Research Article Enhancing Algal Growth by Stimulation ...downloads.hindawi.com/journals/jnm/2015/531352.pdf · culture device Biomechatronics experiment set-up Planning of Taguchi

Research ArticleEnhancing Algal Growth by Stimulationwith LED Lighting and Ultrasound

Shao-Yi Hsia1 and Shiuh-Kuang Yang2

1Department of Mechanical and Automation Engineering Kao Yuan University Kaohsiung City 82151 Taiwan2Department of Mechanical and Electro-Mechanical Engineering National Sun Yat-sen University Kaohsiung City 80424 Taiwan

Correspondence should be addressed to Shao-Yi Hsia syhsiacckyuedutw

Received 6 October 2014 Accepted 7 December 2014

Academic Editor Antonios Kelarakis

Copyright copy 2015 S-Y Hsia and S-K Yang This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Algae are not only rich in natural nutrients but are also a high-priced health food An important constituent called ldquogrowth factorrdquois extracted from algae and used as an ingredient in medical drugs foods cosmetics and other products Its enormous potentialmarket should not be taken lightly Algae are mostly found near coastal areas and their habitats are limited by a number of naturalfactors leading to large labor and financial expenditures to harvest This report describes our study of indoor algae productionusing LED lights and ultrasound and manipulating other growth factors at different temperatures Ultrasound treatment at thealgarsquos natural resonant frequency was varied to determine optimal algal growth using the Taguchi method to plan and to analyzethe experiments The results were very satisfying showing an 823 increase in the growth rate by the fifth day due to ultrasoundtreatment and an amazing 2701 growth rate due to biomechanical stimulation

1 Introduction

Algae present themselves to many substantial potentialmarkets They contain rich nutritive elements that can beextracted from fronds for use in medical drugs foodsand cosmetics As food algae are considered an ideal andbalanced healthy food because of their protein vitamin B

12

120573-carotene unsaturated fatty acids various minerals andother bioactive substances In medicine algae can generatelarge quantities of unique extracts during cell division thatare effective at maintaining health and curing diseases

Spolaore et al [1] discussed the first utilization ofmicroal-gae in China more than 2000 years ago when fat choy wasused during famine However microalgae biotechnology wasnot actually developed until the last century and now thereare many commercial applications For instance they playa critical role in aquaculture and their cellular compoundsare used in human food animal feed and cosmetics Whatis more microalgae contain high-value elements that areapplied to products such as the polyunsaturated fatty acid oilsthat are added to infant formula milk powder and nutrition

supplements Future research should focus on improvingproduction systems and genetically modifying bacterialstrains Chiu [2] cultivated algal cells in low (8 times 105 cellsmL)and high (8 times 106 cellsmL) concentrations and showedthat high initial cell concentrations under a carbon dioxideenvironment present faster growth rates and that the CO

2

tolerance of microalgae increases with increasing algal celldensities To understand growth and CO

2removal efficien-

cies three semicontinuous culture strategies were tested forbiomass capacity (1) a quarter of the culture media beingreplaced every two days (2) a third of the culturemedia beingreplaced every three days and (3) half of the culture mediabeing replaced every eight days These experiments showedthat microalgae biomass capacity increased to 061 gLdwhen one quarter of the culturemediawas replaced every twodays

The application of light to research on cultivation canbe traced back to 1961 when Pipes and Koutsoyannis [3]established a Chlorella family cultivation model and provedthat cell density is proportional to irradiation time Fromthis study the irradiated Chlorella appeared with the amount

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2015 Article ID 531352 11 pageshttpdxdoiorg1011552015531352

2 Journal of Nanomaterials

of growth 0275mL confirming theoretical predictionsTamulaitis et al [4] used LED lamps with four wavelengthsfor comparison with traditional experiments using high-pressure sodium lamps for cultivating radish and lettuceseedlings (1) 455 nm which is closely related to pho-totropism (2) 660 nm for photosynthesis (3) 735 nm (farinfrared) for changing plant growth type and (4) newlyproduced 640 nm LED lamps Their experiments revealedthat LED light sources outperformed high-pressure sodiumlamps at photosynthesis and growth Advantageously thenew LEDs cost much less than early LED lamps The exper-iment proved the critical effects of wavelengths 640 nm and660 nm on photosynthesis and plant shape showed obviouschanges under far infrared irradiation Fu et al [5] utilizedan LED photo bioreactor in 2012 to enhance the biologicalproduction and cell density ofChlorella Such research provedthe feasibility of using LED lighting and carbon dioxidein microalgae biotechnology to further improve biologicalproductivity and other benefits

Reviewing the previous research of ultrasound on bio-logical effects Wang et al [6] cultivated Chlorella cellsin Petri dishes under ultraviolet irradiation for 0 20 4060 80 100 and 120 sec in order to study the effects ofultraviolet irradiation onChlorella growth reproduction andchlorophyll content finding that irradiation for 20ndash60 secappeared to stimulate monocell division and enhance cellgrowth Nevertheless when ultraviolet irradiation was pro-longed formore than 80 sec cell density was sharply reducedUltraviolet irradiation for 20ndash80 sec did not reveal obviouseffects on the Chlorella chlorophyll content but obvious andreduced effects did occur after 100 sec Increasing the solarultraviolet irradiation time apparently directly affects algalgrowth reproduction and chlorophyll content and indirectlyinfluences photosynthetic efficiency Rajasekhar et al [7]utilized 20 kHz ultrasound for irradiating poisonous cyanbacteria and microcystisaeruginosa in a lagoon to reduce thealgal growth rate finding that it inhibited the growth of threetypes of microalgae (circinalis gt microcystisaeruginosa gtChlorella) and that the highest reduction rate occurred duringthe initial 5min Their study proved that ultrasound canselectively remove harmful cyanobacteria from a lagoonHsia and Chou [8] attempted to combine dark-fermentationbiohydrogen production and ultrasound by determining thenumber of experiments with Taguchi methods to establishthe factors of the experiment and achieve optimization condi-tions to develop rapid and effective biohydrogen productionThey discovered that ultrasound energy and frequency irra-diation time and starch concentration affect the biohydrogenproduction system by changing the hydrogen productionefficiency and hydrogen production rate Under optimizationconditions dark-fermentation biohydrogen production effi-ciency increased by 3229

In this study freshwater Chlorella was used to evaluateLED lighting cultivation temperature and ultrasound expo-sure using the Taguchi method orthogonal array to searchfor optimal conditions for enhancing freshwater Chlorellaproduction and further contribute to cultivation techniquesand environmental protection A schematic depicting algalgrowth under stimulation by LEDs and ultrasound is shown

Ultrasound

Algae-biomechanical stimulation

An ideal healthy food

LED

Figure 1 Schematic of algae growth with stimulation by LEDs andultrasound

Preparation of culture device

Biomechatronicsexperiment set-up

Planning of Taguchi method

Optimal experimental combination

Conclusions

Algal growth experiment

TemperatureLEDPumpingUltrasound

Experiment confirmation

Yes

No

Figure 2 Flowchart of algal growth experiment

in Figure 1 and the experimental flowchart is noted inFigure 2

2 Research Methodology

21 Brief Introduction to Chlorella and Its CultivationChlorella (Chlorella sp) is a spherical or oval-shapedmicroor-ganism with a diameter of 2ndash12120583m depending on varietyCell size changes with variety cultivation time nutritionand environment Chlorella is 40 protein 10 fat 10carbohydrate 6 ash and 34 chlorophyll and Chlorellaextract

