novel denitrifying bacteria pseudomonas stutzeri strain … no4_p403-415_oct-de… · (ik-vk)...

Chemical Industry & Chemical Engineering Quarterly

Available on line at Association of the Chemical Engineers of Serbia AChE www.ache.org.rs/CICEQ

Chem. Ind. Chem. Eng. Q. 25 (4) 403−415 (2019) CI&CEQ

403

ANA VIDAKOVIĆ1

OLJA ŠOVLJANSKI1 DAMJAN VUČUROVIĆ1

GORDANA RACIĆ2 MILAN ĐILAS3

NATAŠA ĆURČIĆ4 SINIŠA MARKOV1

1University of Novi Sad, Faculty of Technology, Novi Sad, Serbia

2Faculty of Environmental Protection, Educons University,

Sremska Kamenica, Serbia 3Institute of Public Health of

Vojvodina, Centre for Microbiology, Novi Sad, Serbia

4Institute of Food Technology, University of Novi Sad, Novi Sad,

Serbia

SCIENTIFIC PAPER

UDC 578/579:66.095.828.504

NOVEL DENITRIFYING BACTERIA Pseudomonas stutzeri STRAIN D1 – FROM ISOLATION TO THE BIOMASS PRODUCTION

Article Highlights • A novel aerobic denitrifier, P. stutzeri D1 was isolated • P. stutzeri D1 has great capability to fully reduce 3g/L of nitrate in aerobic conditions • The optimal conditions for biomass scale-up was determined • The scale-up of biomass production of P. stutzeri ATCC 17588 and D1 strain was

performed Abstract

An aerobic denitrifier was newly isolated and identified by VITEK® 2 Compact System and MALDI-TOF MS as P. stutzeri strain D1. Sequence amplification indicates that the denitrification genes napA, nirS, norB and nosZ are present in a novel strain D1, as well as in reference strain ATCC 17588. Strain D1 had capability to fully remove 3 g/L of nitrate (as KNO3) in 48 h, while the reference strain completed this task in 60 h. Single factor experiments indicate that the optimal conditions for biomass production were: temperature of 30 °C, pH value of 7 and inoculum volume of 5 vol.%. Scaling up of biomass production of both denitrifiers was successfully performed in 3 and 7 L laboratory biore-actors by reaching 9 log CFU/mL of the viable cells. The results demonstrate the feasibility of using investigated P. stutzeri strains in denitrification pro-cesses and the simplicity of the up-scaling of biomass production for the treat-ment of large areas contaminated with nitrate.

Keywords: Pseudomonas stutzeri, denitrification, identification, scale-up, biomass production.

Although the nitrogen cycle has been spon-taneously and continuously carried out in nature for about 2.7 billion years, there has been a significant increase in nitrate concentration due to expansion of industrialization over the past several decades [1]. Moreover, progressively frequent use of inorganic pesticides and fertilizers in agriculture, as well as dis-charge of insufficiently treated wastewaters into river flows, has led to nitrate accumulation in the biosphere [1,2]. Accumulation of nitrate in water usually results in eutrophication and deterioration of water quality [3]. It has been proven that high level of nitrate in drinking water may cause human health problems such as Correspondence: A. Vidaković, University of Novi Sad, Faculty of Technology Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia. E-mail: [email protected] Paper received: 11 January, 2019 Paper revised: 15 May, 2019 Paper accepted: 2 July, 2019

https://doi.org/10.2298/CICEQ190111018V

methemoglobinemia in infants and stomach cancer in adults [4]. Also, high concentration of nitrate in groundwater may induce an increase in nitrate levels in various buildings, leading to degradation of mat-erials due to salt crystallization [5].

Generally, there are three methods for removing nitrate, including physical, chemical and biological, although physical and chemical methods do not eli-minate nitrate and nitrite completely from contamin-ated sites [6]. Consequently, the most efficient way of nitrate elimination from contaminated areas is usage of biological processes, such as denitrification, thanks to its selectivity and ability of total nitrate removal [4]. Biological denitrification has been traditionally defined as a process of reducing nitrates and nitrites to nitro-gen oxides and molecular nitrogen under anaerobic conditions, even though this process also occurs under aerobic conditions if it is driven by heterotrophic aerobic denitrifiers [7]. A denitrification process man-aged under aerobic conditions reduces its cost and

A. VIDAKOVIĆ et al.: NOVEL DENITRIFYING BACTERIA P. stutzeri… Chem. Ind. Chem. Eng. Q. 25 (4) 403−415 (2019)

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complexity. For this purpose, extensive studies have been conducted in terms of isolation and character-ization of novel heterotrophic denitrifies, capable of complete nitrate reduction under aerobic conditions [7-9].

According to their denitrification potential, the following members of the Pseudomonas genus are especially distinguished: P. aeruginosa, P. stutzeri, P. picketti, P. mallei, etc. [10]. The latest studies in the field of aerobic denitrification usually include Pseudo-monas stutzeri as a model microorganism due to its ability of nitrate reduction up to molecular nitrogen [7,9,10]. P. stutzeri is commonly isolated from ground-water, river, sea, wastewater [11-13], as well as from sludge and the ground [7,12]. Also, recent studies have been focused on enzyme systems and corres-ponding genes involved in biological denitrification processes in aerobic conditions [8,9,14-16]. Gener-ally, the reduction of nitrate to molecular nitrogen is catalyzed by the following enzymes: nitrate reduct-ase, nitrite reductase, nitric oxide reductase and nit-rous oxide reductase [17]. The investigation of these reductases is quite restricted and analyses of gene fragments of the reductases from the genome of denitrifiers are necessary for understanding the pos-sible denitrification pathway.

