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Inactivation of microalgae in ballast water with pulse intense light treatment
Marine Pollution Bulletin xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Inactivation of microalgae in ballast water with pulse intense light treatment Daolun Feng ⇑, Jidong Shi, Dan Sun College of Ocean Science and Engineering, Shanghai Maritime University, Shanghai 201306, PR China
a r t i c l e
i n f o
Article history: Available online xxxx Key words: Ballast water Pulse intense light Microalgae Energy consumption
a b s t r a c t The exotic emission of ballast water has threatened the coastal ecological environment and people’s health in many countries. This paper firstly introduces pulse intense light to treat ballast water. 99.9 ± 0.09% inactivation of Heterosigma akashiwo and 99.9 ± 0.16% inactivation of Pyramimonas sp. are observed under treatment conditions of 350 V pulse peak voltage, 15 Hz pulse frequency, 5 ms pulse width and 1.78 L/min flow rate. The energy consumption of the self-designed pulse intense light treatment system is about 2.90–5.14 times higher than that of the typical commercial UV ballast water treatment system. The results indicate that pulse intense light is an effective technique for ballast water treatment, while it is only a competitive one when drastic decreasing in energy consumption is accomplished. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Ballast water is used to maintain the stability, trim and structural integrity of unladen ships (Hua and Liu, 2007). During voyage, ballast water is frequently loaded into a ship in one port, and discharged in another exotic port, which poses substantial risk of introducing nonindigenous species (Drake et al., 2007). The introduced nonindigenous species may adversely affect ecological system, cause economical losses and threat people’s health (Tsolaki and Diamadopoulos, 2010). Therefore, in order to eliminate those negative impacts, the International Maritime Organization formulates ‘‘The International Convention for the Control and Management of Ship’s Ballast Water and Sediments’’ (IMO, 2004). Subsequently, different treatment methods were proposed to inactivate viable microorganisms in ballast water (Tsolaki and Diamadopoulos, 2010). Normally, the treatment process consists of two stage treatment: firstly, physical solid–liquid separation techniques, mainly hydrocyclone and filtration that may be enhanced by chemical coagulation, are introduced to remove sands and viable microorganisms with large size. Subsequently, physical or chemical treatment methods are incorporated to inactivate the residual viable microorganisms in ballast water (Lloyd’s Register, 2010). However, none of them can meet ⇑ Corresponding author at: Shanghai Maritime University, 1550 Haigang Avenue, Pudong New District, Shanghai 201306, PR China. Tel.: +86 (021) 38284332; fax: +86 (021) 38284342. E-mail address: [email protected] (D. Feng).
the needs of safety, environmental friendliness, easy maintenance, low cost and effectiveness simultaneously and strictly (Gregg et al., 2009; Nengye and Frank, 2012), and more concerns should be focused on the environmental soundness of the treatment methods (Bowmer and Linders, 2010). Pulse intense light (PIL) is considered to be one of the most promising non-thermal sterilization techniques (Gómez-López et al., 2007; Oms-Oliu et al., 2010) in food industry. PIL technology use short time pulses (100–400 ls) with an intense broad spectrum between 100 and 1100 nm (Ferrario et al., 2013) to inactivate microorganisms. Even though the peak power of each pulse is high, the total pulse energy is relatively low because of its short duration (Barbosa-Canovas et al., 2011). In comparison with the traditional UV inactivation, PIL technique shows several extra advantages: firstly, no toxic substances in xenon lamp in contrast to that of mercury in standard UV lamps (Schaefer et al., 2007); secondly, the UV irradiance of xenon lamp is about three or four orders of magnitude higher than that of UV lamp (Schaefer et al., 2007), which means faster and stronger inactivation ability; thirdly, microorganisms that expose to pulse intense light exhibit no tailing to their survival curves (Dunn et al., 1997; Martínez et al., 2013). The vast research on PIL technology is focused on the inactivation of bacteria, yeast, fungi, viruses in food, food container, food package, etc. (Gómez-López et al., 2007; Elmnasser et al., 2007; Oms-Oliu et al., 2010). Recently, degradation of organic compounds in wastewater (Baranda et al., 2012; Moreau et al., 2013)
http://dx.doi.org/10.1016/j.marpolbul.2014.09.006 0025-326X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Feng, D., et al. Inactivation of microalgae in ballast water with pulse intense light treatment. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.09.006
2
D. Feng et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
and sterilization for final cleaning in hospital (Levin et al., 2013) are also researched. While, to the best of our knowledge, there is no research on applying PIL technique to treat ballast water. For ballast water that undergoes a typical pre-filtering treatment is colorless and transparent, which is an ideal liquid for PIL inactivation (Barbosa-Canovas et al., 2011; Gómez-López et al., 2007; Krishnamurthy et al., 2007). Therefore, PIL technique shows the potential to replace the widely used continuous UV treatment system to inactivate viable microorganisms in ballast water. The work here is to introduce PIL technique into ballast water treatment. In addition, since there is no research on the inactivation of microalgae that is naturally different from previous investigated microorganisms (Gómez-López et al., 2007; Elmnasser et al., 2007; Oms-Oliu et al., 2010) and is the predominant species in ballast water (Steichen et al., 2012), two typical red tide microalgae: Heterosigma akashiwo and Pyramimonas sp. are chosen to test the treatment efficacy.
inactivation. Also the treated water temperature is recorded by an OMEGAÒ thermometer (HH500RA, Omegadyne, Sunbury Ohio).