FreshwaterChlorella which is classified into the kingdomProtista phylumChlorophyta class Trebouxiophyceae orderChlorellales and family Chlorellaceae was cultivated in thisstudy Cells were spherically shaped with diameters of 2ndash10 120583m flagellate and required sunlight water and carbondioxide for growth Its great reproductive ability allows

Journal of Nanomaterials 3

Chlorella to divide into four cells every 20 h By convertingsolar energy in the cell large amounts of oxygen are releasedduring reproduction and photosynthesis is 10 times higherthan in other plants Because Chlorellarsquos photosynthesisachieves 8 efficiencyChlorella could be a potential source offood and energy like other efficient crops such as sugarcaneIt is also attractive for its potential as a food source due toits high content of protein and other necessary nutrientsIn the dry form it contains about 45 protein 20 fat20 carbohydrate 5 fiber and 10 vitamins and mineralsTherefore Chlorella is suggested as a cheap protein foodsupplement to control weight prevent cancers and reinforceimmune systems

Chlorella cultivation is divided into outdoor and indoorcultivation Outdoor cultivation suffers more from weatherso indoor cultivation is utilized to overcome that Gen-erally speaking many nutritional sources could be usedto grow algae For instance Boldrsquos Basal Medium Walnersquosmedium and the recipe provided by Tungkang Biotech-nology Research Center are used as culture media Thecultivation process can be divided into two stages First aconical flask or serum bottle is pasteurized before adding thecultivated algal source Initial culturing takes 1-2 weeks beingartificially stopped before cell density gets too high Secondlythe water is disinfected with bleach and then neutralized withsodium sulfite Algae are then cultivated in a larger containeruntil the desired density is reached

22 CultureMedia Culturemedia generally contain a carbonsource nitrogen source inorganic salts and microelementsA carbon source provides the carbon required for growth andreproduction Nitrogen which can be organic or inorganicwas used in this research There are nine major elementscarbon hydrogen oxygen nitrogen phosphorus potassiumsulphur calcium andmagnesiumThemicroelements boronmanganese zinc molybdenum cobalt iodine and copperare also required by Chlorella

Nutrient salts in culture media have great effects on algalgrowth A lack of nutrient salts results in unexpected inter-mediate products during cellmetabolismandphotosynthesisNutrient salts contain (a) the major elements of nitrogenphosphorus iron silicon magnesium and sodium (b) ionsof copper manganese zinc molybdenum and cobalt and(c) vitamins The percentages and varieties of carbon andnitrogen sources directly affect algal growth and compositionPrevious research shows that algae largely store fat whenlimited by nitrogen The culturing fertilizer used in ourexperiments was Walnersquos medium (Table 1)

23 Development of the LED and Its Application to Bioindustry[9] Light-emitting diode (LED) products were developedearly in 1968 although they had low light intensity andwere not available in a full palette of colors LEDs havesince been developed in various colors Their usage hasexpanded greatly in automobiles communication productsinformation products traffic lights irradiation and bioin-dustry among which the latter has recently become popularin agricultural production and biomedicine

Table 1 Walnersquos medium

Stock solution I

NaNO3 100 gNaH2PO4sdot2H2O 20 g

Na2EDTA 45 gH3BO3 336 g

MnCl2sdot4H2O 036 gFeCl3sdot6H2O 13 g

Stock solution II 1mLH2O 1 L

Stock solution II

ZnSO4sdot7H2O 44 gCoCl2sdot6H2O 2 g

(NH4)6Mo7O24sdot4H2O 09 gCuSO4 sdot5H2O 2 g

H2O 100mL

In-use H2O 1 LStock solution I 1mL

Tubular fluorescent lamps (TFL) and high-pressuresodium lamps are the most common artificial light sourcesfor agricultural production The key research focus is onfill-in light enhancing fill-in light uniformity adjustinglight quality and developing artificial light sources havinghigher efficiencies Progress in electrooptical technology hasenhanced the brightness and efficiency of light-emittingdiodes such that these light sources are feasible for agri-cultural production Light-emitting diodes have high pho-tovoltaic conversion efficiencies small volumes long servicelives fixed wavelengths and low heat production and useonly direct current These advantages offer adjustable lightintensity adjustable light quality (percentages of redblue orredfar-infrared) low cooling load and the ability to enhanceproduction per unit area in comparison to currently used sys-tems that rely on fluorescent lamps or high-pressure sodiumlamps as artificial light sources As a result it is a suitableartificial light source for indoor and controlled agriculturalproduction environments (like plant tissue culture and plantgrowth chambers)

24 Biological Effects of Ultrasound [10 11] Many studiesshow that ultrasound transmits energy via particle vibrationwithin propagation media to form an ultrasound field Whenultrasound irradiates biological media at a distinct frequencyand intensity various physical effects appear in ultrasoundenergy and matter particles These are divided into ther-mal and nonthermal effects and are further divided intomechanical effects and cavitation effects Cavitation effectsare regarded as nonthermal effects and have the greatestimpact on biological tissue as ultrasound transmits a waveof condensation and rarefaction in liquids that results incavitation Cavitation happens in a liquid when the liquidgenerates tiny bubbles due to the positive and negativepressures of a moving ultrasound wave or tiny bubbles occurin the liquid due to ultrasound vibration growth contractionor crashingWhen cells experience the high shear wave that isgenerated from vibrated bubbles or bubble crashing a seriesof biological reactions occurs Rayleigh-Plesset announced


a mathematical model of inner cavitation vibration of anincompressible liquid in 1949 [7] Applying this theory tocalculate the appearance of dark-fermentation hydrogen-producing rod bacteria and the internal tissue can result ina natural frequency by setting different pulse intensity for thevibration The radius 119886 surface tension 120590 heat capacity ratio120574 density 120588 viscosity coefficient 120578 and pressure119875

0of bacteria

are substituted in the following equation to calculate naturalfrequency

(1205961015840119903)2

=1

1205881198862[3120574 (119875

0+

2120590

119886) minus

2120590

119886] minus (

2120578

1205881198862)2

(1)

25 Ultrasound Scattering within a Sphere [12] SinceChlorella is sphere-like irradiatingChlorellawith ultrasoundit can be regarded as sonic irradiation within a sphere andthus the equation for ultrasound within a sphere can be usedThe incident wave is quantified as

119901119894=

119860

119903119861119890minus1198861199032119890119894119896119903 (2)

where 119860 and 119861 are amplitude and beam of the wave 119903is the displacement vector and 119896 is the wave number Allparameters except the displacement vector are assumed to beconstant around the target object The wave is scattered onceit reaches the perimeter of the target because of the sphericalshape of the target object Generally speaking a scatteredwave does not show isotropy because the target object is ananisotropic sounddistributor From this viewpoint the soundpressure distribution of a scattered wave at the perimeter ofthe target object is

119901119904=

119860119904

1199031

119861119904119890minus119886119903121198901198941198961199031 (3)

Here the lower case and 1 stand for the parameter andcoordination is related to the target object Solving targetobject characteristics with this equation is similar to solvingparameters 119860

119904and 119861

119904(amplitude and beam of the scattered

wave)In regard to the interaction between the sound wave and

scatter power the ratio of scattered power (119882119904) and incidence

sound intensity (1198680) can be measured in consideration of

the generated sound wave and the scattering efficacy of thescattered power

120590119904=

119882119904

1198680

(4)