Available scientific papers in this field mostly deal with the efficiency of denitrification process driven by P. stutzeri concerning the influence of C/N ratio, pH value, temperature, dissolved oxygen and addition of carbon source, etc. [8,18]. It is confirmed that on a laboratory level P. stutzeri can be successfully used in wastewater treatment with 99% efficiency in rem-oving 200 mg/mL NO3 [11]. Also, the reference strains of P. stutzeri have verified their effectiveness in bio-cleaning of cultural heritage materials on a laboratory level and restricted zones on the objects [19-21].

When denitrifiers are used in wastewater treat-ment, there are no complicated preparation proce-dures that precede the treatment. Namely, when the inoculum of the chosen denitrifier is inserted in the tank for wastewater treatment, the growth and multi-plication of cells spontaneously occurs, because the wastewaters are naturally rich in organic and inorg-anic substances [11]. These conditions are favorable for cell growth and metabolic activities, so the process of nitrate removal can be carried out for a long period of time. On the other hand, when it comes to the application of denitrifiers in some other areas, such as cultural heritage objects, it is necessary to obtain enough active biomass for the bioremediation treat-ment, because the cells are usually applied onto the nutrient-poor substrates (wall paintings, bricks, stone

materials, etc.). For example, the suspension of denit-rifier biomass is used as a constitutive part of bio-active poultices [21] or it is directly applied onto the cultural heritage objects [19]. About 2 L of denitrifier suspension (1010 CFU/mL) is necessary to efficiently remove nitrate from one square meter of wall painting [20]. It is obvious that for an implementation of effect-ive denitrifiers, such as P. stutzeri, on wider contam-inated areas, it is necessary to produce sufficient bio-mass. In the available literature, there are no papers focusing on the optimization of bioprocess para-meters for the biomass production of denitrifiers, or the attempts of scaling up the biomass production.

Toward this end, the aim of this work was to present a procedure of isolation, selection and ident-ification of efficient novel denitrifier P. stutzeri strain D1. Moreover, the influence of the selected biopro-cess parameters on the biomass production was investigated and the biomass of the reference strain P. stutzeri ATCC 17588 and isolate P. stutzeri D1 was produced in laboratory-scale bioreactors for the first time.

EXPERIMENTAL

Sampling

In June 2015, water samples were collected from the Danube River and Danube-Tisa-Danube (DTD) Canal in Novi Sad, Serbia. In order to isolate denitrifies, the sampling was performed three times from five sites in the Danube (ID-VD) and DTD Canal (IK-VK) (Figure 1). Average values of physicochem-ical parameters of the water samples are presented in Table 1. The analysis of the water samples was per-formed according to the Standard methods SR EN 1899-2:2002 for determination of BOD, SR EN ISO 6878:2005 for determination of total phosphorus, ISO 15705:2002 for determination of COD, SR EN ISO 11905-1:2003 for determination of nitrogen from nit-rate. pH value and temperature of the samples were measured with Hanna HI 99161 pH/temperature meter. All samples were transported to the laboratory at 4 °C.

Isolation and strain selection

Each water sample was homogenized and an aliquot of 0.1 ml was spread on PCA (HiMedia, Mum-bai, India) plates (n = 3) and incubated at 30 °C for 72 h. After the incubation period, bacterial colonies with different macromorphological characteristics were purified via repeating streaking with sterile platinum loop onto fresh PCA plates. The obtained isolates were stored on the PCA slant medium at 4 °C.


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Figure 1. Sampling sites in the Danube and DTD Canal.

The aim of the isolation procedure was to find bacteria with similar characteristics to the P. stutzeri ATCC 17588, which has been reported as a model microorganism for studying the denitrification process due its high capacity of reducing nitrate to molecular nitrogen [10]. Besides, it is well known that wild strains of the same bacteria are usually more adap-tive to different conditions and often more efficient than the reference strain.

With a purpose to obtain an isolate with similar characteristics to P. stutzeri ATCC 17588, a model denitrifier, all isolates were subjected to catalase and oxidase tests, KOH string test [22] and Gram straining.

Moreover, in order to select isolates closely rel-ated to P. stutzeri ATCC 17588, purified isolates were streaked on the chosen selective culture media MacConkey Agar (Torlak, Belgrade, Serbia) and Cet-rimide Agar (Merck, Darmstadt, Germany).

Bacterial strain identification

All selected bacterial strains were stored in Nut-rient Broth (HiMedia, Mumbai, India) with the addition of glycerol (Lach-ner, Neratovice, Czech Republic) as a cryoprotectant and kept in the freezer for ultra-low temperatures (Snijders Labs, Tilburg, the Nether-lands) at -70 °C. Prior to identification procedure, the chosen bacterial strains were grown on PCA plate at 30 °C for 24 h. Thereafter, 3 mL of bacterial suspen-sions in sterile physiological solution were made to reach the concentration of approximately 3×108 CFU/ml (corresponding to McFarland No.1 standard). Then, VITEK® 2 GN ID cards were inserted into pre-pared suspensions and the isolates were identified by VITEK® 2 Compact System (BioMérieux, Carpone, France).

Also, the reference culture (P. stutzeri ATCC 17588) and the isolate identified as P. stutzeri (in further text P. stutzeri D1) by VITEK® 2 Compact Sys-tem were subjected to the additional identification with MALDI-TOF analysis.