2.3. Determination of viable microalgae concentration Flow Cytometry (Cyflow Cube 6, Partec GmbH, Münster, Germany) is used to determine the low viable microalgae concentration in ballast water accurately. A GuavaÒ ViaCountÒ reagent (Guava Technologies Inc., Millipore, USA) is used to distinguish the viable and non-viable cells, nucleus-containing debris, and other impurities based on the differential permeability of DNAbinding dyes in the ViaCountÒ reagent. 10 mL water sample is added into 40 mL centrifugal tube and is well agitated to keep it homogeneous. Subsequently, 400 lL homogeneous sample and 400 lL ViacountÒ reagent are added into a sample tube, and is well agitated. After 30 min staining in the dark and under temperature below 10 °C, the stained sample is diluted by 200 lL 0.2 lm-filtered sea water, and then numerated by flow cytometry.
2. Materials and methods 2.4. Calculation of inactivation percentage 2.1. Microalgae and culture medium H. akashiwo and Pyramimonas sp. are purchased from Ocean University of China. f/2 medium is applied to culture both microalgae (Guillard and Ryther, 1962), while there is no Na2SiO39H2O for Pyramimonas sp.
Inactivation percentage ð%Þ ¼ ðI DÞ=I 100% where I is the concentration of viable microalgae before inactivation (cell/mL) and D is the concentration of viable microalgae after inactivation (cell/mL).
2.2. Pulse intense light inactivation experiments Inactivation of microalgae is performed in a self-designed PIL inactivation set-up (Fig. 1). The experimental process is the following: Firstly, culture medium with H. akashiwo and Pyramimonas sp. at their logarithmic growth stage is diluted with artificial sea water, and stored in raw water tank as untreated ballast water. Then, dosing pump (GM0120PQ1MNN, Milton Roy Industrial (Shanghai) Co., LTD, China) is started to drive the untreated ballast water into a cylindrical treatment chamber with inner diameter of 4.15 cm and length of 20.80 cm, where the viable microalgae is inactivated by PIL generated from a xenon lamp (outer diameter 0.8 cm and lighting length 12.0 cm). The hydraulic retention time of the ballast water in treatment chamber is about 0.53 s for a flow rate of 1.74 L/min. Theoretically, turbulent flow is predominant within the treatment chamber. The xenon lamp is triggered by an ordered pulse power source. The treated water is finally collected by the treated water tank. Samples are taken from two points to test the viable microalgae concentration before and after
Sampling point 1
The inactivation percentage is calculated by the following equation:
2.5. Calculation of energy consumption The energy consumption (Ws, J/m3) for PIL inactivation is calculated by the following equation:
W s ¼ ðF f Þ=Q 60 1000 where F is the input single-pulse power (J/pulse), f is the pulse frequency (Hz) and Q is the volumetric flow rate (L/min).
2.6. Statistical analysis All experiments are performed in triplicate and data are expressed as mean ± standard deviation (SD). Analysis of SD is performed using OriginPro 7.5 software.
Dosing pump Sight port Optical glass
Raw water tank
Inlet Temperature probe
Outlet Xenon lamp Drain
Sampling point 2 Treated water tank
Pulse light power source Fig. 1. Schematic of pulse intense light inactivation set-up.
Please cite this article in press as: Feng, D., et al. Inactivation of microalgae in ballast water with pulse intense light treatment. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.09.006
D. Feng et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
3
disintegration of the cell wall resulted from pulse effect (Cheigh et al., 2012; PSU, 2014).
3. Results and discussion 3.1. Impacts of pulse peak voltage on microalgae inactivation
3.2. Impacts of pulse frequency on microalgae inactivation Inactivation of H. akashiwo is performed with different pulse peak voltage, and the results are shown in Fig. 2. The inactivation percentage increases rapidly with pulse peak voltage lower than 260 V. When the pulse peak voltage is 210 V, there is only 1.60 ± 0.42% inactivation, while for that of 260 V, the inactivation reaches 81.49 ± 0.31%. In addition, for 350 V pulse peak voltage, a maximal 99.90 ± 0.09% inactivation is obtained. The same experiment is performed for Pyramimonas sp., and the results are also presented in Fig. 2. In comparison with H. akashiwo, an almost equal inactivation of 99.9 ± 0.16% is observed with 350 V pulse peak voltage. The results indicate that PIL is effective for ballast water treatment as in food industry sterilization. On the other hand, the inactivation percentage of Pyramimonas sp. is much higher than that of H. akashiwo when pulse peak voltage is below 260 V. E.g.: Pyramimonas sp. yields 70.39% higher inactivation than that of H. akashiwo with 220 V pulse peak voltage. Hence, H. akashiwo seems more tolerant to PIL radiance than Pyramimonas sp. During the inactivation process, a maximal temperature elevation of 8 °C in treated ballast water is observed with 350 V pulse peak voltage, and the final temperature of treated ballast water is smaller than 28 °C. Thus, here heat is unlikely to contribute to the inactivation of viable microalgae (Elmnasser et al., 2007). Hereinafter, if not specially mentioned, the temperature elevation for treated ballast water is smaller than 8 °C. The concentration assay results of viable H. akashiwo without and with treatment are typically presented in Fig. 3. After high percentage inactivation, the sum of the concentration of dead cell and its nucleus-containing debris is about 41.84% higher than that before treatment, which indicates at least the same percentage of nucleus-containing debris generate after treatment. For PIL treatment, normally, UV radiance is the most important mechanism that is responsible for microalgae inactivation (Caminiti et al., 2012; PSU, 2014), while this process mainly destroy cell’s DNA and other components inside the cell, the cell structure is still intact and hence hardly to cause the leakage of nucleus matters (Cheigh et al., 2012). On the other hand, pulse effect generated by PIL irradiance is confirmed to be another important inactivation mechanism. In the above mentioned inactivation process, the generation of nucleus-containing debris is most likely due to the
Heterosigma akashiwo Pyramimimonas sp. 300
3.4. Impacts of pulse width on H. akashiwo inactivation
Inactivation percentage (%)
80
60
40
20
200
225
250
275
3.3. Impacts of input power on H. akashiwo inactivation Inactivation of H. akashiwo is performed with different input power, and the results are shown in Fig. 5. The inactivation percentage increases with input power. For input power of 30 W, there is inactivation of 22.43 ± 3.51%; while for input power of 70 W, 91.88 ± 0.23% inactivation is yielded. Under treatment condition of 1.78 L/min flow rate, 74 s hydraulic retention time and 1.58 W/cm2 radiant intensity (corresponding to 70 W input power), 1.09 log reductions of H. akashiwo is obtained (corresponding to 91.88 ± 0.23% inactivation). In comparison, in a similar treatment chamber, at least 5.5 log reductions of Bacillus subtilis spores in distilled water can be achieved under treatment condition of 2 L/min flow rate, 88 s hydraulic retention time and 0.34 W/cm2 radiant intensity (Krishnamurthy, 2006). The results indicated that the tested microalgae seems more resistant to PIL than that of B. subtilis spores, and is expected to be more resistant to PIL than that of typical bacteria, pathogen and viruses emerged in food and wastewater (Elmnasser et al., 2007; Oms-Oliu et al., 2010). However, low average radiant intensity per pulse, which is 6.37 J/cm2/s/pulse for H. akashiwo and 3148.15 J/cm2/s/ pulse (Krishnamurthy, 2006) for B. subtilis spores, may also be an important reason for low log reduction. The energy consumption of the self-designed PIL treatment system based on 90% inactivation is about 2359550.56 J/m3 (data from Fig. 5), which is about 2.90–5.14 times higher than that of the typical commercial UV ballast water treatment system. The high energy consumption may due to three factors: firstly, the low conversion percentage of electricity to light (PSU, 2014); secondly, the high resistance of tested microalgae and different treating requirement; lastly, no optimization to the treatment chamber and parameters.