The parameter 120590119904is called the scattering cross section

(expressed inm2)Thephysicalmeaning of120590119904can be regarded

as the effective scattering area between the scattered powerand incidence sound wave Apparently 120590

119904and the object

appear to have a close relationship with the orientation of theincidence sound wave

Sphere circumference is far smaller than the wavelength(1198961198860

lt 1) and the function between the sound wave andsphere is scattering When the sound wave is incident to a

0004 0008 0012 0016 00200E + 00

50E minus 08

10E minus 07

15E minus 07

20E minus 07

25E minus 07

ka0

120590sb120587

a2 0

Figure 3 Relations of cross-sectional area 120590119904119887

versus 1198961198860

small rigid sphere with a radius 1198860 the ratio of scattered

sound intensity and incidence sound intensity is

119868119904

1198680

=12058721198812

119903211205824

(1 minus3

2cos 120579119904)2

=(1198961198860)4

11988620

911990321

(1 minus3

2cos 120579119904)2

1198961198860≪ 1

(5)

119881 = (43)12058711988630represents sphere volume The ratio of inverse

scattering cross section and geometric cross-sectional area is

120590119904119887

12058711988620

=25

9(1198961198860)4

1198961198860≪ 1 (6)

This happens when 1198961198860le 05 and the inverse scattering

cross section sharply increases (ray scattering) by a fourthpower equation and approaches 1 when 119896119886

0ge 10 This

presents complex oscillation changes in 1 lt 1198961198860lt 10 because

of the creeping wave being diffracted along the surface In theexample of Chlorella 119886

0is 1sim4 120583m and the speed of sound

in water is 1473ms Substituting 1MHz ultrasound exposureinto the equation sound wave velocity 119888 = 119891 times 120582 andthe acquired wavelength 120582 is 1473 times 10minus6m When 119896119886

0=

(2120587120582)1198860 the final 426 times 10minus3 lt 119896119886

0lt 171 times 10minus2 belongs

to ray scattering and the relationship of 1198961198860versus cross-

sectional area 120590119904119887

is indicated in Figure 3

26 Taguchi Quality Method In the Taguchi experimentaldesign quantified experimental results are called qualitycharacteristics and can achieve an ideal by determining thecontrolling factors in the experimental design In order tosolve a problem an engineer needs to fully understand thecharacteristics of product quality and problems and organizethe levels of quality with a fishbone diagram or equivalentTo conduct the experiment at the least cost an orthogonalarray should be selected based on control factors and thelevels needed to achieve the required quality with the mostprecision The experimental data in this study were further


analyzed with a factor reaction analysis and analysis ofvariance to adjust the control factors allowing the qualitycharacteristics of the arranged function to enable an optimaldesign Traditionally the orthogonal array is named 119871

119887(119888119889)

representing 119889 factors with 119888 levels in each factor for 119887experiments

261 Signal to Noise Ratio (119878119873 Ratio) Different 119878119873 ratioequations are available according to the objectives of qualityand are divided into Nominally the Best Smaller is Betterand Larger is Better Larger is Better was applied to ourexperiment [8 13]

119878119873LB = minus10 log[

[

sum119899

119894=1(1 (119910

119894)2

)

119899]

]

(7)

where 119910119894is the measured value and 119899 is the number of

repeated measurements

262 Response Table and Additive Equation The factorresponse table and reaction diagram were constructed inorder to understand the effects of the factors on the objectiveAmeanwas calculated for the 119878119873 ratio of each factor at eachlevel and further transformed into the reaction diagram

119872119894119895

=sum119873

119896=1(119878119873119894119895119896

)

119873 (8)

119872119894119895is the mean of the 119878119873 ratio containing 119894 factors and 119895

levels 119896 is the 119896th 119878119873 ratio with 119894 factors and 119895 levels and119873is the number of experiments with 119894 factors and 119895 levels

After completing the response table the importance ofthe control factors at each level of quality can be evaluatedby

120578 (119860119894 119861119895 119862119896119863119897 )

= 120578119860119894

+ 120578119861119895

+ 120578119862119896

+ 120578119863119897

+ sdot sdot sdot minus (119899 minus 1) 120578(9)

where 120578 is the quality objective and 120578119860119894and 120578 are the response

value and the mean respectively in the response table Theadditive equation (9) will be used as the prediction model toforesee any combination that we want to know

3 Experimental Framework

31 Experimental Plan Using a microscope the numberof freshwater Chlorella per mL was first calculated with ahemolytic meter After dilution the OD (optical density)relationship diagram and concentration were drawn A highconcentration of freshwater Chlorella was generated withmass culturing methods A spectrophotometer was used formeasuring dailyODwhichwas compared to theODrelation-ship diagram and concentration and a growth curve drawnFreshwater Chlorella was then observed with a microscope(Figure 4) at 1000x The alga used in this experiment waspurchased from Tungkang Biotechnology Research Center(Figure 5)

Figure 4 Microscope of the freshwater Chlorella

32 Cultivation Experiment

321 Culture Volume Culture volume was of primary con-cern during cultivation as the larger the volume the smallerthe percentage of the surface being irradiated For this reasonalgae were initially cultivated in small volumes and thenmoved to larger containers after 1-2 weeks

322 Cultivation Methods Cultivation is generally classifiedinto static culture shaking culture and pumping cultureStatic culture is used when there is no shaker or when apumping culture is not needed Shaking culture is oftenutilized for high concentrations of algae which will notbe experimented on in short periods of time hence thealgae culture is shaken for slow growth Pumping cultureis generally used to add new algae sources into a cultureenvironment when growth to the target concentration inthe shortest time is required At the same time freshwaterChlorella with three initial concentrations was cultivated inWalnersquosmediumwithODs of 002 004 and 006 for five daysGrowth was recorded and the growth curve shows that algaewere not easily cultivated inODs below 006 Algae inWalnersquosmedium with ODs above 006 were used for the Taguchiexperiment

33 LED Irradiation Experiment The light source in theexperiment was white LEDs (replacing the original white flu-orescent lights) in a constant-temperature shaking incubatorequipped with an irradiation timer A 12 hr irradiation periodtends to imitate the general situation for freshwater Chlorellagrowth A 24 hr irradiation period is also discussed herein forcomparison

34 Ultrasound Exposure Experiment Themechanical effectsof ultrasound exposure were stimulated on the experimentto discuss the effects of natural frequency and nonnaturalfrequency of freshwaterChlorella sound intensity and irradi-ation time on the cultivation In the ultrasound experimentthe natural ultrasound frequency of freshwater Chlorella wasfirst calculated in order to set the transducer frequencyso that the freshwater Chlorella would resonate optimallyfor biological effect Considering freshwater Chlorella as abacterial microorganism the data were selected according to


(a) Freshwater Chlorella (b) Mass culture

Figure 5 Original freshwater Chlorella and after mass culture

StatusValue

Measure P1 width P2 ampl P3 ampl

125 kHz 435V 20949mV 215V

P4 max

233 120583s TriggerStopEdge Green

C1 DC896mV

x1 = minus1003975120583sx2 = minus3975ns

C1 BwL DC50A

290mV offset830mVdiv

minus1237Vminus1237V

0mV

1

Δy

(math) (C1) (C1)(math)

darruarr

P5 P6

200 120583sdiv20GSs

Δx = 1000000 120583s1Δx = 100000 kHz

400ks

Time base

C1

Figure 6 Ultrasound waveform induced by function generator

120590 = 7275 dyncm 120574 = 14 120588 = 1 gcm3 120578 = 9197 times

10minus3 gcmsdotsec 1198750

= 760 torr and 119886 = 2ndash8 120583m Finally theabove parameters were substituted into (1) resulting in thenatural resonance frequency of freshwater Chlorella beingcalculated as 05ndash35MHz