Tested cultures were grown overnight on Col-umbia agar +5% sheep blood (BioMérieux, Carpone, France) at 37 °C and analyzed using the standard Bruker's direct transfer sample preparation procedure for MALDI-TOF MS. Single bacterial colony was spot-ted directly onto a 96-spot MALDI target plate (Bruker Daltonics, Bremen, Germany), allowed to dry, and immediately overlaid with 1.0 μL of the matrix solution (Bruker Matrix HCCA; α-cyano-4-hydroxycinnamic acid).

MALDI-TOF mass spectra were obtained by using MicroflexLT/SHBioTyper spectrometer (Bruker Daltonics, Billerica, MA, United States) equipped with a nitrogen laser (337 nm) under control of Flexcontrol software ver. 3.1 (Bruker Daltonics, Billerica, MA, United States). Spectra acquisition in the mass range of 2 to 20 kDa were collected using the Auto Execute option by accumulating 240 laser shots (laser

Table 1. Physicochemical parameters of the water samples of Danube River and DTD Canal

Sampling site COD (mg/L) BOD (mg/L) N-NO3 (mg/L) Ptotal (mg/L) pH Temperature (°C)

ID 10.6 2.1 10.21 0.63 7.85 21.4

IID 8.6 2.0 10.43 0.33 8.12 21.3

IIID 9.4 1.9 9.61 0.47 8.00 21.4

IVD 9.1 2.2 9.41 0.39 7.95 21.2

VD 11.7 2.1 9.02 0.52 8.03 21.5

IK 25.4 2.5 10.44 0.77 8.15 21.8

IIK 18.6 2.2 10.29 0.68 7.88 21.7

IIIK 17.4 2.0 9.52 0.57 7.96 21.5

IVK 18.8 2.1 10.08 0.63 8.04 21.5

VK 20.9 2.5 10.12 0.70 8.12 21.7


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frequency, 60 Hz; ion source 1 voltage, 19.9 kV; ion source 2 voltage, 18.53 kV; lens voltage, 6 kV) acquired at 30–40% of maximum laser power.

Assessment of aerobic denitrification capability

In order to determine the possibility of complete reduction of nitrate to molecular nitrogen in aerobic conditions, P. stutzeri, reference strain and isolate, were grown overnight on PCA plates at 30 °C. An aliquot of 0.2 ml of freshly prepared suspensions (approx. density 3×108 CFU/ml) was transferred into 3 ml of Nitrate Broth (DifcoTM Nitrate Broth, Becton, Dickinson and Company, France). Kinetics of aerobic denitrification was monitored at 30 and 37 °C for 60 h.

Amplification of denitrification genes

Genomic DNA of both P. stutzeri strains was extracted using a DNA Isolation Kit (Agilent Techno-logies, Santa Clara, CA), according to the manufac-turer’s instruction. DNA quantity was measured spec-trophotometrically by NanoDrop™One Microvolume UV-Vis spectrophotometer (Thermo Scientific). To confirm the presence of denitrification genes (napA, nirS, norB and nosZ ) in genome of P. stutzeri ATCC 17588 and D1 strains, PCR amplification was carried out with gene-specific primers (Table 2) in 25 μl react-ions with 1 μl of DNA template, 12.5 μl of 2x PCR Master Mix (Thermo Scientific, Latvia) and 0,6 µM of each primer. All PCR reactions were performed in duplicate. The napA gene was amplified under the fol-lowing conditions: 5 min at 95 °C (initial denaturation); 35 cycles for 30 s at 95 °C, 40 s at 59 °C (primer annealing), 1 min at 72 °C (primer extension) and 10 min of final extension at 72 °C. For the amplification of other denitrification genes nirS, norB and nosZ, PCR was carried out under the following conditions: 95 °C for 5 min; followed by 35 cycles of denaturation at 95 °C for 40 s, annealing at X °C (Table 2) for 40 s, extension at 72 °C for 1.5 min; followed by the final extension at 72 °C for 10 min. Visualization of the PCR products was carried out by the capillary Lab- -on-a-Chip electrophoresis at 2100 Bioanalyzer (Agil-ent Technologies, USA).

Impact of selected process parameters on maximizing the biomass production

In order to examine the influence of tempera-ture, initial pH value and inoculum volume on the bio-mass production, the one factor at a time method was employed. The values of selected factors were set up as follows: temperature (22, 30 and 37 °C), pH value (5, 6 and 7) and inoculum volume (1, 3, 5 and 10 vol.%). When one parameter was varied, the other two were constant: temperature at 30 °C, pH at 7 and inoculum volume at 10%.

Suspensions of 24-h cultures of P. stutzeri ATCC 17588 and P. stutzeri D1 were transferred into Erlenmeyer flasks (300 ml) containing 90 ml of Nitrate Broth. Biomass production was carried out under the described experimental conditions. The sampling was performed at six chosen intervals: 0, 5, 12, 18, 24 and 32 h.

