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J. Kosel, et al.
in these two real water samples di ff ered from the standard colony counting technique. Firstly, 5-fold serial dilutions were prepared and 1 mL of each dilution was poured into a screw-cap tube (20 × 150 mm) containing 10 mL of freshly autoclaved, still molten (at 50 °C in a water bath), iron-sulphite agar medium (Merk ™ , 10 g/L of pancreatic digest of casein, 1 g/L of Na 2 SO 3 , 0.1 g/L of iron powder and 2% agar). During anaerobic incubation, the tubes were almost completely fi lled with media and were tightly sealed with screw caps. Additionally, the iron inside the media combined with any dissolved oxygen and thus pro- vided an anaerobic environment. The strongest serial dilution that still proved positive (black colouration) after a 7 days long incubation at 37 °C was determined as the concentration of sulphate reducing bac- teria and was presented in Log 10 CFUmL − 1 according to its logarithmic order of dilution. All values reported in this paper are the mean of at least two independent biological treatments and three replicates for each treatment. The average values and standard deviations are given. To evaluate the impact of cavitation on the overall growth reduc- tion, a speci fi c decay rate constant ( μ ) was calculated as follows:
direction, but with a slight angle to the camera. The camera was fo- cused perpendicularly to the rotor ’ s teeth in axial direction of the rotor. The motor ’ s power and energy consumption were monitored with a Power Analyzer Norma 4000.
2.2. Microbiological measurements
2.2.1. Working suspension preparation B. subtilis ATCC ® 6633 ™ acquired from the Veterinary Faculty at the University of Ljubljana was cultured at 37 °C on standard count agar plates (SCA, Merck ™ ; 3,0 g/L of meat extract, 5,0 g/L of peptone from casein, 5 g/L of sodium chloride, and 12 g/L of agar). For the hydro- dynamic cavitation experiments colonies from fresh culture plates (24 h old) were harvested, suspended and diluted in sterile Ringer solution (Merck ™ ) until a concentration of around 5 Log 10 CFU mL − 1 (high initial bacterial titer) or 2 Log 10 CFUmL − 1 (low initial bacterial titer) was achieved. Bacterial concentration was determined by optical den- sity measurements at 650 nm (OD 650 ). The prepared suspension was then stored on ice in a Styrofoam box and just before the cavitation run, the bacterial culture was further diluted 100 and 10,000 times to a fi nal working concentration of around 1.0 × 10 5 CFU mL − 1 (high initial titer) and of around 1.0 × 10 2 CFUmL − 1 (low initial titer). The sample volume for the rotation generator was 2 L. 2.2.2. Sampling and quanti fi cation Apart from the experiments with the B. subtilis working suspension, 5 L samples were also taken from real technological process waters isolated from a board paper mill plant. These samples were collected from the individual pool (recycled water or RW) or from the central pool (central recycled water or CRW) of an enclosed water recycle system and were analysed for the presence of anaerobic sulphate re- ducing bacteria, aerobic bacteria, bacterial spores as well as for yeasts and moulds (Fig. 4). The original pH value of RW and CRW samples was 7.6. All samples were kept refrigerated (4 °C) until analysis. Before each experiment, 2 L of sample were introduced into the feeding reservoir and then cavitated for a predetermined time (30 and 60 min for both developed cavitation and supercavitation). The samples for analysis were taken prior, during and after the experiments, and for each sample 40 mL of suspension were released from the device through the sampling valve and poured back into the cavitation device through the entry valve. This ensured that the trapped dead volume inside the sampling pipe (that was not cycled through the cavitation device) was not analysed. Then the next 10 mL were released for the same sampling pipe and were stored in 50 mL tubes on ice in a Styrofoam box. The impact of hydrodynamic cavitation on the destruction of bac- teria B. subtilis was monitored by colony counts. For this, samples of 1 mL were plated on the