100
0
Inactivation of H. akashiwo and Pyramimonas sp. are performed with different pulse frequency, and the results are shown in Fig. 4. For H. akashiwo, a drastic elevation in inactivation percentage with pulse frequency is observed. When pulse frequency is 15 Hz, a low inactivation of 1.60 ± 0.41% is observed. And when pulse frequency elevates to 60 Hz, 90.11 ± 0.19% inactivation is yielded. In comparison, less tolerance of Pyramimonas sp. to PIL is observed again, e.g.: Pyramimonas sp. obtains 67.45% higher inactivation than that of H. akashiwo with 20 Hz pulse frequency. Nevertheless, the inactivation percentage of Pyramimonas sp. increases slower than that of H. akashiwo within 15–60 Hz range. Essentially, the increasing in pulse frequency is equivalent to elevating input energy. When input energy is low and there are enough amounts of viable microalgae in ballast water, input energy is the limiting factor, and a relative linear relation between pulse frequency and inactivation can be expected as that of H. akashiwo shown in Fig. 4. While if there is insufficient amount of viable microalgae in ballast water as that of Pyramimonas sp. presented in Fig. 4, the amount of viable microalgae becomes the limiting factor, and a certain amount of increasing in pulse frequency yields less and less increasing in inactivation percentage.
325
350
Pulse peak voltage (V) Fig. 2. Effects of peak voltage of pulse intense light on inactivation of seawater microalgae. Initial viable Heterosigma akashiwo and Pyramimimonas sp. concentration are 66,688 and 41,141 cell/mL respectively; the treating flow rate, temperature, pulse frequency and width are 1.78 L/min, 20 °C, 15 Hz and 5 ms respectively.
The inactivation percentage of H. akashiwo with different pulse width is determined, and the analytical results are shown in Fig. 6. The increasing in pulse width obviously elevates the inactivation percentage. For pulse width of 3 ms, an inactivation
Please cite this article in press as: Feng, D., et al. Inactivation of microalgae in ballast water with pulse intense light treatment. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.09.006
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D. Feng et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
A
B
Viable cell: 66688 cell/mL
Dead cell and its nucleus-containing debris: 586 count/mL
Viable cell: 67 cell/mL
Dead cell and its nucleus-containing debris: 95357 count/mL
Inactivation percentage (%)
Inactivation percentage (%)
100 80 60 40 20
Heterosigma akashiwo Pyramimimonas sp.
0 15
30
45
60
75
90
90
18
80
15
70
12
60
9
50
6 3
40 3.0
3.5
Fig. 4. Effects of frequency of pulse intense light on inactivation of seawater microalgae. Initial viable Heterosigma akashiwo and Pyramimimonas sp. concentration are 92,796 and 41141 cell/mL respectively; the treating flow rate, temperature, pulse peak voltage and width are 1.78 L/min, 20 °C, 210 V and 5 ms respectively.
4.5
5.0
Pulse width (ms)
105
Pulse frequency (Hz)
4.0
Elevation in Inactivation percentage (%)
Fig. 3. A typical assay results of Heterosigma akashiwo stained with GuavaÒ ViacountÒ by cytometry. (A) Without treatment; (B) with high inactivation percentage, treatment condition: flow rate 1.78 L/min, temperature 20 °C, pulse peak voltage 350 V, pulse frequency 15 Hz and pulse width 5 ms.
Fig. 6. Effects of width of pulse intense light on inactivation of Heterosigma akashiwo. Initial viable Heterosigma akashiwo concentration is 79,354 cell/mL; the treating flow rate, temperature, pulse peak voltage and frequency are 1.78 L/min, 20 °C, 250 V and 20 Hz respectively.
90 105
Inactivation percentage (%)
Inactivation percentage (%)
75 90 75 60 45
60 45 30 15
30 0 15 0.9 32
40
48
56
64
72
1.2
1.5
1.8
2.1
2.4
2.7
Flow rate (L/min)
Input power (W) Fig. 5. Effects of input power on inactivation of Heterosigma akashiwo by pulse intense light. Initial viable Heterosigma akashiwo concentration, flow rate and temperature are 37,264 cell/mL, 1.78 L/min and 20 °C respectively. Pulse frequency and width are preset at 50 Hz and 5 ms respectively, while their value may change a little during the inactivation process.
