Effect of cadmium on composition and diversity of bacterial communities in activated sludges

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International Biodeterioration & Biodegradation 55 (2005) 285–291 www.elsevier.com/locate/ibiod

Effect of cadmium on composition and diversity of bacterial communities in activated sludges Yung-Pin Tsaia,, Sheng-Jie Youb, Tzu-Yi Paic, Ko-Wei Chena a

Department of Civil Engineering, National Chi Nan University, 1, University Rd., PuLi, Nantou 545, Taiwan b Department of Bioenvironmental Engineering, Chung Yuan Christian University, Chungli 320, Taiwan c Department of Environmental Engineering and Management, Chaoyang University of Technology, Wufeng, Taichung, 413, Taiwan Received 15 December 2004; received in revised form 23 February 2005; accepted 11 March 2005

Abstract Microbial communities of two kinds of activated sludge for removing carbon, nitrogen and phosphate (nutrient removal sludge) were identified and compared by combining cloning–denaturing gradient gel electrophoresis methods. The sludges were sampled in an anaerobic–anoxic–oxic system operating under the same conditions, except for one without the addition of cadmium (Cd0) and the other with addition of 5 mg cadmium l 1 (Cd5). Bacteria in the phylum Proteobacteria were predominant in both Cd0 and Cd5 sludges (39.6% and 35.1% of total bacteria, respectively). However, bacteria in the class Betaproteobacteria were significantly more abundant in Cd0 than in Cd5 sludge (30.7% and 2.1%, respectively). Species related to nutrient removal, such as nitrifying bacteria (Nitrosomonas communis), floc-forming bacteria (Zoogloea ramigera) and phosphate-accumulating organisms (Rubrivivax gelatinosus), were the predominant species in Cd0 sludge, but were not found in Cd5 sludge. Cadmium was significantly toxic to the bacterial community in nutrient removal sludge, especially to the bacteria in the Betaproteobacteria. The comparison of microbial communities between these two kinds of sludge was further discussed in the paper. r 2005 Elsevier Ltd. All rights reserved. Keywords: Heavy metal; Activated sludge; Cadmium; Microbial community; Nutrient removal

1. Introduction The heavy metal cadmium is commonly associated with water pollution. It is very toxic and its discharge into receiving water causes detrimental effects on human health and the environment. Therefore, the removal of heavy metals from industrial effluents has become mandatory (Dal Bosco et al., 2005). The increasing trend towards combining industrial and municipal wastes for treatment in sewage plants increases the possibility of contamination of the influent by metal ions (Stasinakis et al., 2002). Since microorganisms are key components for removal of nutrients, the effect of metal toxicity on microorganisms has received special attention in recent years. The species and concentrations Corresponding author.

0964-8305/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2005.03.005

of industrial effluents are quite unpredictable, depending on the sources of industrial wastes. Most cadmium concentration of industrial wastewater in Taiwan is below 5 mg l 1. The effects of heavy metals on the activated sludge process are complex and exceedingly difficult to study owing to the diversity of microorganisms present. Some microorganisms can tolerate toxicity of heavy metals, while others cannot. However, which microorganisms have the ability to resist the toxicity is not yet well known. Past studies merely indicate that microorganisms have the ability to remove heavy metals (Macaskie, 1990). However, at a given concentration, a metal may be toxic to one species while serving as a growth stimulant to others (Lester et al., 1979; Dilek and Go¨kcay, 1996). The influence of metals on microbial communities is not precisely known to date, but what