Scale-up of biomass production

The biomass production of the reference and isolate strain of Pseudomonas stutzeri was carried out in 3 L laboratory bioreactor (Biostat® A Plus, Sar-torius AG, Germany) with 2 L of cultivation media, as well as in 7 L laboratory bioreactor (Chemap AG CH- -8604, Switzerland) with 5 L of cultivation media. The 3 L laboratory bioreactor is equipped with two Rush-ton turbines with parallel configuration. Beside the two Rushton turbines, the 7 L bioreactor is equipped with four baffles with internally set up. Cultivation media for biomass production consists of: KNO3 (2 g/L) and peptone (4 g/L) in the case of P. stutzeri ATCC 17588 and glucose (1 g/L), KNO3 (3 g/L) and peptone (4 g/L) in the case of P. stutzeri D1 [39]. Prior to cultivation process, bioreactors with the appropriate culture media were sterilized by autoclaving at 121 °C and pressure of 2.1 bar for 15 min. Inoculum was pre-pared by double passaging of the cultures on PCA plates. Single colonies were suspended in 9 ml of sterile physiological solution to reach approximately 3×108 CFU/ml (McFarland standard No. 1). The pre-pared suspension was then transferred into 450 ml of

Table 2. Target gene, primer sequences (F-forward, R-reverse) and primer annealing (°C) used for polymerase chain reaction

Gene Description Primer sequence Primer annealing (°C) Ref.

napA Periplasmatic nitrat reductase F 5'- TCTGGACCATGGGCTTCAACCA-3' R 5'- ACGACGACCGGCCAGCGCAG-3'

59 [8,30]

nirS Nitrite reductase F 5'- CCTA(Y)TGGCCGCC(R)CA(R)T-3' R 5'- CGTTGAACTT(R)CCGGT-3'

52 [8]

norB Nitrogen monoxide reductase F5'- CG(N)GA(R)TT(Y)CT(S)GA(R)CA(R)CC-3'R 5'- C(R)TA(D)GC(V)CCR(W)AGAA(V)GC-3'

54 [8]

nosZ Nitrogen suboxide reductase F 5'- CCCGCTGCACACC(A/G)CCTTCGA-3' R 5'- CGTCGCC(C/G)GAGATCTCGATCA-3'

58 [8]


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the culture media optimized for P. stutzeri ATCC 17588 and P. stutzeri D1 and 5 vol.% of diluted sus-pension was added into bioreactors with sterile cul-ture media. The biomass production was carried out in batch mode, under aerobic conditions for 32 h, at the temperature of 30 °C and agitation rate 120 rpm. At ten selected time intervals (0, 3, 5, 9, 12, 16, 19, 24, 30 and 32 h) the sampling of cultivation broth was performed. The selection of the main time intervals for sampling (0, 5, 12, 24 and 32 h) was done in accor-dance to the growth curve obtained during the inves-tigation of the impact of selected process parameters on maximizing the biomass production, while the other sampling times were chosen in such a way to obtain at least three samples from lag, log and statio-nary growth phase.

Analytical methods

During the assessment of the denitrification cap-ability the formation of gas was tracked by its entrap-ment in Durham tubes, while the bacterial growth was estimated by comparison of turbidity to McFarland nephelometer. To assess the concentration of nitrate and nitrite in test tubes, Quantofix® test stripes (Mach-erey-Nagel, Düren, Germany) were used. Further-more, Griess reagents were used for qualitative det-ection of nitrite (the limit of detection 2.5 μM) while the presence of ammonia was detected by Nessler reag-ent (the limit of detection 0.1 mg/mL). All experiments were performed in triplicate (n = 3).

The total number of viable bacteria in all taken samples of cultivation broths was determined by spreading of 100 µL of the sample on the PCA plates that were afterwards incubated at 30 °C for 24 h. After the incubation period, the grown colonies were counted.

In experiments in laboratory bioreactors, the

separation of the P. stutzeri cells from the rest of the cultivation broth was carried out by centrifugation at 10000 rpm for 15 min (Hettich Rotina 380 R, Ger-many). The obtained supernatants were collected and subjected to analysis of residual sugar and nitrogen content.

Also, the cultivation broth samples were also analyzed in terms of total nitrogen content by stan-dard Kjeldahl method (Buchu K-314 Kjeldahl line digestion unit) [23]. In the case of P. stutzeri D1, resi-dual sugar content (glucose) in supernatant was det-ermined by Megazyme D-glucose HK assay kit (Megazyme, Wicklow, Ireland). For this purpose, UV- -1800, UV-Vis spectrophotometer (Shimedzu, Kyoto, Japan) at 340 nm was used.

RESULTS AND DISCUSSION

Results of isolation and selection of denitrifying bacteria

A total of 140 colonies (smooth, butyraceous and pale in color) from all sampling sites (Figure 1) were chosen since they had similar macromor-phological characteristics to P. stutzeri ATCC 17588 [10]. All colonies were streaked onto MacConkey and Cetrimid Agar and subjected to further morphological and biochemical analysis. Based on the obtained results only 10 out of 140 isolates closely matches the characteristics of the reference strain (Table 3).

P. stutzeri ATCC 17588 is a rod-shaped, Gram- -negative, catalase-positive and oxidase-positive bac-terium. This bacterium is lactose non-fermenting and it grows well on the MacConkey agar. Furthermore, P. stutzeri ATCC 17588 neither grows nor produces pig-ments on the Cetrimide agar, which is very useful for

Table 3. Biochemical, morphological and culturable characteristics of the selected isolates; the samples are labeled as follows: roman numerals indicate the sampling site (Fig. 1) following by D for the Danube or C for the DTD Canal and /arabic number for differentiation of the colonies from the same sampling site

Isolate Oxydase test

Catalase test

KOH test

Gram straining

Shape MacConkey Cetrimide

Growth Lactose Growth Pigment

P. stutzeri ATCC 17588 + + + - Rod + - - - ID/1 + + + - Rod + + - -

ID/2 + + + - Rod + - - -

ID/3 + + + - Rod + + + -

IID/4 + + + - Rod + - - -

IVD/3 + + + - Rod + + - -

VD/1 - + + - Rod + - - -

IVC/2 + + + - Rod + + - -

IVC/3 + + + - Rod + - - -

IVC/4 + + + - Rod + - + -

VC/1 + + + - Rod + + + -


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differentiation from Pseudomonas aeruginosa [10]. According to the gained results presented in Table 3, only 3 isolates (ID/2, IID/4 and IVC/3) have the same characteristics as the reference strain. All other iso-lates are also rod-shaped, Gram-negative, catalase- -positive and have ability to grow on MacConkey agar, but they have different results for oxidase test, lactose fermentation or growth on Cetrimide Agar (Table 3).