Fig. 7. Effects of flow rate on inactivation of Heterosigma akashiwo by pulse intense light. Initial viable Heterosigma akashiwo concentration is 37,264 cell/mL; the treating temperature, pulse peak voltage, frequency and width are 20 °C, 250 V, 50 Hz and 5.0 ms respectively.
3.5. Impacts of flow rate on H. akashiwo inactivation of 46.07 ± 1.22% is observed, and that for pulse width of 5 ms is 89.0 ± 0.19%. On the other hand, the elevation in inactivation percentage decreases linearly with pulse width (Fig. 6), which indicates a decreasing in energy efficiency with pulse width, and further determination of energy consumption corresponding to specific pulse width is needed to decide the optimal pulse width.
Inactivation of H. akashiwo is performed with different flow rate, and the results are shown in Fig. 7. The inactivation percentage decreases linearly with flow rate. When the flow rate is 1.08 L/ min, an inactivation of 78.85 ± 0.89% is yielded, while when the flow rate is 2.50 L/min, the inactivation reduces to 9.31 ± 4.11%.
Please cite this article in press as: Feng, D., et al. Inactivation of microalgae in ballast water with pulse intense light treatment. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.09.006
D. Feng et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
For continuous flow treatment, the flow rate has a linear relation with average treating time, thus it is reasonable to obtain a negative linear relation between the inactivation percentage and flow rate. While for much higher inactivation percentage, e.g.: 3 orders of magnitude of inactivation, much lower flow rate may require. 4. Conclusions Here PIL is firstly introduced to treat ballast water. Under treatment condition of 350 V pulse peak voltage, 15 Hz pulse frequency, 5 ms pulse width and 1.78 L/min flow rate, 3 log reduction can be achieved for both H. akashiwo and Pyramimonas sp. Under relative low percentage inactivation condition, H. akashiwo seems more tolerant to pulse intense light than that of Pyramimonas sp. Increasing pulse peak voltage, pulse frequency and pulse width all increase the inactivation percentage significantly. The energy consumption of the self-designed PIL treatment system is about 2.90–5.14 times higher than that of the typical commercial UV ballast water treatment system. The results indicate that PIL is effective for ballast water treatment, while energy consumption seems to be a big challenge. Acknowledgements This project has been funded by Shanghai Maritime University. The authors would like to thank the College of Ocean Science and Engineering which offer the laboratory. The author is also grateful for Jidong Shi and Dan sun’s contribution to this work. References Baranda, A.B., Barranco, A., de Marañón, I.M., 2012. Fast atrazine photodegradation in water by pulse light technology. Water Res. 46, 669–678. Barbosa-Canovas, G.V., Schaffner, D.W., Pierson, M.D., Zhang, Q.H., 2011. Pulse light technology. J. Food Sci. 65 (S8), 82–85. Bowmer, T., Linders, J., 2010. A summary of findings from the first 25 ballast water treatment systems evaluated by GESAMP. WMU J. Maritime Affairs 9 (2), 223– 230. Caminiti, I.M., Noci, F., Morgan, D.J., Cronin, D.A., Lyng, J.G., 2012. The effect of pulse electric fields, ultraviolet light or high intensity light pulses in combination with manothermosonication on selected physico-chemical and sensory attributes of an orange and carrot juice blend. Food Bioprod. Process. 90 (3), 442–448. Cheigh, C., Park, M., Chung, M., Shin, J., Park, Y., 2012. Comparison of intense pulse light- and ultraviolet (UVC)-induced cell damage in Listeria monocytogenes and Escherichia coli O157:H7. Food Control 25 (2), 654–659.
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Drake, L.A., Doblin, M.A., Dobbs, F.C., 2007. Potential microbial bioinvasions via ship’s ballast water, sediment and biofilm. Mar. Pollut. Bull. 55, 333–341. Dunn, J., Burgess, D., Leo, F., 1997. Investigations of pulse light for terminal sterilization of water for injection filled blow/fill polyethylene seal containers. J. Parent. Sci. Technol. 51, 111–115. Elmnasser, N., Guillou, S., Leroi, F., Orange, N., Bakhrouf, A., Ederighi, M., 2007. Pulse-light system as a novel food decontamination technology: a review. Can. J. Microbiol. 53, 813–821. Ferrario, M., Alzamora, S.M., Guerrero, S., 2013. Inactivation kinetics of some microorganisms in apple, melon, orange and strawberry juices by high intensity light pulses. J. Food Eng. 118 (3), 302–311. Gómez-López, V.M., Ragaert, P., Debevere, J., Devlieghere, F., 2007. Pulse light for food decontamination: a review. Trends Food Sci. Technol. 18, 464–473. Gregg, M., Rigby, G., Hallegraeff, G.M., 2009. Review of two decades of progress in the development of management options for reducing or eradicating phytoplankton, zooplankton and bacteria in ship’s ballast water. Aquat. Invasions 4 (3), 521–565. Guillard, R.R.L., Ryther, J.H., 1962. Studies of marine planktonic diatoms I. Cyclotella nana Hustedt and Detonula confervacea (Gleve) Gran. Can. J. Microbiol. 