on the medium containing 1,000 lg/l of silver though the growth was thus hardly showed up: after 3 days of cultivation, a diameter of colonies was 0.3 mm. One of the possible reasons of increase of microbial resistance to silver at formation of microbial community is an ordered structure of granules of modeled activated sludge. At concentration of 100 lg/l of Ag? the culture of F.n, most resistance to silver prevailed in a cover of an activated sludge granule, and the core of the granule consisted mainly of the culture of F.o which was least resistant to silver; whereas the culture of P.g was localized on the average granule layer. Such spatial distribution of the microbial cultures in the activated sludge modeled resulted from natural selection of the microorganisms more tolerant to silver in the cover of the granule and also provided the survival of the cultures less resistant to silver in its internal layers.
Discussion Comparison of toxicity of salts of uranium and silver Among noble metals, silver has the greatest bactericidal action and its wide application for disinfection in medicine is based on this. Therefore, when metals get into waste waters of industrial enterprises, silver causes the greatest damage to natural ecosystems. There are some data on toxic influence of silver, first of all, on nitrogen-fixing and denitrifying bacteria [9, 11]. As there are a very limited number of the microorganisms fixing nitrogen in nature, their inhibition slows down growth of the majority of types
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of soil and activated sludge communities, and the death of denitrifiers, on the contrary, leads to an eutrophication of reservoirs. It is possible to assume that presence of silver in waste waters of enterprises also breaks a nitrogen exchange in microbial community of activated sludge or wastewater treatment plants that leads if not to death of all community but, at least, to deterioration of water treatment quality and decrease in its rate. At solid-phase cultivation, an oppressing impact of silver on the culture of A.t arose already at concentration of 3 lg/l of metal, and almost complete inhibition of growth was observed at concentration of 50 lg/l of silver (Fig. 4). The other three cultures showed much higher level of resistance to silver salts than nitrogen-fixing ones did. The inhibiting effect started being shown at concentration of 50 lg/l of metal, and almost complete inhibition of growth took place at concentration of 600 lg/l of silver (equivalent to 5.6 lM). Thus, the results of the experiments confirmed the data on hypersensibility of nitrogen-fixing bacteria A.t to effect of silver: for the activated sludge studied critical lethal concentration of Ag? is about 10 times lower for nitrogen-fixing A.t than for the other three dominating cultures [9, 11]. By results of cultivation in the liquid medium, (Fig. 5, Table 3) levels of stability of the cultures studied to silver could be rowed as follows: A.t F.o \ P.g \ F.n, unlike the experiments on the agar medium in which cultures of F.n, F.o and P.g showed approximately an identical resistance to silver. Such difference could be connected with the fact that an aeration level increased greatly at liquid-phase cultivation, so did the total metabolic rate, therefore the extent of resistance to silver could decrease, and so microbial sensitivity to metal in the liquid medium increased in comparison with the agar medium. So, for example, tolerance of the cultures of F.n and P.g to silver decreased three times at submerged cultivation in comparison with surface cultivation, and tolerance of the cultures of A.t and F.o decreased ten times. In these experiments, another interesting regularity was found: at growth in the medium with high concentration of silver (causing almost complete inhibition of growth of the cultures), the cells of the inoculating material formed large agglomerates and they were granulated; no increase of a biomass was practically observed. When the concentration of complete inhibition of growth was reached, granulation of microorganisms did not occur that allowed establishing a lethality threshold quite accurately even visually. On this basis, it was suggested that formation of a usual type of granules required rather a high level of metabolism uninhibited by silver ions. Therefore, silver, breaking granulation of activated sludge, could provoke its inflation and reduce its resistance to other toxicants and so the efficiency of wastewater treatment.