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is known is that heavy metals can change the microbial structure of activated sludge by modifying both cell density and species richness. Lester et al. (1979) found that in a mixed culture of four strains isolated from activated sludge, Alcaligenes was the most susceptible and Brevibacterium the most resistant organism to heavy metal inhibition. Badar et al. (2000) found that Pseudomonas stutzeri, isolated from a foundry soil, could resist the toxic effect of chromate up to 1 mM and anaerobically reduce Cr(VI) up to 100 mM. Dilek and Go¨kc- ay (1996) utilized activated sludge to treat the heavy metals Ni and Cr, and found that the dominant strains changed with organic loadings. When the system was under low carbon loading and contained 5 or 10 mg l 1 Ni, Acinetobacter sp. was the dominant bacterium, but when the system was under high carbon loading at the same Ni concentration Arthrobacter simplex, Pseudomonas mendocina, Bacillus cereus and Klebsiella sp. were the dominant bacteria. Toxicity of heavy metals to the mixed culture in activated sludge is dependent on many factors. Past studies have shown that acclimated sludge maintains a high removal efficiency of dissolved organic matter even though exposed to constant input of heavy metals (Chang et al., 1986). This indicates that acclimation can reduce the negative effects of toxic substances on biological reactions, and then some microbial groups can become predominant. Another study considered that shock loads produce remarkable effects on sludge whether it is acclimated or not (Battistoni et al., 1993). Since the characteristics of microorganisms in a complex activated sludge system for nutrient removal are not yet clearly known, the study of the effects of toxic metal on activated sludge becomes important, especially (1) which kinds of acclimated microorganisms can resist the toxicity of heavy metals and (2) what levels they can tolerate. The objective of this work was resolution of differences in the microbial communities, and predominant strains, in nutrient removal sludge acclimated to different cadmium conditions. An anaerobic–anoxic–oxic (A2O) pilot plant was used to acclimatize the activated sludge samples. Mixed culture samples of activated sludge were analyzed by a method based on 16S rDNA to determine the diversity of microbial communities.

2. Materials and methods 2.1. Experimental protocol and analyzing methods The A2O pilot plant used to chronically acclimate activated sludge to different levels of cadmium (Fig. 1) was composed of an anaerobic tank, an anoxic tank,

an aerobic tank and a sedimentation tank. The wastewater containing cadmium solution (if necessary) flowed first into the anaerobic tank and then followed the above sequence. The influent pattern was continuous and the reactors could be modelled as a continuously stirred tank reactor under the conditions of the study. The biochemical reactions of hydrolysis of organic compounds and phosphate release occurred in the anaerobic tank. The nitrates produced by oxidation of ammonium in the aerobic tank were reduced to nitrogen gas in the anoxic tank. This is the denitrification reaction. The organic carbon removal and nitrification reactions were mainly fulfilled in the aerobic tank. The clarifier was responsible for the separation of activated sludge from mixed liquor. The operational conditions of the system are shown in Table 1 and represent the normal conditions of a nutrient removal system. Synthetic wastewater was used as the influent stream of the pilot plant and its corresponding water qualities

Influent Aerobic Phase (48L)

Anoxic Phase(32L)

Anaerobic (16L)

Return Supernatant

Effluent Clarifier (35L)

Return Sludge Fig. 1. Schematic diagram of A2O process.

Table 1 Operational conditions of A2O process Operational parameters

Values

Inflow rate (ml min 1) Return sludge flowrate (ml min 1) Return supernatant flowrate (ml min 1) Hydraulic retention time-anaerobic (h) Hydraulic retention time-anoxic (h) Hydraulic retention time-aerobic (h) Sludge retention time (days) Total hydraulic retention time (h) Controlled dissolved oxygen (mg l 1) Controlled pH value of aerobic tank Organic loading (g COD g MLSS 1 d 1)

