Batch Phytoremediation Of Aquaculture Wastewater Biology Essay

Microalgae could be utilized as an option to take inorganic foods from effluent and makes them a utile phytoremediation tool in effluent intervention procedure. Phytoremediation is a procedure that utilised works such as microalgae to handle the alimentary pollution in the aquaculture effluent. Chlorella sp. ‘s morphology as unicellular micro-organism could let it to absorb food more expeditiously as compared to tellurian works. Therefore, this survey was performed to measure the public presentation of Chlorella sp.

at different vaccination concentrations to find its kinetic growing at the same clip their alimentary remotion efficiency specifically entire ammonium hydroxide N ( TAN ) and entire P ( TP ) . Silver Barramundi, Lates calcarifer effluent was inoculated with microalgae civilization with different concentrations of 10 % ,15 % , 20 % , 25 % , 35 % and 40 % ( v/v ) in 14-days batch intervention period at room temperature ( 25 A± 2A°C ) . Determination of Chlorella sp. growing public presentation, TAN and TP remotion was performed daily based on the APHA ‘s standard method of scrutiny of H2O and effluent. In this survey, consequences indicated that the best alimentary remotion was in the scope of 15 – 20 % ( v/v ) Chlorella sp. vaccination concentrations which yield over 90 % alimentary remotion with concluding wastewater of 0.08 mg/L TAN and 0.01 mg/L TP.

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The Chlorella sp. growing dynamicss fitted the Monod theoretical accounts and the alimentary remotion fitted with the first order dynamicss. It could be concluded that Chlorella sp. is a possible campaigner to handle aquaculture effluent and served as green engineering to extenuate the greenhouse-related jobs.Cardinal words: Chlorella sp. , Lates calcarifer, batch phytoremediation, entire ammonium hydroxide N, entire P, Monod Model, First Order Kinetics


Phytoremediation is a biological intervention using any type of works either tellurian or marine works.

This type of biological intervention has gained its popularity due to its function in rectifying pollution such as heavy metals, high alimentary concentration and besides contributed positively to the environmentCho-Ruk et al. , 2006Al-Farraj and Al-Wabel, 2007El-Sheekh and Fathy, 2009. Microalgae is recognized as the most promising campaigner for bioprocess due to a multiplicity of reactions ( Rawat et al. , 2011 ) .

The usage of microalgae is desirable since they are able to function a multiple function such as bioremediation every bit good as bring forthing biomass for biofuel production with attendant C segregation ( OlguA±in, 2003 ; Mulbry et al. , 2008 ) . In add-on, effluent redress by microalgae is besides an eco-friendly procedure since it does non let go of any secondary pollution every bit long as the biomass produced is continuously reused and efficient food recycling is maintained ( Munoz and Guieysse, 2006 ; Pizarro et al. , 2006 ; MulHYPERLINK “ # _ENREF_18 ” bHYPERLINK “ # _ENREF_18 ” ry et al. , 2008 ) .

The release of untreated agricultural, industrial and municipal effluent poses serious environmental challenges to the receiving H2O organic structures ( Arora and SaxeHYPERLINK “ # Arora, A. and S. Saxena,2005 ” nHYPERLINK “ # Arora, A. and S. Saxena,2005 ” a, 2005 ; De-Bashan and Bashan, 2010 ) . The major consequence of let go ofing effluent which is rich in organic compounds and inorganic chemicals such as phosphates and nitrates is mainly eutrophication ( OlguA±iHYPERLINK “ # _ENREF_20 ” nHYPERLINK “ # _ENREF_20 ” , 2003 ; Pizarro et alHYPERLINK “ # _ENREF_22 ” .

HYPERLINK “ # _ENREF_22 ” , 2006 ; Mulbry eHYPERLINK “ # _ENREF_18 ” tHYPERLINK “ # _ENREF_18 ” al. , 2008 ; Godos et al. , 2009 ; De-Bashan et al. , 2002 ) . Harmonizing to Rawat et al.