SCA agar medium using the 10-fold successive dilution method in saline solution. Colonies were counted after a 48 h long incubation period at 37 °C and results were expressed in Log 10 CFU mL − 1 . For the selective isolation and quanti fi cation of yeasts from RW and CRW samples, 1 mL was plated on the Sabouraud Dextrose Agar plates (SDA, Merck ™ , 10 g/L of peptone, 40 g/L of dextrose, 2.0% agar) and colonies were counted after a 7 days long aerobic incubation at 28 °C. For the quanti fi cation of aerobic bacteria from RW and CRW samples, 1 mL was plated on SCA Petri plates and colonies were counted after 48 h at 37 °C. The bacterial spore count was performed in the same manner as for the aerobic bacteria with the exception of ad- ditional thermal pre-treatment of samples (80 °C for 20 min) before plating on solid SCA plates. The thermal shock destroys the vegetative portion of cells and only spores survive. Mould colony counts for the RW and CRW samples were performed by adding 1 mL of sample into a screw-cap tube (20 × 150 mm) containing 30 mL of freshly autoclaved, still molten (at 50 °C in a water bath) SDA agar medium. The content was vortexed, poured into a Petri plate and incubated at 25 °C for 5 days [37]. Quanti fi cation of the anaerobic sulphate reducing bacteria
− ln ln f f
− X X t t
0
μ
=
0 (1) Speci fi c decay rate (1/h) is the slope of the microbial growth curve and is negative when cells start dying [38]. X 0 is colony count per millilitre at the beginning of treatment; X f is colony count per millilitre at the end of treatment; t 0 is time at the beginning of treatment and t f is time at the end of treatment. To ensure that the hydrodynamic device was free of microorgan- isms, before and after each hydrodynamic cavitation experiment, the device was cleaned using a washing protocol. This consisted of one rinse with tap water (running the hydrodynamic cavitation device fi lled with tap water for 5 min), two 15 min long rinses with 0.5% organic peroxide (peracetic acid, Persan ® S15, Belinka Perkemija, d.o.o., Slovenia), and fi nally six successive device volume rinses with tap water (each lasting 5 min). The rinsed water was disposed after an overnight exposure to active chlorine. To determine the e ff ectiveness of washing between cavitation experiments, the tap water from the last rinse was sampled and quanti fi ed by colony counts. Additionally, be- fore each cavitation run, the e ff ect of possible bacterial attachment on the interior surfaces of the cavitation reactor was tested. For this pur- pose, samples were taken immediately before (sampled directly form the fl ask containing the prepared bacterial suspension) and after fi lling the reactors with tap water containing around 5 Log 10 CFUmL − 1 or 2 Log 10 CFU mL − 1 of bacteria B. subtilis , and colony counts were com- pared. If compared values were similar (before and after fi lling), no signi fi cant bacterial attachment was present.
2.3. Physicochemical analysis
Organic matter (chemical oxygen demand, COD), was measured using COD kits (Hach Lange LCK 314 for samples with a COD value between 15 mgO 2 /L − 150 mgO 2 /L and LCK 714 for samples with a COD value between 100 mgO 2 /L − 600 mgO 2 /L) and a spectro- photometer DR3900 Hach Lange. pH value, redox potential and dis- solved oxygen level were determined during sampling on site, using a Multi 340i analyser (WTW, Germany). Redox potential values (Pt electrode) measured in the fi eld with an Ag/AgCl reference electrode were normalized to 25 °C and referenced to the standard hydrogen electrode (Eh). Settleable solids were analysed according to the Deutsches Institut fur Normung DIN 38409 – 2 [39], in which settleable substances were shaken and timed sedimentation was determined in a measuring container. Insolubles were determined according to SIST ISO 11,923 [40], for which Sartorius Glass-Micro fi bre Discs GMF3 fi lter paper was used for fi ltration. After drying the fi lter at 105 °C, the weight of the residual mass on the fi lter was measured. Colour intensity measurements were carried out in terms of the spectral absorption
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