100 50 250 2.66 5.33 8 10 16 2 7.2 0.27

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were (l 1): total chemical oxygen demand (COD), 276729 mg; soluble COD, 236728 mg; total nitrogen (TN), 42.476.6 mg; organic nitrogen, 18.976.2 mg; ammonia nitrogen, 21.673.7 mg; nitrate nitrogen, 2.0171.06 mg; nitrite nitrogen, 0.6470.75 mg; total phosphorus (TP), 4.6170.42 mg; and orthophosphate, 4.0570.68 mg. Such wastewater strength simulated the settled wastewater properties of the Taipei Min-Sheng Municipal Wastewater Treatment Plant. Chemical CdCl2 was used as the source of cadmium in the influent wastewater. The cultured activated sludge originated from the fullscale nutrient removal plant of National Chi-Nan University (NCNU). The aerobic tank in the A2O system was automatically maintained at pH 7.270.1 by addition of a NaOH–NaHCO3 solution, and the dissolved oxygen (DO) concentration was also automatically controlled at 2.070.2 mg l 1. At the beginning of the study, the activated sludge was acclimatized under without-cadmium conditions for 69 days to stabilize the nitrogen and phosphate removal ability and the microbial community of the system. Then, 2 mg cadmium l 1 (final concentration of influent wastewater) was continuously added to A2O system for further acclimation for about 72 days, followed by addition of 5 mg cadmium l 1 for a further 140 days. During the acclimation periods, influent and effluent water qualities were analyzed twice per week mainly according to APHA Standard Method (APHA, 1998), including total COD (5220-C), soluble COD (5220-C), 3 NH+ 4 (4500-NH3-H), total nitrogen (4500-N-B), PO4 (4500-P-G), mixed liquid volatile suspended solid (MLVSS) (2540-E), mixed liquid suspended solid (MLSS) (2540-D), total phosphorus (4500-P-I), NO3 (4500-NO3-B), NO2 (4500-NO2-B), alkalinity (2320-B), effluent suspended solid (SS) (2540-D), sludge volume index (SVI) (2710-D) and cadmium (3111-B). The Cd concentration was analyzed by the atomic absorption spectrometry (Shimadzu AA-6200). All analyses were in duplicate. 2.2. DNA extraction Sludge samples acclimated at 0 and 5 mg cadmium l 1 were separately taken from A2O pilot system, and then the DNA was extracted by the phenol–chloroform method. Approximately 5 mg biomass (dry weight) was suspended in 750 ml lysis buffer (100 mM Tris–HCl, 100 mM EDTA, 0.75 M sucrose). Lysozyme (1.0 mg l 1) and achromopeptidase (100 mg l 1) were added, and then the suspension solution was incubated at 37 1C for 30 min. After adding proteinase K (200 mg l 1) and sodium docecyl sulfate (1%, w/v), the samples were incubated at 37 1C for 2 h again. During incubation, each tube was gently inverted several times every 30 min. The mixtures were then subjected to three types of

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successive freeze–thaw cycle. Hexadecyltrimethyl ammonium bromide and sodium chloride were added to give final concentrations of 1.0% (w/v) and 0.7 M, respectively. And then, these mixtures were incubated at 65 1C for 20 min to precipitate the polysaccharides and residual proteins. The precipitate was extracted in an equal volume of phenol–chloroform–isoamyl alcohol (25:24:1), followed by extraction with chloroform–isoamyl alcohol (24:1). DNA was recovered by addition of one volume isopropanol, followed by centrifugation at 10,000g for 10 min. The pellets were washed with 70% ethanol, dried and resuspended in 50 ml distilled water. Finally, they were stored at 20 1C for construction of the cloning library. 2.3. PCR, cloning, denaturing gradient gel electrophoresis and sequence Before constructing the 16S rDNA cloning library, the extracted DNA was used as template for PCR reaction with the primer set 11f and 1512r, as shown in Table 2. The PCR products were then used for the cloning reaction according to the manufacture’s protocol (TA cloning kit, Invitrogen). The primer set M13f/ M13r was used to amplify the correct inserted rDNA fragments carried on the plasmid. Then, the correct amplified M13f/M13r PCR product was used as the DNA template in subsequent PCR reactions with the denaturing gradient gel electrophoresis (DGGE) primer set, 968fgc and 1392r. Table 2 shows the PCR primer sequences used in this study. The DGGE pattern was performed by Biorad Dcode system at 200 V and 60 1C for 3.5 h with denaturant gradients ranging from 40% to 60%. The bands on the gel were then stained with silver nitrate after DGGE screening. The bands with the same position (operation taxonomy unit, OTU) were further sequenced using ABI 3730 XL DNA Sequencer (Applied Biosystems, CA, USA) and the sequences were then compared to the known 16S rRNA sequences in the GenBank using the NCBI BLAST program (You et al., 2003).