, HYPERLINK “ # _ENREF_23 ” ( HYPERLINK “ # _ENREF_23 ” 201HYPERLINK “ # _ENREF_23 ” 1 ) , this planetary job could be solved by using microalgae whereby the effluent is utilised as provender for the care of microalgae growing. Throughout the intervention period, there will be attendant accretion of biomass for downstream processing enabling the production of biodiesel feedstock ( Munoz and GHYPERLINK “ # _ENREF_19 ” uHYPERLINK “ # _ENREF_19 ” ieysse, 2006 ; PizaHYPERLINK “ # _ENREF_22 ” rHYPERLINK “ # _ENREF_22 ” Ro et al. , 2006 ; Pittman et al.

, 2011 ) . Cellular N is chiefly used to construct proteins, aminic acids and nucleic acids while P is largely constitutional of nucleic acids and phospholipids ( Geider and Roche, 2002 ) . These findings has proven that microalgae are able to take foods from effluent to run into the stringent demands harmonizing to international criterions ( Rawat eHYPERLINK “ # _ENREF_23 ” tHYPERLINK “ # _ENREF_23 ” al. , 2011 ) .This research is motivated by the increasing involvement in application of biotechnology and the execution of environmentally friendly tools in handling effluent ( Yoshimoto et al. , 2005 ; Concas et al. , HYPERLINK “ # Concas, A.

, M. Pisu and G. Cao,2010 ” 2HYPERLINK “ # Concas, A. , M. Pisu and G.

Cao,2010 ” 010 ; Sato et al. , 2010 ) . Chlorella sp. was classified as a biological tool in effluent intervention to cut down the nowadays of alimentary content ( Sydney et al. , 2010 ) . At the same clip, biomass of Chlorella sp.

which is the byproduct of the intervention procedure could be marketed as high-value merchandises ( Brennan and Owende, 2010 ) . In add-on, intervention of phytoremediation could besides assist in the consumption of assorted heavy metals and nursery gasses present in the effluent ( Davis HYPERLINK “ # Davis, T.A. , B.

Volesky and A. Mucci,2003 ” eHYPERLINK “ # Davis, T.A. , B. Volesky and A. Mucci,2003 ” T al. , 2003 ) .

This would lend to get the better of environmentally related job of domestic effluent reported around the universe ( Godos et aHYPERLINK “ # Godos, I.d. , V.

A. Vargas, S. Blanco, M.C.G. Gonzalez and R. Soto & lt ; I & gt ; et al & lt ; I/ & gt ; .

,2010. “ lHYPERLINK “ # Godos, I.d. , V.A. Vargas, S. Blanco, M.

C.G. Gonzalez and R. Soto & lt ; I & gt ; et al & lt ; I/ & gt ; .,2010. “ . , 2010 ; Listowski etHYPERLINK “ # Listowski, A. , H.

Ngo, W. Guo, S. Vigneswaran, H. Shin and H.

Moon,2011 ” HYPERLINK “ # Listowski, A. , H. Ngo, W. Guo, S. Vigneswaran, H. Shin and H. Moon,2011 ” al.

, 2011 ; Rawat et al.HYPERLINK “ # _ENREF_23 ” , HYPERLINK “ # _ENREF_23 ” 2011 ) .The purpose of this survey was to find the possibilities of microalgae Chlorella sp. in handling aquaculture effluent.

This was implemented through monitoring of TP and TAN remotion public presentation in aquaculture effluent by phytoremediation utilizing Chlorella sp. The most suited vaccination concentration in batch manner and the growing dynamicss of Chlorella sp. throughout the intervention period were investigated.

Materials and methods


The pure strain of green algae Chlorella sp. was obtained from the Institute of Tropical Aquaculture ( AKUATROP ) of University Malaysia Terengganu on October 2011. It was cultivated for 30 yearss to bring forth secondary civilization for up-scaling. The medium used for civilization was Conway media with autoclaved and filtered saltwater.

