Chiang Mai J. Sci. 2015; 42(3) 549

Chiang Mai J. Sci. 2015; 42(3) : 549-559http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Detention Time Study of Algal Biomass Productionwith Natural Water MediumRameshprabu Ramaraj*[a], David Da-Wei Tsai [b] and Paris Honglay Chen [b][a] School of Renewable Energy, Maejo University, Sansai, Chiang Mai 50290, Thailand.[b] Department of Soil and Water Conservation, National Chung-Hsing University, Taichung, 402, Taiwan.*Author for correspondence; e-mail: rrameshprabu@gmail.com; rameshprabu@mju.ac.th

Received: 15 August 2014Accepted: 27 November 2014

ABSTRACTAlgae are very important ecologically since they comprised the major portion of

primary producers in the aquatic environment. Currently, algal biomass had attracted muchinterest for the production of food, bioactive compounds and bioenergy. This study utilizedthe natural water medium to cultivate the algal biomass of the mixed culture with differentdetention time in the photo-bioreactors. The environmental parameters were monitoredcontinuously during growth period. The study showed that natural water resource had suitablenutrients for micro-algal growth. The best biomass productions were observed in detentiontimes of 10 days and 7 days by the total suspended solids (TSS) average 0.15g/L and0.14g/L respectively. In the short detention time of 4 days, there was less biomass yieldwith large amount of water medium consumption. Consequently, we proved the productionof algal biomass by the natural water was prospectively feasible in the ecological sense.The study results of the different detention time which represented the various types ofnatural water bodies could help us to understand the algal growth in the ecosystem better.

Keywords: microalgae, biomass, natural water medium, detention time

1. INTRODUCTIONMicroalgae were the dominant primary

producers in freshwater and marineecosystems. They represented a diverse groupof microscopic prokaryotic and eukaryoticphotosynthetic organisms of tremendousecological importance, because they werethe beginning of the food chain for otheranimals [1]. Microalgae played an importantrole in self-purification of contaminatednatural waters and offered an alternativefor advance nutrition removal in water

or wastewater. The idea to incorporatemicroalgae as an agent of bioremediationwas firstly proposed by Oswald and Gotaasin 1957 [2]; the biomass recovered wasconverted to methane, which was a majorsource of energy [3]. Hence, algae providedthe basis of the aquatic food chain and theywere fundamental to keep CO2 of carboncycle via photosynthesis as a substantial rolein biogeochemical cycles [1]. Most algae werephotoautotrophic, converting solar energy

550 Chiang Mai J. Sci. 2015; 42(3)

into chemical forms through photosynthesis[4]. The mechanisms of algal photosynthesiswere very similar to photosynthesis in higherplants and their products are molecularlyequivalent to conventional agricultural crops[5]. The main advantages of culturingmicroalgae as a source of biomass wereas follows: (1) high photosynthetic yields(up to a maximum of 5-6% conversion oflight c.f. 1-2% for the majority of terrestrialplants); (2) the ability to grow in fresh, saltand wastewater; (3) high oil content; (4) theability to produce nontoxic and biodegradablebiofuels; (5) many species of algae can beinduced to produce particularly highconcentrations of chosen compounds -proteins, carbohydrates, lipids and pigments- that are of commercial value; (6) the abilityto be used in conjunction with wastewatertreatment [1, 6]. Since algae was a key primaryproducer global-wide, algae biomass wasessential biological natural resources whichplayed an important role in nutrient, food,fertilizer, pharmaceutics and biofuel.

Regarding biofuel production,microalgae can provide different types ofbiofuels, including: methane (produced byanaerobic digestion of algal biomass);biodiesel (from algal fatty acids); ethanol(produced by fermentation of starch); andhydrogen (produced biologically) [1, 7, 8].Some studies had also indicated theimportance of algae in carbon dioxidefixation [9, 10].

For the successful algae biomasscultivation, the detention time was a veryimportant factor. Algae required sufficienttime to grow and multiply through binaryfission. Each growth unit should provide aminimum retention time to avoid prematurecell washout [11]. Detention time will alsoinfluence the achievement of the anticipatedlevel of algal density. This study used natural

water with natural algae culture to mimic thenatural system to accomplish the basic aimof the understanding of the nature betterthrough different detention time. Currently,there was limited paper focused on naturalwater medium [8, 12]. The major aim ofthe present study was to investigate thefeasibility of maintaining continuous culturesof fresh water microalgae growth in naturalwater medium and to provide practicalguidance to run such types of cultures.Consequently, this paper was to investigatethe microalgae biomass production onnatural water medium with different detentiontime.