Table 2 Primer sequences employed Primers

Sequences

11f 1512r M13( 20) M13r 968fgc

GTTTGATCCTGGCTCAG GG(TC)TACCTTGTTACGACTT GTAAAACGACGGCCAG CAGGAAACAGCTATGAC CGCCCGGGGCGCGCCCCGGGCGGGGCGGGG GCACGGGGGGAACGC CAAGAACCTTAC ACGGGCGGTGTGTAC

1392r

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3. Results and discussion 3.1. Comparison of treating efficiencies with and without the addition of Cd(II) The experimental results showed that with the A2O system acclimatized under the conditions of nutrient removal the removal efficiency of the heavy metal cadmium was high (498%). Cd(II) in effluent water could not be detected at an influent level of 2 mg l 1, and only o0.2 mg Cd(II) l 1 was detected in effluent water at an influent level of 5 mg l 1. The variation in MLVSS in the reactor and the effluent SS and COD concentrations are shown in Fig. 2. Increasing Cd(II) concentration up to 5 mg l 1 resulted in a slight decrease in sludge concentration (MLVSS). Effluent SS concentration did not show a significant difference between Cd(II) 0 and 2 mg l 1. However, both the concentration and variability of SS increased significantly at 5 mg Cd(II) l 1. Effluent total and soluble COD concentrations at the 2 mg Cd(II) l 1 level were similar to those at without Cd(II). However, the former and its variability were increased at 5 mg Cd(II) l 1 due to the increase in the effluent SS concentration. The efficiency of denitrification and phosphate removal in the A2O system (Fig. 3) and the corresponding effluent concentration of nitrogen and phosphate (Fig. 4) reveal that the efficiency of the ammonification reaction, i.e. the transformation of organic nitrogen to ammonia nitrogen, was not influenced by the addition of Cd(II). The presence of cadmium at 2 mg Cd(II) l 1 did not affect nitrification efficiency either. The effluent ammonia concentration was o0.1 mg l 1. However, nitrification efficiency was obviously depressed to 29%, when the cadmium concentration was 5 mg l 1. The denitrification efficiency was 475% irrespective of Cd(II) level; it appeared not to be affected by cadmium. However, it cannot be concluded that the denitrification reaction was not inhibited. The maintenance of the

Fig. 3. Efficiency of nitrogen and phosphate removal in A2O process at different cadmium concentrations.

Fig. 4. Effluent concentration of nitrogen and phosphate at different cadmium concentrations.

higher denitrification efficiency might result from the extremely low production of nitrate due to the inhibition of nitrification reaction. The experimental results also showed that the efficiency of phosphate removal was decreased with the increase of Cd(II) level. 3.2. Microbial community analysis

Fig. 2. COD and SS concentration of effluent water and the MLVSS concentration in reactor of A2O process at different cadmium concentrations.

After the screen of 200 clones with DGGE, 45 and 17 OTUs were obtained from the Cd0 and Cd5 sludges, respectively. This revealed that the microbial diversity was significantly affected in Cd5 sludge. Tables 3, 4 and Fig. 5 show the microbial structure of Cd0 and Cd5 sludges. In agreement with other studies (Bond et al., 1995; Snaidr et al., 1997), it was observed that the predominant bacterium in both Cd0 and Cd5 sludge samples was in the phylum Proteobacteria. And, the ratios of that bacterium to total bacteria were 39.6% and 35.1%, respectively.

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Table 3 Classification of bacteria in Cd0 sludge Division

Proteobacteria

Family

Genus

Species

Rhodospirillaceae

Azospirillum

sp.