Although the natural frequency of freshwater Chlorellawas 05ndash35MHz the frequency calculated by mean cellsize was 1MHz The 05 and 10MHz single-crystal straight-beam longitudinal-wave immersion transducers were there-fore selected as the natural frequency irradiation transducersA power amplifier was utilized in the ultrasound exposureexperiment for amplifying the modulated waveform gener-ated from the signal generator so as to control the ultrasoundintensity of the induced transducer for better parameter con-trol Figure 6 shows the induced ultrasoundwaveform whichis the modulated waveform combined with a 10 kHz squarewave and sine wave Based on the irradiation frequency of thetransducer the sine wave frequency was adjusted to 05 and

1MHz Since the ultrasound transducer presents Gaussiandistribution differences in irradiation intensity are inevitableHowever in order to observe the freshwater Chlorella changeunder irradiation possibly overlapped intensities shouldbe avoided when setting irradiation intensity Moreover inconsideration of the irradiation intensity consistency whenfrequencies are 05 and 1MHz in the Taguchi experimentultrasound exposure intensities of 55 and 185 volts wereutilized in this study

There has been no related research on the effects ofultrasound exposure time on freshwater Chlorella Hsia andChou [8] utilized ultrasound to irradiate anaerobic sludgefor generating hydrogen in 2014 and discovered that anonoff schedule of 1515min was best in comparison withno irradiation and full-time irradiation and that full-timeirradiation results in inhibition Shao-Yi et al [14] usedultrasound for generating hydrogen in 2012 in order toenhance biomass hydrogen production efficiency and found


Table 2 Seven factors and two levels of the orthogonal array 1198718(27)

Factors Specifics Level 1 Level 2119860 Temperature (∘C) 25 30119861 LED irradiation intensity (lux) 3000 8000119862 LED irradiation time (hr) 12 24119863 Pumping intensity (ccmin) 0 2000119864 Ultrasound frequency (MHz) 05 1119865 Ultrasound voltage (Volt) 55 185119866 Ultrasound exposure time 10 s 15min

that the best enhancement occurred with irradiation for10 sec every 8 h Irradiation times for this experiment weretherefore set at 10 sec and 15min every 8 h

35 Taguchi Experiment Temperature LED irradiationintensity LED irradiation time pumping intensity ultra-sound frequency ultrasound voltage and ultrasound expo-sure time were the factors selected for the Taguchi exper-iment (Table 2) Referring to the 119871

8(27) orthogonal array

the control factors and levels required for this experimentare also included in Table 2 The temperature of the algalgrowth culture experiments was 25sim30∘C The experimentalflowchart in Figure 2 depicts the experimental process Firstthe algal culturing device was constructed and the ultrasoundexperiment was set up The factors and parameters werethen selected and Taguchi methods utilized for planningthe experiment Experimental data was used to look for theoptimal combination assessed with analysis of variance andfinally the experiment was confirmed The Larger is Better119878119873 in equation (7)was used for the optimization experimentto search for maximum freshwater Chlorella productionBased on the control factors within given levels the meanof 119878119873 ratios was calculated to assess the effects of thefactorslevels on quality and then construct a response tableAn analysis of variance was utilized to evaluate experimentalerror evaluate which control factors were meaningful andcompensate for the insufficiency of the reaction diagramwithstatistics Finally control factors with low importance valueswere considered to be part of the experimental error

4 Results and Discussion

41 Freshwater Algae Experiment In order to determine theinitial concentration suitable for freshwater Chlorella culti-vation three concentrations were used for the experimentOD 002 (106 times 106cellscc) OD 0041 (209 times 106cellscc)and OD 0063 (320 times 106cellscc) Figure 7 shows the growthcurves resulting from each concentration which indicate thatstable growth appeared at an OD of 0063 but growth didnot proceed when OD was lt 0041 Although slight growthappeared on the secondday concentrations dropped from thethird day The following experiments are therefore discussedbased on the initial OD concentration of 0063 being optimal

42 Taguchi Experiment Freshwater Chlorella in Walnersquosmedium was used in this experiment Eight sets of exper-imental data (Table 3) on the amount of growth and

000

004

008

012

1 2 3 4 5

OD

val

ue

Time (day)

OD = 002

OD = 0041

OD = 0063

Figure 7 Freshwater Chlorella growth curves for different initialconcentrations

00

10

20

30

1 2 3 4 5

OD

val

ue

Test 1Test 2Test 3Test 4


Time (day)

Figure 8 FreshwaterChlorella growth curves for eight experiments

the growth rate over five days were used to prepare growthtrend diagrams (Figures 8 and 9) OD in the eight sets ofexperiment data increased with increasing numbers of dayswith the fourth set showing the most growth followed by thesixth setThe second and eighth sets presented similar growthlevels while the first third fifth and seventh sets grew theleast (Figure 8) Growth rates increased in the fourth sixthand eighths sets on the second third and fourth days butdecreased on the fifth day (Figure 9)The fourth set decreasedthe most the second continued growing and the first thirdfifth and seventh sets barely increasedThe fourth set which


Table 3 119878119873 ratios and maximal amounts of freshwater chlorella growth using the Taguchi method

Exp 1199101

1199102

1199103

Average (106 cellscc) Standard deviation 119878119873 (dB)1 21863 21366 20372 21200 759 46522 728426 730414 728923 729254 1034 77263 60619 60122 60619 60454 287 55634 1153755 1151768 1149283 1151602 2241 81235 67576 67576 68569 67907 574 56646 826808 827802 828299 827636 759 78367 54160 53663 54160 53994 287 54658 768176 768673 769170 768673 497 7771

Ave= 460090 Ave= 6600

Table 4 Growth response of freshwater chlorella for different 119878119873 ratios

119860 119861 119862 119863 119864 119865 119866

Level 1 6516 6469 6403 5336 6455 6552 6519Level 2 6684 6730 6796 7864 6744 6647 6681Range 168 261 393 2528 289 095 162Rank 5 4 2 1 3 7 6

0

1000

2000

3000

4000

5000

1 2 3 4 5



Time (day)

Gro

wth

rate

(106ce

llsc

cd

ay)

Figure 9 Freshwater Chlorella growth rates for eight experiments

increased the most had the largest growth rate in the firstfour days but slowed down on the fifth day possibly becauseof cell density saturation (Figures 8 and 9) The primaryobjective of the experiment was to reduce the variability infreshwater Chlorella cultivation and only then consider othercharacteristics In this case analysis of variance for 119878119873 wasfirst taken into account that is analysis of variance of themean of freshwater Chlorella followed by the quality charac-teristics Each of the previous experiments was repeated threetimes Growth was organized for calculating the 119878119873 ratiosfor control factor levels The Larger is Better 119878119873 ratio wasused to determine the maximal amount of growth with theequation (Table 3) Table 4 shows the factor reaction which