8, 229– 239. Hua, J., Liu, S.M., 2007. Butyltin in ballast water of merchant ships. Ocean Eng. 34, 1901–1907. IMO, 2004. International Convention for the Control and Management of Ships’ Ballast Water and Sediments. International Maritime Organization, London. Krishnamurthy, K., 2006. Decontamination of Milk and Water by Pulse UV-light and Infrared Heating. PhD thesis. Pennsylvania State University, p. 138. Krishnamurthy, K., Demirci, A., Irudayaraj, J.M., 2007. Inactivation of Staphylococcus aureus in milk using flow-through pulse UV light treatment system. J. Food Sci. 72, 233–239. Levin, J., Riley, L.S., Parrish, C., English, D., Ahn, S., 2013. The effect of portable pulse xenon ultraviolet light after terminal cleaning on hospital-associated Clostridium difficile infection in a community hospital. Am. J. Infect. Control 41 (8), 746–748. Lloyd’s Register, 2010. Ballast Water Treatment Technology: Current Status. Lloyd’s Register, London. Martínez, L.F., Mahamud, M.M., Lavín, A.G., Bueno, J.L., 2013. The regrowth of phytoplankton cultures after UV disinfection. Mar. Pollut. Bull. 67, 152–157. Moreau, M., Lescure, G., Agoulon, A., Svinareff, P., Orange, N., Feuilloley, M., 2013. Application of the pulse light technology to mycotoxin degradation and inactivation. J. Appl. Toxicol. 33 (5), 357–363. Nengye, L., Frank, M., 2012. The European union and the international maritime organization: EU’s external influence on the prevention of vessel-source pollution. J. Marit. Law Commer. 41, 581–594. Oms-Oliu, G., Martín-Belloso, O., Soliva-Fortuny, R., 2010. Pulse light treatments for food preservation. A review. Food Bioproc. Technol. 3, 13–23. Pennsylvania State University (PSU). Pulse Light and PEF. (access on 06.02.14). Schaefer, R., Grapperhaus, M., Schaefer, I., Linden, K., 2007. Pulse UV lamp performance and comparison with UV mercury lamps. J. Environ. Eng. Sci. 6 (3), 303–310. Steichen, J.L., Windham, R., Brinkmeyer, R., Quigg, A., 2012. Ecosystem under pressure: ballast water discharge into Galveston Bay, Texas (USA) from 2005 to 2010. Mar. Pollut. Bull. 64, 779–789. Tsolaki, E., Diamadopoulos, E., 2010. Technologies for ballast water treatment: a review. J. Chem. Technol. Biotechnol. 85, 19–32.
Please cite this article in press as: Feng, D., et al. Inactivation of microalgae in ballast water with pulse intense light treatment. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.09.006
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Inactivation of microalgae in ballast water with pulse intense light treatment Daolun Feng ⇑, Jidong Shi, Dan Sun College of Ocean Science and Engineering, Shanghai Maritime University, Shanghai 201306, PR China
a r t i c l e
i n f o
Article history: Available online xxxx Key words: Ballast water Pulse intense light Microalgae Energy consumption
a b s t r a c t The exotic emission of ballast water has threatened the coastal ecological environment and people’s health in many countries. This paper firstly introduces pulse intense light to treat ballast water. 99.9 ± 0.09% inactivation of Heterosigma akashiwo and 99.9 ± 0.16% inactivation of Pyramimonas sp. are observed under treatment conditions of 350 V pulse peak voltage, 15 Hz pulse frequency, 5 ms pulse width and 1.78 L/min flow rate. The energy consumption of the self-designed pulse intense light treatment system is about 2.90–5.14 times higher than that of the typical commercial UV ballast water treatment system. The results indicate that pulse intense light is an effective technique for ballast water treatment, while it is only a competitive one when drastic decreasing in energy consumption is accomplished. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Ballast water is used to maintain the stability, trim and structural integrity of unladen ships (Hua and Liu, 2007). During voyage, ballast water is frequently loaded into a ship in one port, and discharged in another exotic port, which poses substantial risk of introducing nonindigenous species (Drake et al., 2007). The introduced nonindigenous species may adversely affect ecological system, cause economical losses and threat people’s health (Tsolaki and Diamadopoulos, 2010). Therefore, in order to eliminate those negative impacts, the International Maritime Organization formulates ‘‘The International Convention for the Control and Management of Ship’s Ballast Water and Sediments’’ (IMO, 2004). Subsequently, different treatment methods were proposed to inactivate viable microorganisms in ballast water (Tsolaki and Diamadopoulos, 2010). Normally, the treatment process consists of two stage treatment: firstly, physical solid–liquid separation techniques, mainly hydrocyclone and filtration that may be enhanced by chemical coagulation, are introduced to remove sands and viable microorganisms with large size. Subsequently, physical or chemical treatment methods are incorporated to inactivate the residual viable microorganisms in ballast water (Lloyd’s Register, 2010). However, none of them can meet ⇑ Corresponding author at: Shanghai Maritime University, 1550 Haigang Avenue, Pudong New District, Shanghai 201306, PR China. Tel.: +86 (021) 38284332; fax: +86 (021) 38284342. E-mail address: [email protected] (D. Feng).