Rhodocyclaceae Rhodocyclaceae Rhodocyclaceae Rhodocyclaceae

Propionivibrio Dechloromonas Azovibrio Zoogloea

limicola sp. sp. ramigera

Comamonadaceae

Rubrivivax

gelatinosus

Nitrosomonadaceae Uncultured sludge bacterium Unidentified beta proteobacterium

Nitrosomonas

communis

Gamma

Thiotrichaceae

Thiothrix

Nivea

5.4

Delta

Polyangiaceae

Polyangium

vitellinum

1.5

Epsilon

N.D.

Alpha

Beta

Sub. total (%) 2.0

39.6

30.7

Flexibacteraceae

0 Uncultured Cytophagaceae bacterium

2.5

Bacteroidetes (CFB group)

4.0 CFB group bacterium

Planctomycetes

Total (%)

Planctomycetales Uncultured bacterium

Above subtotal Other nonsequenced clones Total

1.5 Uncultured planctomyete

2.0

2.0

27.6

27.6 73.2 26.8 100

N.D.: not detected.

Among bacteria in the phylum Proteobacteria, those in the classes of Betaproteobacteria and Gammaproteobacteria were predominant bacteria in Cd0 and Cd5 sludges, respectively. Other classes, Alphaproteobacteria, Deltaproteobacteria and Epsilonproteobacteria, were minor in both sludge samples. The Betaproteobacteria was considered to have a positive role in nutrient removal in activated sludge processes (Bond et al., 1995; You et al., 2000). The percentage of Betaproteobacteria in Cd0 sludge (30.7%) was significantly greater than that in Cd5 sludge (2.1%). In Cd0 sludge, the major families in the class Betaproteobacteria included Rhodocyclaceae, Comamonadaceae and Nitrosomonadaceae. Several bacteria in the Betaproteobacteria that are helpful to nutrient removal in activated sludge were also observed in Cd0 sludge, e.g. Zoogloea ramigera (floc-forming bacterium), Rubrivivax gelatinosus (phosphate-accumulating organism) and Nitrosomonas communis (nitrifier). By contrast, the percentage of

Betaproteobacteria in Cd5 sludge was only 2.1%, and only the family Rhodocyclaceae still survived. The average of ammonia concentration in Cd0 and Cd5 effluent were 0.07 and 22.1 mg l 1, respectively. And, the NOx (nitrate plus nitrite) concentration in Cd0 and Cd5 effluent were 8.50 and 2.26 mg l 1, respectively (Table 5). These data agreed with the percentages of nitrifier in Cd0 and Cd5 sludges (Tables 3 and 4, respectively). In Cd0 sludge, the N. communis was the predominant microorganism in the class Betaproteobacteria. However, no nitrifier was found in Cd5 sludge. The disappearance of nitrifier in Cd5 sludge resulted in the reduction of nitrification efficiency, i.e. the oxidization of ammonia to nitrate. Thus, the concentration of ammonia increased substantially in Cd5 effluent. The same trend could also be found in phosphate removal performance. The phosphate concentration in Cd0 and Cd5 effluent were 0.94 and 3.08 mg l 1, respectively. At the same time, the percentage of phosphate removal

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Table 4 Classification of bacteria in Cd5 sludge Division

Family

Proteobacteria

Genus

Species

Alpha

N.D.

Beta

Rhodocyclaceae

Azospira

oryzae

Thiotrichaceae

Thiothrix

eikelboomii

Thiotrichaceae

Thiothrix

nivea

Sub. total (%) 0

Gamma

Total (%)

35.1

2.1

25.1

Delta

N.D.

Epsilon

Campylobacteraceae

Arcobacter

sp.

7.9

Sapropiraceae

Haliscomenobacte

hydrossis

4.2

Flexibacteraceae

Cytophaga

sp.