Normal air filtered with 40 Aµm air filter was provided as unfertile aeration to forestall any bacterial taint. Microalgae civilizations were maintained at room temperature of about 25A±2 EsC under a light strength of 4100 lx from white fluorescent visible radiation for 24 h photoperiod. Care of secondary civilization was done for six months throughout the experimental period.

Fish civilization

The aquaculture effluent was supplied from the civilization of Lates calcarifer besides known as Barramundi or Silver Sea bass as farm animal. Lates calcarifer was chosen because of their ability to last in H2O at assorted salt degrees, runing from 0 ppt. up to 30 ppt..

In this survey, 30 fishes were reared in a armored combat vehicle under controlled temperature of 27 A± 2 A°C with uninterrupted aeration. The same type of provender was given based on 3 % of organic structure weight to command P and N content in the wastewater. Foods content in the wastewater were considered upper limit on the 4th twenty-four hours of rise uping period. At this clip, the effluent produced was channeled for phytoremediation intervention by microalgae.

Microalgae phytoremediation

The intervention of effluent was conducted in batch manner utilizing 5000 milliliter flasks. This intervention commenced with the vaccination of Chlorella sp.

civilization into the effluent. Six samples with different concentration of Chlorella sp. civilizations i.e.

, 10 % , 15 % , 20 % , 25 % , 35 % , and 40 % ( v/v ) , were examined in this survey. Illumination was provided continuously from the top of the flask. The experiments were conducted at controlled room temperature of 25 A± 2 A°C for 14 yearss.

Analytic methods

Three parametric quantities of entire phosphate ( TP ) , entire ammonium hydroxide N ( TAN ) and biomass of Chlorella sp. concentrations were analyzed. The alterations of concentrations in the alimentary content and Chlorella sp. biomass were monitored daily for a period of 14 yearss.

Alimentary concentrations ( phosphate and N ) were the chief concerned for measuring removal efficiency in conformity to the growing of Chlorella sp. biomass as kinetic survey.

Chlorella sp. biomass concentration analysis

Analysis of Chlorella sp. biomass concentration was performed at the same time with alimentary concentrations.

Daily samples withdrawn from flasks were centrifuged at 4 A°C to divide algae biomass from H2O. Two-hundred-milliliters sample from each flask were poured into the four 50 milliliter extractor tubings and so they were centrifuged at 5000-6000 revolutions per minute for about 30 min to divide the Chlorella sp. cells from H2O. Then, 10 mL sample of algal suspension was once more centrifuged at 3000 revolutions per minute and the supernatant was discarded. The algae were suspended in 3 milliliter methyl alcohol and heated for approximately 5 min in a H2O bath. The samples were cooled to room temperature and so the volume was made up to 5 milliliters by adding methyl alcohol. The Chlorophyll a concentration in the infusion was determined utilizing the Equation 1 with the reading of soaking up ( A ) of the pigment infusion in a spectrophotometer at the given wavelength, I» ( 650nm and 665nm ) against a solvent space ( Becker, 2008 ) .Chlorophyll a ( mg/L ) = ( 16.

5 A- A665 ) – ( 8.3 A- A650 ) ( 1 )

TP and TAN concentration analysis

Analysis of TP and TAN concentrations were carried out by utilizing HACH DR2400 kit. A 200 milliliter samples were day-to-day withdrawn from the flasks. Then, the samples were centrifuged at 5000-6000 revolutions per minute to divide algae in order to obtain a clear supernatant. Measurements of TP and TAN were executed in clear supernatant by colorimetric methods i.e.

, Ascorbic Acid Method and Salicylic Acid Method, severally. These methods were adapted from Standard Method for Examination of Water and Wastewater ( AHYPERLINK “ # APHA,2012 ” PHYPERLINK “ # APHA,2012 ” HA HYPERLINK “ # APHA,2012 ” 2HYPERLINK “ # APHA,2012 ” 0HYPERLINK “ # APHA,2012 ” 1HYPERLINK “ # APHA,2012 ” 2 ) . They were tantamount to USEPA Method 365.