the needs of safety, environmental friendliness, easy maintenance, low cost and effectiveness simultaneously and strictly (Gregg et al., 2009; Nengye and Frank, 2012), and more concerns should be focused on the environmental soundness of the treatment methods (Bowmer and Linders, 2010). Pulse intense light (PIL) is considered to be one of the most promising non-thermal sterilization techniques (Gómez-López et al., 2007; Oms-Oliu et al., 2010) in food industry. PIL technology use short time pulses (100–400 ls) with an intense broad spectrum between 100 and 1100 nm (Ferrario et al., 2013) to inactivate microorganisms. Even though the peak power of each pulse is high, the total pulse energy is relatively low because of its short duration (Barbosa-Canovas et al., 2011). In comparison with the traditional UV inactivation, PIL technique shows several extra advantages: firstly, no toxic substances in xenon lamp in contrast to that of mercury in standard UV lamps (Schaefer et al., 2007); secondly, the UV irradiance of xenon lamp is about three or four orders of magnitude higher than that of UV lamp (Schaefer et al., 2007), which means faster and stronger inactivation ability; thirdly, microorganisms that expose to pulse intense light exhibit no tailing to their survival curves (Dunn et al., 1997; Martínez et al., 2013). The vast research on PIL technology is focused on the inactivation of bacteria, yeast, fungi, viruses in food, food container, food package, etc. (Gómez-López et al., 2007; Elmnasser et al., 2007; Oms-Oliu et al., 2010). Recently, degradation of organic compounds in wastewater (Baranda et al., 2012; Moreau et al., 2013)
http://dx.doi.org/10.1016/j.marpolbul.2014.09.006 0025-326X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Feng, D., et al. Inactivation of microalgae in ballast water with pulse intense light treatment. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.09.006
2
D. Feng et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
and sterilization for final cleaning in hospital (Levin et al., 2013) are also researched. While, to the best of our knowledge, there is no research on applying PIL technique to treat ballast water. For ballast water that undergoes a typical pre-filtering treatment is colorless and transparent, which is an ideal liquid for PIL inactivation (Barbosa-Canovas et al., 2011; Gómez-López et al., 2007; Krishnamurthy et al., 2007). Therefore, PIL technique shows the potential to replace the widely used continuous UV treatment system to inactivate viable microorganisms in ballast water. The work here is to introduce PIL technique into ballast water treatment. In addition, since there is no research on the inactivation of microalgae that is naturally different from previous investigated microorganisms (Gómez-López et al., 2007; Elmnasser et al., 2007; Oms-Oliu et al., 2010) and is the predominant species in ballast water (Steichen et al., 2012), two typical red tide microalgae: Heterosigma akashiwo and Pyramimonas sp. are chosen to test the treatment efficacy.
inactivation. Also the treated water temperature is recorded by an OMEGAÒ thermometer (HH500RA, Omegadyne, Sunbury Ohio).
2.3. Determination of viable microalgae concentration Flow Cytometry (Cyflow Cube 6, Partec GmbH, Münster, Germany) is used to determine the low viable microalgae concentration in ballast water accurately. A GuavaÒ ViaCountÒ reagent (Guava Technologies Inc., Millipore, USA) is used to distinguish the viable and non-viable cells, nucleus-containing debris, and other impurities based on the differential permeability of DNAbinding dyes in the ViaCountÒ reagent. 10 mL water sample is added into 40 mL centrifugal tube and is well agitated to keep it homogeneous. Subsequently, 400 lL homogeneous sample and 400 lL ViacountÒ reagent are added into a sample tube, and is well agitated. After 30 min staining in the dark and under temperature below 10 °C, the stained sample is diluted by 200 lL 0.2 lm-filtered sea water, and then numerated by flow cytometry.
2. Materials and methods 2.4. Calculation of inactivation percentage 2.1. Microalgae and culture medium H. akashiwo and Pyramimonas sp. are purchased from Ocean University of China. f/2 medium is applied to culture both microalgae (Guillard and Ryther, 1962), while there is no Na2SiO39H2O for Pyramimonas sp.
Inactivation percentage ð%Þ ¼ ðI DÞ=I 100% where I is the concentration of viable microalgae before inactivation (cell/mL) and D is the concentration of viable microalgae after inactivation (cell/mL).
2.2. Pulse intense light inactivation experiments Inactivation of microalgae is performed in a self-designed PIL inactivation set-up (Fig. 1). The experimental process is the following: Firstly, culture medium with H. akashiwo and Pyramimonas sp. at their logarithmic growth stage is diluted with artificial sea water, and stored in raw water tank as untreated ballast water. Then, dosing pump (GM0120PQ1MNN, Milton Roy Industrial (Shanghai) Co., LTD, China) is started to drive the untreated ballast water into a cylindrical treatment chamber with inner diameter of 4.15 cm and length of 20.80 cm, where the viable microalgae is inactivated by PIL generated from a xenon lamp (outer diameter 0.8 cm and lighting length 12.0 cm). The hydraulic retention time of the ballast water in treatment chamber is about 0.53 s for a flow rate of 1.74 L/min. Theoretically, turbulent flow is predominant within the treatment chamber. The xenon lamp is triggered by an ordered pulse power source. The treated water is finally collected by the treated water tank. Samples are taken from two points to test the viable microalgae concentration before and after
Sampling point 1
The inactivation percentage is calculated by the following equation:
2.5. Calculation of energy consumption The energy consumption (Ws, J/m3) for PIL inactivation is calculated by the following equation:
W s ¼ ðF f Þ=Q 60 1000 where F is the input single-pulse power (J/pulse), f is the pulse frequency (Hz) and Q is the volumetric flow rate (L/min).
2.6. Statistical analysis All experiments are performed in triplicate and data are expressed as mean ± standard deviation (SD). Analysis of SD is performed using OriginPro 7.5 software.
Dosing pump Sight port Optical glass
Raw water tank
Inlet Temperature probe
Outlet Xenon lamp Drain
Sampling point 2 Treated water tank
Pulse light power source Fig. 1. Schematic of pulse intense light inactivation set-up.
Please cite this article in press as: Feng, D., et al. Inactivation of microalgae in ballast water with pulse intense light treatment. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.09.006
D. Feng et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
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disintegration of the cell wall resulted from pulse effect (Cheigh et al., 2012; PSU, 2014).