1.6

Bacteroidetes (CFB group)

0

14.7

Uncultured Bacteroidetes bacterium

8.9

Fusobacteria

Uncultured Fusobacteria bacterium

7.9

7.9

Firmicutes

Peptostreptococcaceae

1.6

1.6

33.4

33.4

Fusibacter

Uncultured bacterium Above subtotal Other non-sequenced clones Total

Paucivorans

92.7 7.3 100

N.D.: not detected. 2 <1

Proteobacteria Alpha Proteobacteria Beta

30.7

2.1 5.4

Proteobacteria Gamma 1.5 <1 <1

Proteobacteria Delta Proteobacteria Epsilon

25.1

7.9 4

Bacteroidetes 2 <1 <1

Planctomycetes Fusobacteria

14.7

Cd0 7.9

Cd5

<1 1.6

Firmicutes

0

5

10

15

20

25

30

35

40

percentage Fig. 5. Comparison of microbial communities in Cd0 and Cd5 sludges.

Table 5 Average effluent concentrations of nitrogen and phosphate compounds Cd0

Cd5

NH4-N NOx-N

(mg l 1) (mg l 1)

0.07 8.50

22.1 2.26

TP PO4-P

(mg l 1) (mg l 1)

1.12 0.94

3.51 3.08

related microorganisms (Rhodocyclaceae) was also substantially decreased from 16.9% in Cd0 sludge to 2.1% in Cd5 sludge. It revealed that the presence of 5 mg Cd(II) l 1 was markedly harmful to the bacterial species relating to the nitrification and phosphate removal performance. In both Cd0 and Cd5 sludges, the only family in the class Gammaproteobacteria appearing was Thiotrichaceae, a type of filamentous bacterium. However, in Cd0 sludge it was significantly less abundant than in Cd5 sludge (5.4% vs. 25.1%, respectively). That is why bulking phenomenon occurred in Cd5 sludge, as observed in acclimatization periods. Only Thiothrix nivea (5.1%) in the class Gammaproteobacteria appeared in Cd0 sludge, while both T. nivea (10.0%) and Thiothrix eikelboomii (15.1%) appeared in Cd5 sludge. If Haliscomenobacter hydrossis (4.2%) of the CFB group is included, 29.3% of all bacteria in Cd5 sludge were filamentous, agreeing with the observation of Kim et al. (2002) and revealing that the tolerance of filamentous bacteria to heavy metal cadmium is significantly greater than that of floc-forming bacteria. Furthermore, the percentage of the CFB group (Bacteroidetes) in Cd0 sludge (4.0%) was somewhat less than that in Cd5 sludge (14.7%). It is believed that the CFB group is harmful to the biochemical process of phosphate removal (Bond et al., 1995; You et al., 2000).

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In the study by Bond et al. (1995), the percentage of the CFB group in phosphate removal and non-phosphate removal processes were 5.2% and 13%, respectively. In this study, phosphate removal efficiency was about 78% in system of Cd0 sludge with a CFB group percentage of about 4%. However, the comparative removal efficiency of phosphate was reduced to about 22% in the Cd5 sludge system due to a significant 10% increase of the CFB group. This result revealed that the bacteria of CFB group could tolerate 5 mg Cd(II) l 1. And, in addition to the reduction of the amount of phosphateaccumulating organisms, the predominant propagation of the CFB group in the Cd5 sludge system was one of the important reasons for the biochemical process of phosphate removal being inhibited.

4. Conclusions Microbial diversity and richness were reduced in A2O system owing to the existence of the heavy metal cadmium. In both Cd0 and Cd5 sludge samples, members of the Proteobacteria were collectively predominant but the percentage of bacteria in classes Alphaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria was small. The percentage of Betaproteobacteria in Cd0 sludge, which is predominant in nutrient removal system, was markedly greater than in Cd5 sludge (30.7% vs. 2.1%, respectively). Several bacteria known to be helpful to nutrient removal in activated sludge, such as Z. ramigera (floc-forming bacterium), R. gelatinosus (phosphate-accumulating organism) and N. communis (nitrifier) could not be found in Cd5 sludge. The results of transitions in microbial species were in agreement with the results for nutrient removal efficiency, as evaluated by effluent water quality. Filamentous bacteria, including T. nivea (10.0%), T.x eikelboomii (15.1%) and H. hydrossis (4.2%), were the predominant species of activated sludge in A2O system if the influent wastewater contained heavy metal cadmium.

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