Identification of potential denitrifying bacteria

All selected isolates were subjected to identific-ation by VITEK® 2 compact system. The gained results of identification are presented in Table 4.

According to the obtained results, even six isolates are identified as Aeromonas sobria, two as Aeromonas hydrophila, one as Citrobacter freundii and only one as Pseudomonas stutzeri (Table 4). Generally, members of Aeromonas genus are incomplete denitrifiers; therefore, they have ability to reduce nitrate to nitrite or to ammonia [24]. Moreover, bacteria from the Aeromonas genus are pathogenic for humans (A. hydrophila) and fishes (A. sobria), so they should not be used for bioremediation processes. Similarly, isolate VD/1 is identified as C. freundii, bacterium responsible for the conversion of nitrate to nitrite in various niches [25]. However, C. freundii is a well-known opportunistic pathogen; hence its usage in denitrification process is restricted. As it can be seen from Table 4, only one isolate is identified as P. stutzeri (in further text P. stutzeri D1). According to the available literature, P. stutzeri

belongs to the group of complete denitrifiers and it is one of the most efficient bacteria in denitrification processes [10,11,24].

In order to confirm the identification by VITEK® 2 compact system, P. stutzeri D1, as well as the refer-ence strain P. stutzeri ATCC 17588 were subjected to MALDI-TOF analysis. Namely, techniques based on protein analysis, proteomics, are currently applied in bacterial identification and taxonomy and within them matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) has been the most promising technique [26]. It has been proven that MALDI-TOF MS is a powerful technique for rapid and accurate bacteria identification, including Pseudomonas genus [27]. The obtained results from MALDI-TOF analysis are given in Table 5.

Based on the results of the MALDI-TOF MS analysis (Table 5), it is evident that the both tested microorganisms are identified as P. stutzeri with a very high score value for the best match and even for the organism second best match. The results obtained by MALDI-TOF MS analysis are in excellent correl-ation with the identification obtained by VITEK® 2 compact system.

Capability of aerobic denitrification

As shown in Table 6, both strains start the pro-cess of denitrification after 24 h of the incubation period, which can be noticed by the reduction of nit-rate and nitrite content, as well as by the formation of gas in Durcham tubes. In the case of P. stutzeri D1, the complete reduction of nitrate (3 g/L as KNO3) was

Table 4. Identification of the selected isolates by VITEK® 2 Compact System

Isolate Identified microorganism Probability of identification (%) Confidence level

ID/1 Aeromonas sobria 98 Excellent identification ID/2 Aeromonas sobria 98 Excellent identification ID/3 Aeromonas hydrophila 95 Very good identification IID/4 Pseudomonas stutzeri 99 Excellent identification IVD/3 Aeromonas sobria 98 Excellent identification VD/1 Citrobacter freundii 99 Excellent identification IVC/2 Aeromonas sobria 94 Very good identification IVC/3 Aeromonas sobria 97 Excellent identification IVC/4 Aeromonas sobria 98 Excellent identification VC/1 Aeromonas hydrophila 95 Very good identification

Table 5. Results of the MALDI-TOF MS identification; meaning of the score values: I) 2.300-3.000 – highly probable species identific-ation; II) 2.000-2.299 – secure genus identification, probable species identification; III) 1.700-1.999 – probable genus identification; IV) 0.000-1.699 – not reliable identification

Tested microorganism Organism (best match) Score value Organism (second best match) Score value

P. stutzeri ATCC 17588 Pseudomonas stutzeri 2.278* Pseudomonas stutzeri 2.173 P. stutzeri D1 Pseudomonas stutzeri 2.226 Pseudomonas stutzeri 2.116


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observed after 48 h. On the other hand, P. stutzeri ATCC 17588 requires more time to finish the denitri-fication process, resulting in complete nitrate reduct-ion after 60 h. In both cases, the process of denitri-fication was independent of examined temperatures. According to the obtained results, P. stutzeri D1 was more efficient in removing the nitrate from nitrate broth over time, compared to the reference strain. A high efficiency of denitrification process (99.24%) at 30 °C driven by P. stutzeri has been noticed even under high oxygen atmosphere (92%), although this process lasted for 96 h [28]. In addition, P. stutzeri ATCC 17588 and D1 strain have capability to fully remove high concentrations of nitrate under stationary conditions without accumulation of nitrite as inter-mediate (Table 6), which is very important for use in bioremediation processes. A similar denitrification pattern was also observed in the case of P. stutzeri ASM-2-3, isolated from the Ariake Sea Tideland, Japan [29].