3. Results and discussion 3.1. Impacts of pulse peak voltage on microalgae inactivation
3.2. Impacts of pulse frequency on microalgae inactivation Inactivation of H. akashiwo is performed with different pulse peak voltage, and the results are shown in Fig. 2. The inactivation percentage increases rapidly with pulse peak voltage lower than 260 V. When the pulse peak voltage is 210 V, there is only 1.60 ± 0.42% inactivation, while for that of 260 V, the inactivation reaches 81.49 ± 0.31%. In addition, for 350 V pulse peak voltage, a maximal 99.90 ± 0.09% inactivation is obtained. The same experiment is performed for Pyramimonas sp., and the results are also presented in Fig. 2. In comparison with H. akashiwo, an almost equal inactivation of 99.9 ± 0.16% is observed with 350 V pulse peak voltage. The results indicate that PIL is effective for ballast water treatment as in food industry sterilization. On the other hand, the inactivation percentage of Pyramimonas sp. is much higher than that of H. akashiwo when pulse peak voltage is below 260 V. E.g.: Pyramimonas sp. yields 70.39% higher inactivation than that of H. akashiwo with 220 V pulse peak voltage. Hence, H. akashiwo seems more tolerant to PIL radiance than Pyramimonas sp. During the inactivation process, a maximal temperature elevation of 8 °C in treated ballast water is observed with 350 V pulse peak voltage, and the final temperature of treated ballast water is smaller than 28 °C. Thus, here heat is unlikely to contribute to the inactivation of viable microalgae (Elmnasser et al., 2007). Hereinafter, if not specially mentioned, the temperature elevation for treated ballast water is smaller than 8 °C. The concentration assay results of viable H. akashiwo without and with treatment are typically presented in Fig. 3. After high percentage inactivation, the sum of the concentration of dead cell and its nucleus-containing debris is about 41.84% higher than that before treatment, which indicates at least the same percentage of nucleus-containing debris generate after treatment. For PIL treatment, normally, UV radiance is the most important mechanism that is responsible for microalgae inactivation (Caminiti et al., 2012; PSU, 2014), while this process mainly destroy cell’s DNA and other components inside the cell, the cell structure is still intact and hence hardly to cause the leakage of nucleus matters (Cheigh et al., 2012). On the other hand, pulse effect generated by PIL irradiance is confirmed to be another important inactivation mechanism. In the above mentioned inactivation process, the generation of nucleus-containing debris is most likely due to the
Heterosigma akashiwo Pyramimimonas sp. 300
3.4. Impacts of pulse width on H. akashiwo inactivation
Inactivation percentage (%)
80
60
40
20
200
225
250
275
3.3. Impacts of input power on H. akashiwo inactivation Inactivation of H. akashiwo is performed with different input power, and the results are shown in Fig. 5. The inactivation percentage increases with input power. For input power of 30 W, there is inactivation of 22.43 ± 3.51%; while for input power of 70 W, 91.88 ± 0.23% inactivation is yielded. Under treatment condition of 1.78 L/min flow rate, 74 s hydraulic retention time and 1.58 W/cm2 radiant intensity (corresponding to 70 W input power), 1.09 log reductions of H. akashiwo is obtained (corresponding to 91.88 ± 0.23% inactivation). In comparison, in a similar treatment chamber, at least 5.5 log reductions of Bacillus subtilis spores in distilled water can be achieved under treatment condition of 2 L/min flow rate, 88 s hydraulic retention time and 0.34 W/cm2 radiant intensity (Krishnamurthy, 2006). The results indicated that the tested microalgae seems more resistant to PIL than that of B. subtilis spores, and is expected to be more resistant to PIL than that of typical bacteria, pathogen and viruses emerged in food and wastewater (Elmnasser et al., 2007; Oms-Oliu et al., 2010). However, low average radiant intensity per pulse, which is 6.37 J/cm2/s/pulse for H. akashiwo and 3148.15 J/cm2/s/ pulse (Krishnamurthy, 2006) for B. subtilis spores, may also be an important reason for low log reduction. The energy consumption of the self-designed PIL treatment system based on 90% inactivation is about 2359550.56 J/m3 (data from Fig. 5), which is about 2.90–5.14 times higher than that of the typical commercial UV ballast water treatment system. The high energy consumption may due to three factors: firstly, the low conversion percentage of electricity to light (PSU, 2014); secondly, the high resistance of tested microalgae and different treating requirement; lastly, no optimization to the treatment chamber and parameters.
100
0
Inactivation of H. akashiwo and Pyramimonas sp. are performed with different pulse frequency, and the results are shown in Fig. 4. For H. akashiwo, a drastic elevation in inactivation percentage with pulse frequency is observed. When pulse frequency is 15 Hz, a low inactivation of 1.60 ± 0.41% is observed. And when pulse frequency elevates to 60 Hz, 90.11 ± 0.19% inactivation is yielded. In comparison, less tolerance of Pyramimonas sp. to PIL is observed again, e.g.: Pyramimonas sp. obtains 67.45% higher inactivation than that of H. akashiwo with 20 Hz pulse frequency. Nevertheless, the inactivation percentage of Pyramimonas sp. increases slower than that of H. akashiwo within 15–60 Hz range. Essentially, the increasing in pulse frequency is equivalent to elevating input energy. When input energy is low and there are enough amounts of viable microalgae in ballast water, input energy is the limiting factor, and a relative linear relation between pulse frequency and inactivation can be expected as that of H. akashiwo shown in Fig. 4. While if there is insufficient amount of viable microalgae in ballast water as that of Pyramimonas sp. presented in Fig. 4, the amount of viable microalgae becomes the limiting factor, and a certain amount of increasing in pulse frequency yields less and less increasing in inactivation percentage.
325
350
Pulse peak voltage (V) Fig. 2. Effects of peak voltage of pulse intense light on inactivation of seawater microalgae. Initial viable Heterosigma akashiwo and Pyramimimonas sp. concentration are 66,688 and 41,141 cell/mL respectively; the treating flow rate, temperature, pulse frequency and width are 1.78 L/min, 20 °C, 15 Hz and 5 ms respectively.