2 and Standard Method 4500-PE for effluent.

c. Statistical analysis

Microalgae cell growing, TP and TAN concentration were recorded in Microsoft Office Excel throughout the experimental period.

Graphic analysis was performed utilizing Originlab OriginPro 8.6 whereas statistical analysis affecting ANOVA and Tukey ‘s HSD Test was implemented utilizing Minitab 16. Assurance interval of 95 % was selected in order to purely find the significance of different Chlorella sp. vaccination concentrations on alimentary decrease rate, maximal biomass growing rate, staying alimentary concentration and intervention period for maximal alimentary decrease.


The comparative surveies of the growing public presentation of Chlorella sp.

, TAN and TP concentrations between different vaccinations were determined as shown in Figure 1. The growing dynamicss of Chlorella sp. suited the Monod theoretical accounts in line with the growing phases such as slowdown, log, stationary and worsening stages.

Treatment with 15 % ( v/v ) Chlorella sp. had maximal cell concentration of 49.99 mg/L, 51.

55 mg/L for 20 % ( v/v ) and 48.36 mg/L for 40 % ( v/v ) vaccination concentration. The TP and TAN decreases followed the First Order Kinetics where remotion per centum continued to increase exponentially until making an asymptote where the remotion per centum was higher than 99 % with concluding food concentration of 0.08 mg/L TAN and 0.

01 mg/L TP.In order to farther look into the consequence of different Chlorella sp. vaccination concentrations on TP and TAN remotion, correlativity and arrested development analysis were performed specifically on Day 7 of the intervention period since the most obvious decrease occurred at this point. Figure 2 shows the staying TP and TAN concentrations logarithmically decreased with the increased of Chlorella sp. vaccination concentration. At Day 7 which was the mid-point of intervention period, 0.

23 mg/L TP and 0.08 mg/L TAN still remain for 10 % ( v/v ) vaccination as compared to 0.10 mg/L TP and 0.05 mg/L TAN for 40 % ( v/v ) vaccination concentration. Therefore, a higher vaccination concentration of Chlorella sp. would cut down the staying alimentary concentrations.

Figure 3 shows the correlativity between intervention periods required to accomplish the maximal decrease and different vaccination concentrations of Chlorella sp. The increased of Chlorella sp. vaccination concentration contributed to a shorter intervention period required to make the maximal TP and TAN remotion. A really strong negatively additive relationship with incline of -0.

1335 and adjusted R2 of 0.9057 was established for TP, whereas a similar form of strong relationship with incline of -0.0832 and adjusted R2 of 0.8713 for TAN remotion with the addition of Chlorella sp. vaccination concentration. The higher vaccination concentration contributed to a higher readily available biomass which produces higher initial alimentary decrease. After that, Chlorella sp.

would undergo subsequent mitotic cell division of growing to increase its biomass concentration by using the staying foods exist in the effluent ( De-Bashan and BashHYPERLINK “ # _ENREF_11 ” aHYPERLINK “ # _ENREF_11 ” n, 2010 ) .

Microalgae growing

Batch phytoremediation on aquaculture effluent using Chlorella sp. was successfully performed. During the survey, the concentrations of Chlorella sp. used for phytoremediation was varied at 10 % , 15 % , 20 % , 25 % , 35 % and 40 % ( v/v ) . All samples were treated for a period of 14 yearss after Chlorella sp.

vaccination. Different concentrations of Chlorella sp. have shown a rather similar growing form and suited the Monod Model. As shown in Table 1, the maximal growing rate changeless and half impregnation invariable of TP show decrease with the addition of vaccination concentrations. For 10 % ( v/v ) , the half impregnation invariable, Ks was 0.354 mg/L and diminishing to 0.