Table 5 Initial analysis of variance for different 119878119873 ratios

Factors Sum of square Degree offreedom Variance

119860 566 1 566119861 1365 1 1365119862 3087 1 3087119863 127834 1 127834119864 1668 1 1668119865 180 1 180119866 527 1 527

integrated the 119878119873 ratio of the factors within the same levelFor instance the factor reaction of factor 119860 at level 1 wasthe mean of the 119878119873 ratio of the first four experimentsWhen determining the analysis of variance for 119878119873 ratios119878119873 data were repeated only once (119903 = 1) and substitutedinto the variance equation so that the error vector variabilitywas 0 To estimate error vector variability control factorswith smaller variabilities were assumed to be unimportantand classified as the quadratic sum of error vector so asto continue the analysis of variance Temperature variabilitywas 566 ultrasound voltage variability 180 and ultrasoundexposure time variability 527 all of which were smallerthan other control factors (Table 5) Temperature ultrasoundvoltage and ultrasound exposure time were therefore pooledinto an error vector for the next analysis of variance

After integrating the above control factors into theexperimental error the analysis of variance for the amountof freshwater Chlorella growth and 119878119873 ratio was recalcu-lated (Table 6) LED irradiation time and pumping inten-sity combined totaled gt80 of the confidence index andultrasound frequency was closed Nevertheless key controlfactors were set so that the index of confidence was gt75


Table 6 Analysis of variance for different 119878119873 ratios

Factors Sum of square Degree of freedom Variance 119865 value Probability Confidence level Significancelowast

119860 566 1 566 090 4133 5867 No119861 1365 1 1365 216 2376 7624 Yes119862 3087 1 3087 490 1138 8862 Yes119863 127834 1 127834 20274 008 9992 Yes119864 1668 1 1668 265 2023 7977 Yes119865 180 1 180 029 6301 3699 No119866 527 1 527 084 4280 5720 NoError 1892 3 631Total 135226 10 13523lowastNote at least 75 confidence level

Table 7 Final analysis of variance for different 119878119873 ratios


119860 Pooled119861 1365 1 1365 216 2376 7624 Yes119862 3087 1 3087 490 1138 8862 Yes119863 127834 1 127834 20274 008 9992 Yes119864 1668 1 1668 265 2023 7977 Yes119865 Pooled119866 PooledError 1892 3 631Total 135226 10 13523lowastNote at least 75 confidence level

Table 8 Growth response of freshwater chlorella based on quality characteristics

119860 119861 119862 119863 119864 119865 119866


and control factor variability was reduced An analysis ofvariance of the key control factors was then completed(Table 7) from which LED irradiation intensity LED irra-diation time pumping intensity and ultrasound frequency(which presented a higher index of confidence)were regardedas important control factors in the 119878119873 analysis Pumpingintensity appeared to have the most importance followedby LED irradiation time ultrasound frequency and LEDirradiation intensity According to the maximal reaction ofcontrol factor levels to 119878119873 ratios (Table 4) the optimizedparameters contained B2 C2D2 and E2 that is LED irradi-ation intensity 8000 lux LED irradiation time 24 hr pumpingintensity 2000 ccmin and ultrasound frequency 1MHz hadthe best freshwater Chlorella productivity This combinationof treatments reduced the variability in freshwater Chlorellagrowth

It is not enough to merely reduce variability the qualitythat is the amount of growth should also be taken intoaccount The variability of quality was further analyzed Inother words analysis of variance was done to determine

the mean amount of growth in order to discuss whethertemperature ultrasound exposure intensity and ultrasoundexposure time can be used to adjust quality First the meanamount of growth (Table 3) was calculated to the factorreaction according to the quality characteristics (Table 8)Analysis of variance of the mean (Table 9) was then calcu-lated Key control factors were set for an index of confi-dence gt85 LED irradiation intensity LED irradiation timepumping intensity and ultrasound frequency were appliedto reduce experimental variability but only temperatureultrasound exposure intensity and ultrasound exposure timewere considered in the analysis of variance of quality char-acteristics Indices of confidence for temperature ultrasoundexposure intensity and ultrasound exposure time were gt85and were regarded as the key control factors of qualitycharacteristics (Table 9) The control factor reactions forthe amount of freshwater Chlorella growth temperatureultrasound exposure intensity and ultrasound exposure timewere further selected as the optimal level combination Themaximal amount of growth occurred at 25∘C 55 v ultrasound


Table 9 Analysis of variance of quality characteristics

Factors Sum of square (106) Degree of freedom Variance (106) 119865 value Probability Confidence level Significancelowast

119860 075 1 075 286 1105 8895 Yes119861 189 1 189 723 161 9839 Yes119862 357 1 357 1367 020 9980 Yes119863 13396 1 13396 51273 000 10000 Yes119864 138 1 138 505 391 9609 Yes119865 143 1 143 547 327 9673 Yes119866 229 1 229 877 092 9908 YesError 418 16 026Total 14520 23 631lowastNote at least 85 confidence level

Table 10 Comparison of predictions with and without ultrasoundtreatments

Predictedamountof growth

Withoutultrasound

(106 cellscc)

With ultrasound(106 cellscc)

Enhancedefficiency

()Day 1 34786 34786 000Day 2 131823 161325 2238Day 3 475477 614480 2923Day 4 876335 1087509 2410Day 5 1050015 1186388 1299

exposure intensity and 10 s ultrasound exposure time (A1 F1and G1 in Table 8) The control factors selected on the basisof 119878119873 ratios and quality characteristics were then combinedto conclude that the maximal amount of freshwater Chlorellagrowth was at A1 B2 C2 D2 E2 F1 and G1

To understand the effects of ultrasound on freshwaterChlorella growth additive equation (9) was first utilized forestimating the effects of an optimized combination with orwithout ultrasound on the amount of freshwater Chlorellagrowth (Table 10)The amount of freshwaterChlorella growthwith ultrasound was enhanced with 1299 by the fifth dayand was the highest (2923) by the third day The opti-mized combination was further tested in another experiment(Table 11) The amount of growth increased by 823 afterfive days of ultrasound with the highest (2701) occurringon the third day Finally comparing the difference betweenthe prediction and the experiment the error of the highestamount of growth was about 222 and the difference inthe amount of growth on the fifth day was 476 Theenhancement observed in the experiment was slightly lowerthan the prediction but tended to be consistent (Tables 10and 11) Maximal enhancement appeared on the third daybut gradually decreased afterward possibly because of celldensity saturation (freshwaterChlorella cannot increase in anunlimited fashion within a limited volume)

Most current biodiesel materials are made from plantoils like soybean oil grape seed oil and palm oil whosesupplies are restricted because they are also used as cookingoilsThe search for sustainable and stable sources of biodieselfeedstocks that can satisfy the large demand is essential

Table 11 Comparison of experiments with and without ultrasoundtreatments

Experimentalamountof growth

Withoutultrasound

(106 cellscc)


Enhancedefficiency


Researchers understand the feasibility of using microalgaeas a biodiesel source [15] The oil content of microalgaevaries among species and in fact a lot of microalgaedo not produce oil and general oil production microalgaecontain oil at 20ndash50 of cell weight The oil production ratedepends on the algarsquos growth rate so species with high oilproduction rates are first choices We proved in this studythat the additive equation did not exhibit a large error duringthe experiment confirmation Consequently it is feasible toutilize the additive equationwith biomechatronics to increasefreshwater Chlorella growth

5 Conclusion

This study aimed to enhance indoor freshwater Chlorellacultivation techniques Biomechatronics with Taguchi meth-ods were utilized to systematically look for the optimalcombination of treatments to increase algal production andgrowth rates Our conclusions are as follows