The inactivation percentage of H. akashiwo with different pulse width is determined, and the analytical results are shown in Fig. 6. The increasing in pulse width obviously elevates the inactivation percentage. For pulse width of 3 ms, an inactivation
Please cite this article in press as: Feng, D., et al. Inactivation of microalgae in ballast water with pulse intense light treatment. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.09.006
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D. Feng et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
A
B
Viable cell: 66688 cell/mL
Dead cell and its nucleus-containing debris: 586 count/mL
Viable cell: 67 cell/mL
Dead cell and its nucleus-containing debris: 95357 count/mL
Inactivation percentage (%)
Inactivation percentage (%)
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Fig. 4. Effects of frequency of pulse intense light on inactivation of seawater microalgae. Initial viable Heterosigma akashiwo and Pyramimimonas sp. concentration are 92,796 and 41141 cell/mL respectively; the treating flow rate, temperature, pulse peak voltage and width are 1.78 L/min, 20 °C, 210 V and 5 ms respectively.
4.5
5.0
Pulse width (ms)
105
Pulse frequency (Hz)
4.0
Elevation in Inactivation percentage (%)
Fig. 3. A typical assay results of Heterosigma akashiwo stained with GuavaÒ ViacountÒ by cytometry. (A) Without treatment; (B) with high inactivation percentage, treatment condition: flow rate 1.78 L/min, temperature 20 °C, pulse peak voltage 350 V, pulse frequency 15 Hz and pulse width 5 ms.
Fig. 6. Effects of width of pulse intense light on inactivation of Heterosigma akashiwo. Initial viable Heterosigma akashiwo concentration is 79,354 cell/mL; the treating flow rate, temperature, pulse peak voltage and frequency are 1.78 L/min, 20 °C, 250 V and 20 Hz respectively.
90 105
Inactivation percentage (%)
Inactivation percentage (%)
75 90 75 60 45
60 45 30 15
30 0 15 0.9 32
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Flow rate (L/min)
Input power (W) Fig. 5. Effects of input power on inactivation of Heterosigma akashiwo by pulse intense light. Initial viable Heterosigma akashiwo concentration, flow rate and temperature are 37,264 cell/mL, 1.78 L/min and 20 °C respectively. Pulse frequency and width are preset at 50 Hz and 5 ms respectively, while their value may change a little during the inactivation process.
Fig. 7. Effects of flow rate on inactivation of Heterosigma akashiwo by pulse intense light. Initial viable Heterosigma akashiwo concentration is 37,264 cell/mL; the treating temperature, pulse peak voltage, frequency and width are 20 °C, 250 V, 50 Hz and 5.0 ms respectively.
3.5. Impacts of flow rate on H. akashiwo inactivation of 46.07 ± 1.22% is observed, and that for pulse width of 5 ms is 89.0 ± 0.19%. On the other hand, the elevation in inactivation percentage decreases linearly with pulse width (Fig. 6), which indicates a decreasing in energy efficiency with pulse width, and further determination of energy consumption corresponding to specific pulse width is needed to decide the optimal pulse width.
Inactivation of H. akashiwo is performed with different flow rate, and the results are shown in Fig. 7. The inactivation percentage decreases linearly with flow rate. When the flow rate is 1.08 L/ min, an inactivation of 78.85 ± 0.89% is yielded, while when the flow rate is 2.50 L/min, the inactivation reduces to 9.31 ± 4.11%.
Please cite this article in press as: Feng, D., et al. Inactivation of microalgae in ballast water with pulse intense light treatment. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.09.006
D. Feng et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx
For continuous flow treatment, the flow rate has a linear relation with average treating time, thus it is reasonable to obtain a negative linear relation between the inactivation percentage and flow rate. While for much higher inactivation percentage, e.g.: 3 orders of magnitude of inactivation, much lower flow rate may require. 4. Conclusions Here PIL is firstly introduced to treat ballast water. Under treatment condition of 350 V pulse peak voltage, 15 Hz pulse frequency, 5 ms pulse width and 1.78 L/min flow rate, 3 log reduction can be achieved for both H. akashiwo and Pyramimonas sp. Under relative low percentage inactivation condition, H. akashiwo seems more tolerant to pulse intense light than that of Pyramimonas sp. Increasing pulse peak voltage, pulse frequency and pulse width all increase the inactivation percentage significantly. The energy consumption of the self-designed PIL treatment system is about 2.90–5.14 times higher than that of the typical commercial UV ballast water treatment system. The results indicate that PIL is effective for ballast water treatment, while energy consumption seems to be a big challenge. Acknowledgements This project has been funded by Shanghai Maritime University. The authors would like to thank the College of Ocean Science and Engineering which offer the laboratory. The author is also grateful for Jidong Shi and Dan sun’s contribution to this work. References Baranda, A.B., Barranco, A., de Marañón, I.M., 2012. Fast atrazine photodegradation in water by pulse light technology. Water Res. 46, 669–678. Barbosa-Canovas, G.V., Schaffner, D.W., Pierson, M.D., Zhang, Q.H., 2011. Pulse light technology. J. Food Sci. 65 (S8), 82–85. Bowmer, T., Linders, J., 2010. A summary of findings from the first 25 ballast water treatment systems evaluated by GESAMP. WMU J. Maritime Affairs 9 (2), 223– 230. Caminiti, I.M., Noci, F., Morgan, D.J., Cronin, D.A., Lyng, J.G., 2012. The effect of pulse electric fields, ultraviolet light or high intensity light pulses in combination with manothermosonication on selected physico-chemical and sensory attributes of an orange and carrot juice blend. Food Bioprod. Process. 90 (3), 442–448. Cheigh, C., Park, M., Chung, M., Shin, J., Park, Y., 2012. Comparison of intense pulse light- and ultraviolet (UVC)-induced cell damage in Listeria monocytogenes and Escherichia coli O157:H7. Food Control 25 (2), 654–659.
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Please cite this article in press as: Feng, D., et al. Inactivation of microalgae in ballast water with pulse intense light treatment. Mar. Pollut. Bull. (2014), http://dx.doi.org/10.1016/j.marpolbul.2014.09.006