164 mg/L at 40 % ( v/v ) Chlorella sp.. However, there was no clear tendency between dynamicss coefficients of TAN and different Chlorella sp. vaccination concentrations.

Entire P and entire ammonium hydroxide N remotion

The initial concentrations of TP for all interventions were maintained at 2.50A±0.05 mg/L inorganic phosphate, POa‚„A? — P in order to find the per centum of TP remotion for a period of 14 yearss. The reading for TP concentration was taken daily to detect its decrease in the wastewater.

TP concentration in each intervention decreased aggressively at early period of Chlorella sp. growing due to the rapid addition of Chlorella sp. biomass as indicated in log stage. All samples achieved the high per centum of TP remotion, 99.6 % which indicate that the wastewater contain 0.01 mg/L PO43- except for sample of 10 % ( v/v ) Chlorella sp.

. The concentration of 0.01mg/L PO43- in wastewater is being considered as a safe degree for wastewater to be released to H2O organic structure and being used for H2O recycled in aquaculture. The earliest sample that achieved the maximal removal per centum of more than 99 % of TP is the sample with 35 % ( v/v ) Chlorella sp. , followed by the samples with 20 % and 40 % ( v/v ) Chlorella sp..

Next is the sample with 25 % ( v/v ) Chlorella sp. and the last 1 is the sample with 15 % ( v/v ) of Chlorella sp..

As shown in Table 3, the initial TAN concentrations for all 6 aquaculture effluent samples were about 0.80A±0.05 mg/L before being inoculated with Chlorella sp. Once the intervention of phytoremediation completed, the concluding TAN concentration with 10 % ( v/v ) Chlorella sp. was reduced to 0.12mg/L. Therefore, the optimal per centum of microalgae for phytoremediation at specified intervention period was found to be in the scope of 15 – 25 % ( v/v ) Chlorella sp.

Figure 4 shows the maximal growing rate invariable of Chlorella sp. analyzed utilizing Monod Model and decrease rate analyzed utilizing First Order Kinetics with respect to TAN and TP. The features of log stage were accurately quantified in conformity to the First Order Kinetics. The highest maximal growing rate invariable was at 20 % ( v/v ) vaccination concentration for TP whereas for TAN at 35 % ( v/v ) . However, the highest cumulative alimentary decrease rate was at 15 % ( v/v ) somewhat decreased as vaccination concentration increased to 25 % ( v/v ) . Reduction rate of TP was recorded as the highest within the scope of 15 – 35 % ( v/v ) whereas for TAN at the scope of 10 – 20 % ( v/v ) vaccination concentrations.

Therefore, the most suited concentration of Chlorella sp. vaccination that produced the optimal decrease for both foods was determined in the scope of 15 – 20 % ( v/v ) .


The usage of several species of microalgae as a third effluent intervention was proposed over a decennary ago, and assorted possible interventions continue to be evaluated today ( DHYPERLINK “ # _ENREF_12 ” eHYPERLINK “ # _ENREF_12 ” -Bashan and Bashan, 2010 ) . The implicit in premise is that the microalgae will transform some of the contaminations to non-hazardous stuffs and so the treated H2O can be reused or safely discharged ( Borowitzka and BorowitHYPERLINK “ # Borowitzka, M.A.

and L.J. Borowitzka,1988 ” zHYPERLINK “ # Borowitzka, M.A.

and L.J. Borowitzka,1988 ” Ka, 1988 ) . In this survey, one time the TP and TAN concentrations largely depleted, Chlorella sp.

growing began to worsen. This was besides supported by Droop-based theoretical account suggested by Bougaran et Al. ( 2010 ) . The interaction of TP and TAN concentrations on the growing of microalgae should be considered at the acquisition degree instead than at the assembly degree. Alimentary nowadays in the effluent ( extracellular part ) affect the Chlorella sp.

growing more than those in the intracellular part. Therefore, this phenomenon indicated that the nowadays of these foods in effluent contributed significantly on the growing of Chlorella sp..Based on the old survey, microalgae can be efficaciously used to take high sum of food because they require big measure of N and P for protein ( 45 – 60 % of microalgae dry weight ) , nucleic acid and phospholipids synthesis ( Rawat et alHYPERLINK “ # _ENREF_23 ” .