(1) Small-volume cultivation is required initially to en-hance culture and success rates Pumping can greatlyincrease the culture rate but the resultant waterpollution needs attention

(2) In a 300mL freshwater Chlorella culture volumeshaking culture had the lowest culture concentrationOD of 006 (320 times 106cellscc) this grew 176x afterfive days of cultivation

(3) Optimal culture conditions based on Taguchi meth-ods are 25∘C 8000 lux LED irradiation intensity


24 hr LED irradiation time 2000 ccmin pumpingintensity 1MHz ultrasound frequency 55 v ultra-sound exposure intensity and ultrasound exposuretime of 10 s every 8 hr

(4) The predicted additive equation showed that theamount of freshwater Chlorella growth with ultra-sound increased 1299 after five days and optimal(2923) growth appeared on the third day Confir-mation of the experiment revealed that the amount offreshwater Chlorella growth treated with ultrasoundincreased 823 by day five and was best (2701)on the third day The highest error of the amount ofgrowth was about 222 with the predicted additiveequation and confirmation

Research results were optimized with the additive equa-tion from Taguchi methods and biomechatronics to estimatethe amount and rate of freshwater Chlorella growth It canbe used for increasing the amount of Chlorella or biooilthat can be extracted from Chlorella by contributing to thedevelopment of better cultivation techniques with adequateenvironmental protection

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors thank theMinistry of Science and Technology ofthe Republic of China Taiwan for financially supporting thisresearch partially under Grant NSC 101-2221-E-244-008

References

[1] P Spolaore C Joannis-Cassan E Duran and A IsambertldquoCommercial applications of microalgaerdquo Journal of Bioscienceand Bioengineering vol 101 no 2 pp 87ndash96 2006

[2] S-Y Chiu ldquoEstablishing a microalgae-incorporated photo-bioreactor system for CO

2reduction and microalgal biomass

productionrdquo Agricultural Biotechnology Industry Quarterly2010

[3] W O Pipes and S P Koutsoyannis ldquoLight-limited growth ofChlorella in continuous culturesrdquo Applied Microbiology vol 10no 1 pp 1ndash5 1962

[4] G Tamulaitis P Duchovskis Z Bliznikas et al ldquoHigh-powerlight-emitting diode based facility for plant cultivationrdquo Journalof Physics D Applied Physics vol 38 no 17 pp 3182ndash3187 2005

[5] W Fu O Gudmundsson A M Feist G Herjolfsson S Bryn-jolfsson and B Oslash Palsson ldquoMaximizing biomass productivityand cell density of Chlorella vulgaris by using light-emittingdiode-based photobioreactorrdquo Journal of Biotechnology vol 161no 3 pp 242ndash249 2012

[6] M-H Wang Y-Y Chen and Y-M He Influence of UltravioletRadiation on Growth Reproduction and Chlorophyll Contentof Green Algae Scenedesmus Dimorphus National BiologicalExperimental Instruction Center School of Life Sciences SunYat-Sen University 2008

[7] P Rajasekhar L Fan T Nguyen and F A Roddick ldquoImpactof sonication at 20 kHz on Microcystis aeruginosa Anabaenacircinalis and Chlorella sprdquo Water Research vol 46 no 5 pp1473ndash1481 2012

[8] S-Y Hsia and Y-T Chou ldquoOptimization of biohydrogenproduction with biomechatronicsrdquo Journal of Nanomaterialsvol 2014 Article ID 721267 11 pages 2014

[9] W Fang and J-C Jao ldquoReview of the application of LEDs in bio-industryrdquo Journal of the Agricultural Association of China vol 5no 5 pp 432ndash446 2004

[10] E L Cartensen S Z Child W K Law D R Horwitz and MWMiller ldquoCaviation as amechanism for the biological effects ofultrasound on plant rootsrdquoThe Journal of the Acoustical Societyof America vol 66 no 5 pp 1285ndash1291 1979

[11] E A Neppiras ldquoAcoustic cavitationrdquo Physics Reports Section ofPhysics Letters vol 61 no 3 pp 159ndash251 1980

[12] C-Y Liu Underwater Sound Operating Principles of Underwa-ter Acoustic Systems Wuchou Taiwan 1999

[13] S-Y Hsia ldquoOptimization of microextrusion preforming usingtaguchi methodrdquo Mathematical Problems in Engineering vol2013 Article ID 305797 9 pages 2013

[14] H Shao-Yi Y Shiuh-Kuang C Jyin-Wen and K Huan-Chen ldquoUltrasonic treatment of clostridium on bio-hydrogenproducing effectrdquo in Proceedings of the 29th National Conferenceon Mechanical Engineering no 1509 Kaohsiung Taiwan 2012

[15] Y Chisti ldquoBiodiesel from microalgaerdquo Biotechnology Advancesvol 25 no 3 pp 294ndash306 2007

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


of growth 0275mL confirming theoretical predictionsTamulaitis et al [4] used LED lamps with four wavelengthsfor comparison with traditional experiments using high-pressure sodium lamps for cultivating radish and lettuceseedlings (1) 455 nm which is closely related to pho-totropism (2) 660 nm for photosynthesis (3) 735 nm (farinfrared) for changing plant growth type and (4) newlyproduced 640 nm LED lamps Their experiments revealedthat LED light sources outperformed high-pressure sodiumlamps at photosynthesis and growth Advantageously thenew LEDs cost much less than early LED lamps The exper-iment proved the critical effects of wavelengths 640 nm and660 nm on photosynthesis and plant shape showed obviouschanges under far infrared irradiation Fu et al [5] utilizedan LED photo bioreactor in 2012 to enhance the biologicalproduction and cell density ofChlorella Such research provedthe feasibility of using LED lighting and carbon dioxidein microalgae biotechnology to further improve biologicalproductivity and other benefits

Reviewing the previous research of ultrasound on bio-logical effects Wang et al [6] cultivated Chlorella cellsin Petri dishes under ultraviolet irradiation for 0 20 4060 80 100 and 120 sec in order to study the effects ofultraviolet irradiation onChlorella growth reproduction andchlorophyll content finding that irradiation for 20ndash60 secappeared to stimulate monocell division and enhance cellgrowth Nevertheless when ultraviolet irradiation was pro-longed formore than 80 sec cell density was sharply reducedUltraviolet irradiation for 20ndash80 sec did not reveal obviouseffects on the Chlorella chlorophyll content but obvious andreduced effects did occur after 100 sec Increasing the solarultraviolet irradiation time apparently directly affects algalgrowth reproduction and chlorophyll content and indirectlyinfluences photosynthetic efficiency Rajasekhar et al [7]utilized 20 kHz ultrasound for irradiating poisonous cyanbacteria and microcystisaeruginosa in a lagoon to reduce thealgal growth rate finding that it inhibited the growth of threetypes of microalgae (circinalis gt microcystisaeruginosa gtChlorella) and that the highest reduction rate occurred duringthe initial 5min Their study proved that ultrasound canselectively remove harmful cyanobacteria from a lagoonHsia and Chou [8] attempted to combine dark-fermentationbiohydrogen production and ultrasound by determining thenumber of experiments with Taguchi methods to establishthe factors of the experiment and achieve optimization condi-tions to develop rapid and effective biohydrogen productionThey discovered that ultrasound energy and frequency irra-diation time and starch concentration affect the biohydrogenproduction system by changing the hydrogen productionefficiency and hydrogen production rate Under optimizationconditions dark-fermentation biohydrogen production effi-ciency increased by 3229