PCR amplification of denitrification genes

In this study, four crucial denitrification genes (napA, nirS, norB and nosZ) were successfully amp-lified from the strains ATCC 17588 and D1 of the P. stutzeri (Figure 2). Using the napA primer pair, 875 and 850 bp fragments were amplified from strains

ATCC 17588 and D1, respectively. The presence of napA gene, which regulates the activity of periplasmic nitrate reductase (napA), is essential for the reduction of nitrate to nitrite under the aerobic conditions; therefore, it is commonly used as a functional marker for identification of aerobic denitrifiers [14]. The obtained results (Figure 2a) are consistent with other studies related to the identification of aerobic denit-rifying bacteria, based on the amplification of napA gene [8,14,30].

As shown in Figure 2b, the 1060 and 1065 bp nirS amplification products were observed in the tested ATCC 17588 and D1 strains, respectively. Results show the existence of nirS gene, which regul-ates the activity of nitrite reductase (nirS) during the reduction of nitrite to nitrogen monoxide. Many rep-orts have shown that the fragment length of the nirS gene varies between 800 and 850 bp [6,8,31]. How-ever, according to the relevant literature [32,33], this fragment can be significantly shorter (425 bp). The similar study conducted by Zhao and coworkers [9] showed that the size of the amplified nirS gene frag-ment is above 1000 bp, as the fragments obtained in this study. The large variations in the length of the amplified nirS fragment happen most likely due to the use of complex degenerate primers.

Table 6. Kinetic of denitrification at 30 °C and 37 °C for P. stutzeri ATCC 17588 and P. stutzeri D1

Analysis time (h)

Temperature (°C)

Microorganism Turbidity (McFarland No.)

Gas (+/-)

Quantofix® test stripes Griess reagents

Nessler reagent

Nitrate (mg/L) Nitrite (mg/L) Nitrite (+/-) Ammonia (+/-)

8 30 P. stutzeri ATCC 17588 3 - 500 80 + - P. stutzeri D1 1 - 500 80 + -

37 P. stutzeri ATCC 17588 3 - 500 80 + - P. stutzeri D1 3 - 500 80 + -

24 30 P. stutzeri ATCC 17588 3 + 500 80 + - P. stutzeri D1 3 + 50 1 + -

37 P. stutzeri ATCC 17588 3 + 50 1 + - P. stutzeri D1 3 + 50 1 + -

34 30 P. stutzeri ATCC 17588 3 + 50 1 + - P. stutzeri D1 3 + 10 1 - -

37 P. stutzeri ATCC 17588 3 + 50 1 + - P. stutzeri D1 3 + 50 1 + -

48 30 P. stutzeri ATCC 17588 3 + 10 1 - - P. stutzeri D1 3 +


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To examine whether aerobic denitrification is catalyzed by the nitrogen monoxide reductase (nor), enzyme that regulates the reduction of nitrogen mon-oxide to nitrogen suboxide, the norB gene of the tested strains was amplified. The 675 bp norB ampli-fication product was observed from both examined strains (Figure 2c). The obtained results are in correl-ation with studies related to the amplification of the norB gene [8,33,34].

The final step in the denitrification process imp-lies the activity of nitrogen suboxide reductase (nos) that controls the reduction of nitrogen suboxide to molecular nitrogen. The activity of nos may be sup-pressed in the presence of oxygen, although in some microorganisms the activity of this enzyme is inde-pendent of the oxygen concentration [34]. In this study, 1230 and 1225 bp of the nosZ gene fragments were amplified from the strains ATCC 17588 and D1, respectively (Figure 2d). According to the available literature, the size of the amplified nosZ gene frag-ment should be about 300 bp [8,33,34]. There are indications that the size of the amplified fragment of the nosZ gene depends on the designs of the used complex degenerate primers, as well as PCR con-ditions [35]. Consequently, the large fragments of above 1000 bp of the nosZ gene could be also ampli-fied [9,35], as the fragments obtained in this study.

The results presented in this paper indicate that both tested strains of P. stutzeri possess all four key enzymes (nap, nir, nor and nos), necessary to per-form complete denitrification process in aerobic con-ditions.

The influence of temperature on biomass production

Temperature is one of the most important fac-tors affecting the growth of microorganisms, as well as the efficiency of the denitrifying process. In this study, the influence of temperature on growth of P. stutzeri ATCC 17588 and D1 strain (Figures 3 and 4) was investigated.

At 37 °C, both strains had greater growth rate during the exponential phase, reaching the 9log CFU/ /mL after 24 h. When temperature of 30 °C was examined, the growth was slightly slower in the case of P. stutzeri D1, but after 24 h when the stationary phase starts, the number of viable cells of P. stutzeri ATCC 17588 and D1 strain was approximately the same (9log CFU/mL). The bioprocess done at lower temperature (22 °C), in the case of both strains of P. stutzeri, resulted in the biomass yield between 8 and 9 log CFU/mL. According to the obtained results, the biomass production of reference strain followed the same growth trend at all examined temperatures, Fig-ure 3a. On the other hand, from the results presented in Figure 4a, it can be noticed that D1 strain did not reach the stationary phase at temperature of 22 °C even after 32 h, while the stationary phase in the bioprocess performed at 30 and 37 °C has been achieved after 24 h. The production of biomass at 30 °C should be chosen over 37 °C, because it is simpler and cost efficient to carry out a process at lower temperatures. Toward these results, 30 °C might be an optimal temperature for the production of biomass of the chosen denitrifiers. The obtained results are consistent with P. stutzeri XL-2, P. stutzeri ZF31 and Bacillus YX-6 [6,8,9].

Figure 2. Lab-on-chip electrophoresis of the products from denitrification genes: a) napA; b) nirS; c) norB; d) nosZ; S: 25-1000bp DNA

ladder; lane1 - P. stutzeri strain ATCC 17588; lane 2 - P. stutzeri strain D1.