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It is possible to mention the following most significant results of the research carried out. First, thresholds of toxic influence of silver on the activated sludge cultures which were the most significant under stress were established that gave direct information on viability of the activated sludge studied in the waste waters containing silver ions. Secondly, the experiment confirmed that the culture of A.t, i.e., nitrogen-fixing bacteria, was least steady among the main cultures of the activated sludge under consideration. And if sensitivity of nitrogen fixers to silver ions was 10 times more on the agar nutrient medium than one of the other cultures, this difference increased hundred times in the liquid medium. It is possible to make a conclusion that the culture of A.t which was the strongest nitrogen fixer and most essential for the activated sludge community would die one of the first at polluting of waste waters with silver salts. In that case just this culture would need to be brought additionally periodically into treatment facilities to maintain the microbial community integrity. Soluble salts of U(VI) also made negative impact both on activated sludge, and on microbial communities of soils polluted with this metal. Poisoning effect of U(VI) on the microorganisms started being shown at its concentration more than 25 mg/l (more than 60 mg/l for especially resistant bacteria). Therefore, in the present paper, the influence of salts of uranium on the activated sludge was investigated in detail on the example of two-water uranyl acetate, a reactant often used at the industrial enterprises [18–24]. In the experiments with uranium, the threshold values of toxic effect of uranyl acetate were determined on four pure cultures of microorganisms (Figs. 6, 7; Table 4), and it was also established that nitrogen fixers showed low tolerance concerning other cultures to effect of not only silver but also other metals. The cultures of F.n, F.o and P.g grew actively at concentration up to 300 mg/l of uranium, completely stopping their growth only at 1,500 mg/l (equivalent to 6.3 mM) of uranium, whereas a delay of growth of the culture of A.t began at concentration of 30 mg/l of uranium, and its growth stopping completely at 120 mg/l of uranium. The established thresholds of influence of uranium on the activated sludge cultures allowed predicting approximately the extent of influence of this toxic metal on the activated sludge in case of its getting into sewage, for example, in the form of such a reactant as uranyl acetate often used in chemical laboratories and at the industrial enterprises. As a whole, the experiments carried out on cultivation of four main cultures of the activated sludge showed that toxicity of silver cations was ten thousand times higher than toxicity of the uranium salt. It confirmed the assumption that the microorganisms which were rather
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steady against effect of silver would show their resistance also to heavy metals even at higher concentrations. Resistance of microorganisms to silver can be taken for a universal characteristic of resistance to metals. It is also possible to make a conclusion that as far as silver surpasses uranium so strongly in toxicity, it is silver salts that are the main aspects of the problem connected with polluting waste waters with salts of metals on a number of occasions, and it is expedient to use activated sludge resistant to silver for treatment of waste waters with high content of other toxic metals. Influence of salts of silver on activated sludge and model activated sludge Initial activated sludge surpassed all the pure cultures in resistance to silver that could testify to either symbiotic interactions in the community of microorganisms or existence of one or several cultures especially resistant to effect of silver as a part of the activated sludge and which had not been isolated in a pure form yet. However, the especially resistant isolates were not revealed in the experiments on agar media that testified for existence of symbiotic mechanisms of activated sludge resistance to Ag?. As for the culture of microorganisms being most resistant to effect of silver as a part of the activated sludge and named BI, it showed lower tolerance to metal in its pure form than the pure culture of F.n. Besides, it showed rather slow growth and low accumulation of biomass in comparison with pure cultures that means it is not promising enough for creation of activated sludge effective at treatment of waste waters with toxic metals. Further researches in overcoming the problem of activated sludge resistance to salts of heavy metals were continued by formation of an artificial consortium of activated sludge on the basis of the isolated microorganisms. The prepared mix (denoted as synthetic activated sludge) of the pure cultures of F.n, F.o b P.g, the most tolerant to silver surpassed separate pure cultures in resistance to silver that confirmed the influence of symbiotic relationships of microorganisms on tolerance of the community to effect of metals (Fig. 8). Moreover, such synthetic activated sludge excelled considerably in silver salt resistance and also in biomass accumulation intensity. At formation of a community, one of the possible mechanisms explaining increase of stability of microorganisms to metals is connected with formation of multilayered activated sludge granules with the structure ordered. The most tolerant to silver culture of F.n which was localized in a cover of synthetic activated sludge granule reduced ions of Ag? to elementary silver that led to its precipitation on the surface of a granule and prevented
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penetration of the metal into its core. It increased chances of a survival of less resistant to silver culture of F.o which was a constituent of the center of an activated sludge granule. While the culture of F.n carried out mainly a protective function in the microbial community, the cultures of F.o and P.g could make the contribution to collective resistance of the modeled activated sludge, for example, secreting the enzymes which were responsible for reduction of ions of Ag? to atomic silver. The mechanisms of collective resistance of the modeled activated sludge demand further studying as the data of experiments testify to prospects of purposeful modeling of special activated sludge intended for treatment of sewage with high content of metals by mixing microbial cultures which were most resistant to effect of metals and most actively growing.
Conclusion The promising approach for formation of the activated sludge resistant to effect of silver and uranium salts consisting in selection of the cultures dominating under the conditions of an oxidative stress has been offered. Four cultures of microorganisms (Agrobacterium tumifaciens, Fusarium nivale, Fusarium oxysporum, and Penicillium glabrum) isolated from the activated sludge of treatment facilities and the steadiest against an oxidative stress were investigated on the subject of silver and uranium salts resistance. The lethal doses of silver for these microorganisms were 1 and 600, 50 and 300 lg/l and the lethal doses of uranium were 120, 1,500; 1,000, and 1,000 mg/l, respectively. It was established that nitrogen-fixing bacteria (Agrobacterium tumifaciens) were more sensitive to heavy metals in comparison with micromyces of the activated sludge. The toxic impact of uranium and silver on the activated sludge was compared: the toxicity of the later was ten thousand times higher, silver made a strong impact on the activated sludge already at concentration of 1 lg/l. It was suggested that at selection of activated sludge or its components for treatment of metallic waste waters, it is possible to estimate the resistance of microorganisms to all other metals on their resistance to silver. On the basis of three activated sludge cultures most resistant to silver, the synthetic activated sludge shows resistance to silver, surpassing the stability of any of the pure cultures and native activated sludge. The concentration at which 50 % inhibition of growth of biomass came amounted to 100–110 lg/l for the native activated sludge and came up to 160–170 lg/l for the synthetic modeled activated sludge.
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