In this study freshwater Chlorella was used to evaluateLED lighting cultivation temperature and ultrasound expo-sure using the Taguchi method orthogonal array to searchfor optimal conditions for enhancing freshwater Chlorellaproduction and further contribute to cultivation techniquesand environmental protection A schematic depicting algalgrowth under stimulation by LEDs and ultrasound is shown

Ultrasound

Algae-biomechanical stimulation

An ideal healthy food

LED

Figure 1 Schematic of algae growth with stimulation by LEDs andultrasound

Preparation of culture device

Biomechatronicsexperiment set-up

Planning of Taguchi method

Optimal experimental combination

Conclusions

Algal growth experiment

TemperatureLEDPumpingUltrasound

Experiment confirmation

Yes

No

Figure 2 Flowchart of algal growth experiment

in Figure 1 and the experimental flowchart is noted inFigure 2

2 Research Methodology

21 Brief Introduction to Chlorella and Its CultivationChlorella (Chlorella sp) is a spherical or oval-shapedmicroor-ganism with a diameter of 2ndash12120583m depending on varietyCell size changes with variety cultivation time nutritionand environment Chlorella is 40 protein 10 fat 10carbohydrate 6 ash and 34 chlorophyll and Chlorellaextract

FreshwaterChlorella which is classified into the kingdomProtista phylumChlorophyta class Trebouxiophyceae orderChlorellales and family Chlorellaceae was cultivated in thisstudy Cells were spherically shaped with diameters of 2ndash10 120583m flagellate and required sunlight water and carbondioxide for growth Its great reproductive ability allows








Stock solution I





Stock solution II



H2O 100mL






0of bacteria


(1205961015840119903)2

=1

1205881198862[3120574 (119875

0+

2120590

119886) minus

2120590

119886] minus (

2120578

1205881198862)2

(1)


119901119894=

119860

119903119861119890minus1198861199032119890119894119896119903 (2)


119901119904=

119860119904

1199031

119861119904119890minus119886119903121198901198941198961199031 (3)


119904and 119861






120590119904=

119882119904

1198680

(4)








0004 0008 0012 0016 00200E + 00

50E minus 08

10E minus 07

15E minus 07

20E minus 07

25E minus 07

ka0

120590sb120587

a2 0


versus 1198961198860



119868119904

1198680

=12058721198812

119903211205824

(1 minus3

2cos 120579119904)2

=(1198961198860)4

11988620

911990321

(1 minus3

2cos 120579119904)2

1198961198860≪ 1

(5)



120590119904119887

12058711988620

=25

9(1198961198860)4

1198961198860≪ 1 (6)



0ge 10 This





0=




sectional area 120590119904119887





119887(119888119889)



119878119873LB = minus10 log[

[

sum119899

119894=1(1 (119910

119894)2

)

119899]

]

(7)




119872119894119895

=sum119873

119896=1(119878119873119894119895119896

)

119873 (8)




120578 (119860119894 119861119895 119862119896119863119897 )

= 120578119860119894

+ 120578119861119895

+ 120578119862119896

+ 120578119863119897















StatusValue


125 kHz 435V 20949mV 215V

P4 max


C1 DC896mV


C1 BwL DC50A



0mV

1

Δy


darruarr

P5 P6

200 120583sdiv20GSs

Δx = 1000000 120583s1Δx = 100000 kHz

400ks

Time base

C1


120590 = 7275 dyncm 120574 = 14 120588 = 1 gcm3 120578 = 9197 times
















000

004

008

012

1 2 3 4 5

OD

val

ue

Time (day)

OD = 002

OD = 0041

OD = 0063


00

10

20

30

1 2 3 4 5

OD

val

ue



Time (day)





Exp 1199101

1199102

1199103


Ave= 460090 Ave= 6600


119860 119861 119862 119863 119864 119865 119866


0

1000

2000

3000

4000

5000

1 2 3 4 5



Time (day)

Gro

wth

rate

(106ce

llsc

cd

ay)





119860 566 1 566119861 1365 1 1365119862 3087 1 3087119863 127834 1 127834119864 1668 1 1668119865 180 1 180119866 527 1 527











119860 119861 119862 119863 119864 119865 119866











Withoutultrasound

(106 cellscc)


Enhancedefficiency







Withoutultrasound

(106 cellscc)


Enhancedefficiency



5 Conclusion











Acknowledgment


References

























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










Stock solution I





Stock solution II



H2O 100mL






0of bacteria


(1205961015840119903)2

=1

1205881198862[3120574 (119875

0+

2120590

119886) minus

2120590

119886] minus (

2120578

1205881198862)2

(1)


119901119894=

119860

119903119861119890minus1198861199032119890119894119896119903 (2)


119901119904=

119860119904

1199031

119861119904119890minus119886119903121198901198941198961199031 (3)


119904and 119861






120590119904=

119882119904

1198680

(4)








0004 0008 0012 0016 00200E + 00

50E minus 08

10E minus 07

15E minus 07

20E minus 07

25E minus 07

ka0

120590sb120587

a2 0


versus 1198961198860



119868119904

1198680

=12058721198812

119903211205824

(1 minus3

2cos 120579119904)2

=(1198961198860)4

11988620

911990321

(1 minus3

2cos 120579119904)2

1198961198860≪ 1

(5)



120590119904119887

12058711988620

=25

9(1198961198860)4

1198961198860≪ 1 (6)



0ge 10 This





0=




sectional area 120590119904119887





119887(119888119889)



119878119873LB = minus10 log[

[

sum119899

119894=1(1 (119910

119894)2

)

119899]

]

(7)




119872119894119895

=sum119873

119896=1(119878119873119894119895119896

)

119873 (8)




120578 (119860119894 119861119895 119862119896119863119897 )

= 120578119860119894

+ 120578119861119895

+ 120578119862119896

+ 120578119863119897















StatusValue


125 kHz 435V 20949mV 215V

P4 max


C1 DC896mV


C1 BwL DC50A



0mV

1

Δy


darruarr

P5 P6

200 120583sdiv20GSs

Δx = 1000000 120583s1Δx = 100000 kHz

400ks

Time base

C1


120590 = 7275 dyncm 120574 = 14 120588 = 1 gcm3 120578 = 9197 times
















000

004

008

012

1 2 3 4 5

OD

val

ue

Time (day)

OD = 002

OD = 0041

OD = 0063


00

10

20

30

1 2 3 4 5

OD

val

ue



Time (day)





Exp 1199101

1199102

1199103


Ave= 460090 Ave= 6600


119860 119861 119862 119863 119864 119865 119866


0

1000

2000

3000

4000

5000

1 2 3 4 5



Time (day)

Gro

wth

rate

(106ce

llsc

cd

ay)





119860 566 1 566119861 1365 1 1365119862 3087 1 3087119863 127834 1 127834119864 1668 1 1668119865 180 1 180119866 527 1 527











119860 119861 119862 119863 119864 119865 119866











Withoutultrasound

(106 cellscc)


Enhancedefficiency







Withoutultrasound

(106 cellscc)


Enhancedefficiency



5 Conclusion











Acknowledgment


References

























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





0of bacteria


(1205961015840119903)2

=1

1205881198862[3120574 (119875

0+

2120590

119886) minus

2120590

119886] minus (

2120578

1205881198862)2

(1)


119901119894=

119860

119903119861119890minus1198861199032119890119894119896119903 (2)


119901119904=

119860119904

1199031

119861119904119890minus119886119903121198901198941198961199031 (3)