411

Figure 3. Influence of temperature (a), pH value (b) and inoculum

volume (c) on the growth of P. stutzeri ATCC 17588.

Figure 4. Influence of temperature (a), pH value (b) and inoculum

volume (c) on the growth of P. stutzeri D1.

The influence of an initial pH value on biomass production

According to Lalucat and coworkers [10], none of the P. stutzeri strains is tolerant to acidic conditions and does not grow at pH values lower than 4.5. Con-sequently, the experiments were set up to monitor the influence of pH values 5, 6 and 7 on the biomass pro-duction.

The results presented in Figures 3b and 4b clearly indicate that the initial pH value has no signi-ficant influence on the biomass production of selected P. stutzeri strains. Independent of the initial pH value, the number of viable cells of the tested bacteria at the end of the exponential phase reaches around 9log CFU/mL. Thus, the optimal initial pH value for the

production of biomass might be 7, as it is in nutrient media (nitrate broth) according to the producer requi-rements. In this way, there is no need for the correct-ion of pH value at the starting point of the bioprocess. Moreover, the optimal pH value for the denitrification process is also neutral (in range of 7-7.5), which can be useful for further comparative studies [17]. The obtained results are consistent to the previous work of Bosch-Roig and coworkers [36] who also emphasized the ability of P. stutzeri 5190 to grow at basic pH values (from 5 to 10). In addition, this group of authors confirmed that the addition of urea to the growth medium leads to better tolerance to acid pH of P. stutzeri by the detection of genes encoding urea transporters (urtABCDE) and urease (ureA, ureB, ureC, ureD and ureEFG) in P. stutzeri 5190 genome [36].


412

The influence of inoculum volume on biomass production

Among varied % of inoculums, 5 vol.% of seed culture leads to the maximum (approx. 9.8log CFU/ /mL) of P. stutzeri D1 biomass production (Figure 4c). In the case of P. stutzeri ATCC 17588, maximum number of cells (approx. 9log CFU/mL) was achieved with the addition of 3, 5 and 10 vol.% of the seed culture into nutrient medium (Figure 3c). When the lowest inoculum volume (1%) was used, a small dec-line in the cell number could be noticed (Figures 3c and 4c).

The use of high inoculum volumes (above 10%) in biomass production can cause some difficulties during the process. More precisely, the greater num-ber of cells in production media the more competition for substrate occurs and vice versa [37]. Taking into account the gained results, the selected inoculum size in further analysis for both tested microorganisms is set at 5 vol.%.

Biomass production in laboratory bioreactors

In general, fermentation scale-up is aimed at the manufacture of larger product quantities with a simul-taneous increase or with a consistency of specific yields and product quality [38]. For each individual process, it is necessary to identify and optimize the most relevant process parameters influencing the product yield, so they could be maintained constant as far as possible during the scale-up process. After optimizing the chosen process parameters (temperature, initial pH value and inoculum volume), as well as the nutrient medium composition [39], the process of biomass production of the chosen denitrifiers was scaled up.

In both cases, an obvious lag phase between 0 and 5 h was noticed, indicating the period of adap-tation to the liquid medium (Figures 5a and 6a). In a 3 L laboratory bioreactor, a prominent exponential phase was observed between 5 and 24 h, reaching the maximum number of P. stutzeri ATCC 17588 and P. stutzeri D1 cells (about 9log CFU/mL) at the end of this phase. In a 7 L laboratory bioreactor, the phase between 5 and 24 h is slightly retarded, resulting in a small decline in the number of cells at the end of the exponential growth phase (about 8.8log CFU/mL). A reason for this phenomenon could be explained with the changed geometric conditions and impeller type between the two bioreactors, leading to less favorable mixing conditions. Namely, mixing is not a critical parameter for the production of biomass of P. stutzeri ATCC 17588 and P. stutzeri D1, because they can grow in aerobic and anaerobic conditions, but it is necessary to maintain it in such a way that ensure the

availability of nutrients to all cells in a vessel. A higher mixing rate could result in stress-induced metabolic shifts or the growth retardation [38]. Nevertheless, both denitrifying bacteria reach the maximum number of cells (about 9log CFU/mL) regardless of the type of laboratory bioreactor in which the production of bio-mass was performed. After the 24 h the production of biomass should be stopped, because the cells enter stationary growth phase.

Figure 5. Changes of the cell number (a) and total nitrogen concentration (b) in the case of P. stutzeri ATCC 17588 in

laboratory bioreactors.

Monitoring the cultivation waste stream load has a huge impact on the environment protection and the cost of the bioprocess. Toward this end, residues of nutrients and metabolic products in the cultivation waste stream should always be reduced to a mini-mum.

According to the gained results presented in Figures 5b and 6b, it can be noticed that the nitrogen


413

Figure 6. Changes of the cell number (a), total nitrogen (b) and

glucose concentration (c) in the case of P. stutzeri D1 in laboratory bioreactors.

consumption starts almost immediately after the ino-culation in the case of strain D1 (Figure 6b), while there is a short lag phase of approximately 5 hours in the case of the reference strain (Figure 5b). Initially, cultivation media for P. stutzeri ATCC 17588 con-tained total nitrogen in the amount of 603 mg/mL,

while the determined amount of nitrogen at the start of cultivation of P. stutzeri D1 was 617 mg/mL. From the results shown in Figures 5b and 6b, it can be seen that during the exponential growth phase, when the growth of biomass is the most intensive, total nitrogen content in the cultivation medium considerably dec-reases. In the case of P. stutzeri D1, the consumption of nitrogen is slightly faster than during the cultivation of the reference culture. In the stationary phase, total nitrogen content remains almost constant. At the end of the process, nitrogen content in the cultivation medium for P. stutzeri ATCC 17588 in 3 L bioreactor was 110 mg/mL, with the achieved level of its con-version was 81.76%, while in the 7 L bioreactor the remain nitrogen content was 174 mg/mL, resulting in 71.14% of nitrogen conversion. In the case of P. stu-tzeri D1 the nitrogen content in the cultivation medium reached 26 and 58 mg/mL in 3 and 7 L bioreactors, respectively. The obtained results indicate that the conversion level of nitrogen content in the 3 L bio-reactor was 95.79%, while in the 7 L bioreactor total nitrogen conversion reached 90.60%.