HYPERLINK “ # _ENREF_23 ” , 2011 ) .In this survey, the concentrations of Chlorella sp. used for phytoremediation was varied at 10 % , 15 % , 20 % , 25 % , 35 % and 40 % ( v/v ) . Different concentrations of Chlorella sp.

have shown a rather similar growing form and suited the Monod Model which consists of slowdown stage, log stage, stationary stage and decease stage. After the vaccination, decrease of microalgae growing occurred within a short period of about 2 yearss known as slowdown stage. In this stage, the decrease of growing occurred because microalgae were still accommodating with the new environment ( El-SheHYPERLINK “ # _ENREF_13 ” eHYPERLINK “ # _ENREF_13 ” kh and Fathy, 2009 ) . When microalgae civilization inoculated into the aquaculture effluent, other pollutants present may besides lend to the decrease in its growing. The microalgae growing rate rebounded back one time they were successfully adapted to the new environment. This phase was known as log stage. This is indicated by the rapid addition in the biomass as depicted with exponential growing of microalgae.

Therefore, microalgae utilized the TP and TAN that nowadays in aquaculture effluent as beginning of nutrient lending to the decrease of food.From foods remotions aspect, TP in the signifier of inorganic phosphate was absorbed by Chlorella sp. and being stored as polyphosphates within the cells ( RawHYPERLINK “ # _ENREF_23 ” aHYPERLINK “ # _ENREF_23 ” T et al. , 2011 ) . Under subsequent aerophilic status, the inorganic phosphate is oxidized to bring forth energy and re-accumulation of phosphate into polyphosphate. The energy generated in this transmutation procedure was used by Chlorella sp. for its growing.

However, phosphorus concentration above 6 mg/L will take to explosive growing of algae. It has become a planetary job where pools and lakes bit by bit turn into fens. Algae are considered to be a biological agency of P remotion which helps in observing a possible eutrophication.

Since P is ecologically important in algal productiveness, its remotion from aquatic organic structures is indispensable to forestall the happening of eutrophication jobs.During the experiment, it was found that microalgae started to degrade themselves after all substrate had been used. When debasement of microalgae occurred, the microalgae would let go of the foods that it had absorbed.

Due to this phenomenon, the removal per centum of TAN had decreased after accomplishing its maximal value. The finding of proper vaccination concentration of Chlorella sp. is crucially of import for the execution on the existent scale aquaculture effluent intervention.The concluding TAN concentration has been reduced to 0.08 mg/L after undergoing the full intervention period. In the existent application of phytoremediation, this value would stand for the existent concentration of TAN before it is disposed into the H2O organic structure such as river or sea. As recommended by the USEPA Standard, outflowing TAN concentration released must be less than 0.

2 mg/L to be considered as environmentally safe since the wastewater discharged did non exercise important impact on the vegetations and zoologies. Therefore, this indicated that the phytoremediation intervention of aquaculture effluent was an first-class method in taking alimentary particularly TAN and TP.


It was shown that the used of microalgae Chlorella sp. in cut downing harmful foods in the aquaculture effluent was suited and effectual. Based on the kinetic coefficients utilizing Monod Models and First Order Kinetics on the remotion of both TAN and TP, the optimal concentration of Chlorella sp. biomass occurred in the scope of 15 – 20 % ( v/v ) that contributed over 99 % alimentary remotion with concluding wastewater of 0.08 mg/L and 0.

01mg/L, severally. Microalgae Chlorella sp. did exhibit a normal growing forms with clear differentiation of growing stages.

Aquaculture effluent intervention utilizing microalgae Chlorella sp. besides known as phytoremediation could be considered as a fresh invention of effluent intervention engineering.


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