119904and 119861






120590119904=

119882119904

1198680

(4)








0004 0008 0012 0016 00200E + 00

50E minus 08

10E minus 07

15E minus 07

20E minus 07

25E minus 07

ka0

120590sb120587

a2 0


versus 1198961198860



119868119904

1198680

=12058721198812

119903211205824

(1 minus3

2cos 120579119904)2

=(1198961198860)4

11988620

911990321

(1 minus3

2cos 120579119904)2

1198961198860≪ 1

(5)



120590119904119887

12058711988620

=25

9(1198961198860)4

1198961198860≪ 1 (6)



0ge 10 This





0=




sectional area 120590119904119887





119887(119888119889)



119878119873LB = minus10 log[

[

sum119899

119894=1(1 (119910

119894)2

)

119899]

]

(7)




119872119894119895

=sum119873

119896=1(119878119873119894119895119896

)

119873 (8)




120578 (119860119894 119861119895 119862119896119863119897 )

= 120578119860119894

+ 120578119861119895

+ 120578119862119896

+ 120578119863119897















StatusValue


125 kHz 435V 20949mV 215V

P4 max


C1 DC896mV


C1 BwL DC50A



0mV

1

Δy


darruarr

P5 P6

200 120583sdiv20GSs

Δx = 1000000 120583s1Δx = 100000 kHz

400ks

Time base

C1


120590 = 7275 dyncm 120574 = 14 120588 = 1 gcm3 120578 = 9197 times
















000

004

008

012

1 2 3 4 5

OD

val

ue

Time (day)

OD = 002

OD = 0041

OD = 0063


00

10

20

30

1 2 3 4 5

OD

val

ue



Time (day)





Exp 1199101

1199102

1199103


Ave= 460090 Ave= 6600


119860 119861 119862 119863 119864 119865 119866


0

1000

2000

3000

4000

5000

1 2 3 4 5



Time (day)

Gro

wth

rate

(106ce

llsc

cd

ay)





119860 566 1 566119861 1365 1 1365119862 3087 1 3087119863 127834 1 127834119864 1668 1 1668119865 180 1 180119866 527 1 527











119860 119861 119862 119863 119864 119865 119866











Withoutultrasound

(106 cellscc)


Enhancedefficiency







Withoutultrasound

(106 cellscc)


Enhancedefficiency



5 Conclusion











Acknowledgment


References

























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





119887(119888119889)



119878119873LB = minus10 log[

[

sum119899

119894=1(1 (119910

119894)2

)

119899]

]

(7)




119872119894119895

=sum119873

119896=1(119878119873119894119895119896

)

119873 (8)




120578 (119860119894 119861119895 119862119896119863119897 )

= 120578119860119894

+ 120578119861119895

+ 120578119862119896

+ 120578119863119897















StatusValue


125 kHz 435V 20949mV 215V

P4 max


C1 DC896mV


C1 BwL DC50A



0mV

1

Δy


darruarr

P5 P6

200 120583sdiv20GSs

Δx = 1000000 120583s1Δx = 100000 kHz

400ks

Time base

C1


120590 = 7275 dyncm 120574 = 14 120588 = 1 gcm3 120578 = 9197 times
















000

004

008

012

1 2 3 4 5

OD

val

ue

Time (day)

OD = 002

OD = 0041

OD = 0063


00

10

20

30

1 2 3 4 5

OD

val

ue



Time (day)





Exp 1199101

1199102

1199103


Ave= 460090 Ave= 6600


119860 119861 119862 119863 119864 119865 119866


0

1000

2000

3000

4000

5000

1 2 3 4 5



Time (day)

Gro

wth

rate

(106ce

llsc

cd

ay)





119860 566 1 566119861 1365 1 1365119862 3087 1 3087119863 127834 1 127834119864 1668 1 1668119865 180 1 180119866 527 1 527











119860 119861 119862 119863 119864 119865 119866











Withoutultrasound

(106 cellscc)


Enhancedefficiency







Withoutultrasound

(106 cellscc)


Enhancedefficiency



5 Conclusion











Acknowledgment


References

























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






StatusValue


125 kHz 435V 20949mV 215V

P4 max


C1 DC896mV


C1 BwL DC50A



0mV

1

Δy


darruarr

P5 P6

200 120583sdiv20GSs

Δx = 1000000 120583s1Δx = 100000 kHz

400ks

Time base

C1


120590 = 7275 dyncm 120574 = 14 120588 = 1 gcm3 120578 = 9197 times
















000

004

008

012

1 2 3 4 5

OD

val

ue

Time (day)

OD = 002

OD = 0041

OD = 0063


00

10

20

30

1 2 3 4 5

OD

val

ue



Time (day)





Exp 1199101

1199102

1199103


Ave= 460090 Ave= 6600


119860 119861 119862 119863 119864 119865 119866


0

1000

2000

3000

4000

5000

1 2 3 4 5



Time (day)

Gro

wth

rate

(106ce

llsc

cd

ay)





119860 566 1 566119861 1365 1 1365119862 3087 1 3087119863 127834 1 127834119864 1668 1 1668119865 180 1 180119866 527 1 527











119860 119861 119862 119863 119864 119865 119866











Withoutultrasound

(106 cellscc)


Enhancedefficiency







Withoutultrasound

(106 cellscc)


Enhancedefficiency



5 Conclusion











Acknowledgment


References

























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials













000

004

008

012

1 2 3 4 5

OD

val

ue

Time (day)

OD = 002

OD = 0041

OD = 0063


00

10

20

30

1 2 3 4 5

OD

val

ue



Time (day)





Exp 1199101

1199102

1199103


Ave= 460090 Ave= 6600


119860 119861 119862 119863 119864 119865 119866


0

1000

2000

3000

4000

5000

1 2 3 4 5



Time (day)

Gro

wth

rate

(106ce

llsc

cd

ay)





119860 566 1 566119861 1365 1 1365119862 3087 1 3087119863 127834 1 127834119864 1668 1 1668119865 180 1 180119866 527 1 527











119860 119861 119862 119863 119864 119865 119866











Withoutultrasound

(106 cellscc)


Enhancedefficiency







Withoutultrasound

(106 cellscc)


Enhancedefficiency



5 Conclusion











Acknowledgment


References

























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





Exp 1199101

1199102

1199103


Ave= 460090 Ave= 6600


119860 119861 119862 119863 119864 119865 119866


0

1000

2000

3000

4000

5000

1 2 3 4 5



Time (day)

Gro

wth

rate

(106ce

llsc

cd

ay)





119860 566 1 566119861 1365 1 1365119862 3087 1 3087119863 127834 1 127834119864 1668 1 1668119865 180 1 180119866 527 1 527











119860 119861 119862 119863 119864 119865 119866











Withoutultrasound

(106 cellscc)


Enhancedefficiency







Withoutultrasound

(106 cellscc)


Enhancedefficiency



5 Conclusion











Acknowledgment


References

























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials











119860 119861 119862 119863 119864 119865 119866











Withoutultrasound

(106 cellscc)


Enhancedefficiency







Withoutultrasound

(106 cellscc)


Enhancedefficiency



5 Conclusion











Acknowledgment


References

























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials









Withoutultrasound

(106 cellscc)


Enhancedefficiency







Withoutultrasound

(106 cellscc)


Enhancedefficiency



5 Conclusion











Acknowledgment


References

























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials









Acknowledgment


References

























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



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