In the culture media composition for maximizing the biomass of P. stutzeri D1 one of the components is glucose (1 g/L) [38]. According to the obtained results, it is obvious that the curve of glucose consumption (Figure 6c) completely follows the growth curve (Figure 4b). Namely, during the lag phase (0-5 h) the consumption of glucose is minimal. The concentration of glucose between 5 and 30 h significantly declines, and then, during the stationary phase, remains constant. A total consumption of glucose during the production of biomass of P. stutzeri D1 was about 0.7 mg/mL, regardless of the used laboratory bioreactor.

According to the obtained results, the scale-up of biomass production of the chosen denitrifying bac-teria was successfully carried out by achieving the consistency of the maximum number of cells of approx. 9 log CFU/ml and minimally loaded waste stream of the bioprocess. These results may be very useful when it comes to the pilot and industrial pro-duction of biomass. The production of P. stutzeri bio-mass will have a huge impact on the improving of bio-remediation processes, resulting in the removal of nit-rate from large contaminated areas. Thus, the global accumulation of nitrate would be reduced to a mini-mum, which would have a positive impact on human health and environment protection.

CONCLUSION

P. stutzeri D1 was isolated from the Danube River, Serbia. The isolate was successfully identified


414

by VITEK® 2 compact system and MALDI-TOF MS analysis. P. stutzeri D1 showed great capability in complete nitrate removal, removing 3 g/L (as KNO3) in just 48 h, while the reference strain required about 60 h. The presence of four key denitrification genes was confirmed in genome of the both strains of P. stutzeri. The optimal conditions for biomass product-ion of P. stutzeri ATCC 17588 and strain D1 were temperature of 30 °C, initial pH value of 7 and ino-culums volume of 5 vol.%. The up-scaling process of biomass production in laboratory bioreactors was suc-cessfully conducted, resulting in viable cell production of 9log CFU/mL.

Acknowledgement

The financial support of the Ministry of Edu-cation, Science and Technological Development of the Republic of Serbia (Contract No. III45008) is gratefully acknowledged.

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415

ANA VIDAKOVIĆ1 OLJA ŠOVLJANSKI1

DAMJAN VUČUROVIĆ1

GORDANA RACIĆ2 MILAN ĐILAS3

NATAŠA ĆURČIĆ4 SINIŠA MARKOV1

1Univerzitet u Novom Sadu, Tehnološki fakulteta Novi Sad, Bulevar cara

Lazara 1, 21000 Novi Sad, Srbija 2Fakultet zaštite životne sredine,

Univerzitet Educons, Vojvode Putnika 87, 21208 Sremska Kamenica, Srbija

3Institut za javno zdravlje Vojvodine, Centar za mikrobiologiju, Futoška 121,

21000 Novi Sad, Srbija 4Naučni institut za prehrambene

tehnologije, Univerzitet u Novom Sadu, Bulevar cara Lazara 1, 21000 Novi

Sad, Srbija

NAUČNI RAD

NOVA DENITRIFIKUJUĆA BAKTERIJA Pseudomonas stutzeri SOJ D1 – OD IZOLACIJE DO PROIZVODNJE BIOMASE

Novi denitrifikator je izolovan i identifikovan pomoću Vitek 2 Compact sistema i MALDI--TOF MS kao P. stutzeri soj D1. Amplifikacijom denitrifikujućih gena napA, nirS, norB i nosZ potvrđeno je njihovo prisustvo u genomu novog soja D1, kao i u genomu refe-rentne kulture P. stutzeri ATCC 17588. Pokazano je da soj D1 ima mogućnost da u pot-punosti ukloni 3 g/L nitrata (u obliku KNO3) za 48 h, a referentom soju oko 60 h. U jednofaktorijalnim eksperimentima određeni su optimalni bioprocesni uslovi za proiz-vodnju biomase ispitivanih denitrifikatora: temperatura od 30 °C, pH vrednost 7 i veličina inokuluma 5 zapr.%. Uvećanje razmera proizvodnje biomase oba ispitivana denitrifika-tora je uspešno je izvedeno u laboratorijskim bioreaktorima ukupne zapremine 3 i 7 L, pri čemu je postignut broj vijabilnih ćelija na kraju bioprocesa bio 9log CFU/mL. Posti-gnuti rezultati ukazuju na mogućnost upotrebe ispitivanih sojeva P. stutzeri u denitrifika-cionim procesima i lakoću i jednostavnost proizvodnje njihove biomase u uvećanim raz-merama što omogućava izvođenje bioremedijacionih postupaka na velikim kontaminira-nim površinama.

Ključne reči: Pseudomonas stutzeri, denitrifikacija, identifikacija, uvećanje raz-mera, proizvodnja biomase.

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