2. MATERIALS AND METHODSThe methodology was illustrated in

(Figure 1). The mixed culture microalgaewere obtained from Sustainable Resourcesand Sustainable Engineering research lab(SRSE-LAB), Department of Soil andWater Conservation, National Chung-HsingUniversity, Taichung, Taiwan. The streamwater was used as medium, collected fromthe Green River at Fu-Te Dao temple(24° 7’27.35"N; 120°40’22.79"E) near theUniversity and the water was filtrated by0.45 μm filter paper as feed. The algae weregrown in the autotrophic conditions ofalgae/bacterial units with 10 days, 7 days and4 days detention time following reactors P1,P2 and P3, for 18 months period; the growthsystem was shown in (Figure 2).

We chose the gravimetric methods ofdry weight to measure algae biomassincluding total suspended solids (TSS),fixed suspended solids (FSS) and volatilesuspended solids (VSS) were measuredaccording to the standard method [13].All physico-chemical analyses were carriedout whole growth period and methodswere listed in (Table 1).

Chiang Mai J. Sci. 2015; 42(3) 551

Figure 1. The flowchart of methodology.

Figure 2. Photo-bioreactor.

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3. RESULTS AND DISCUSSION3.1 Physicochemical and Biological Natureof the System

The most important parametersregulating algal growth are nutrientquantity/quality, light, pH and temperature[1, 4, 6]. The mean values of the measuredphysicochemical and biological parametersof the unsupplemented natural fresh watermedium and culture conditions were shownin (Table 2). The microalgal species in theculture were initially microscopicallyidentified, and their succession and survivalwere monitored at regular intervals. In theculture, the species of the genera Anabaena,Chlorella, Oedogonium and Oscillatoria werepresent as the numerically dominantmicroalgae, along with several other minormicroalgae including the species of the generaLyngbya, Scenedesmus, Phytoconis, Coccochloris andPhormidium and a few unidentified microalgae.

The essential nutrition for algae growthis carbon, nitrogen and phosphorous [1].

The carbon, nitrogen and phosphorous(CNP) ratio for feed, rectors, Redfield data[14] and nutrition removal (i.e. nitrogen andphosphorous) displayed in (Table 3). TheCNP ratio of the unsupplemented naturalfreshwater used in the present studywas 20:75:1, and thus our system werecarbon-limited, according to the Redfieldratio [14]. In term of the carbon limitation,the photosynthesis was to provide the drivingforce to withdraw the air CO2, to dissolveCO2 and to compensate the carbon shortagein growth medium [1]. Those phenomenaand processes were exactly same in the naturalecosystem. Consequently, the applicationsof the algal growth which was carbon limitedin freshwater had great potential to utilizeCO2 from atmosphere. In the culture, theefficient microalgal utilization of macronutrients of nitrogen and phosphorus led toeffective removal of nitrogen and phosphorusup to 81% and 44% (in 10 days detentiontime) from the respective pools.

Table 1. Environmental factors and algal biomass parameter.

ParameterGrowth conditionLight intensityWater temperaturespeciesWater quality in medium and reactorpHDOCODNH4

+-NTKNNO2

--NNO3

--NTPAlgal biomassTSSFSSVSS

Equipment or method

LI-COR light meter (LI-250)thermometermicroscope

Method 423 (Standard Methods)Method 421B (Standard Methods)Method 508B (Standard Methods)Method 417D (Standard Methods)Method 420A (Standard Methods)Method 419 (Standard Methods)Method 418A (Standard Methods)Method 424D (Standard Methods)

Method 209 (Standard Methods)Method 209 (Standard Methods)Method 209 (Standard Methods)

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Table 2. Algae culture conditions.

ParameterGeneral parameterCulturesMediumContinuousOperational parameterPhoto-bioreactorFeedingLight intensityDetention timeGrowth environmental parameterTemperatureDetention timepHDO (mg/L)COD (mg/L)NO3

- -N(mgN/L)NO2

--N(mgN/L)TKN(mgN/L)TN(mgN/L)TP(mg/L)

Conditions

MixedRiver water15 months

CSTRBatch FeedAvg. 30.12 [μmol-1m-2 per μA]10 days, 7 days and 4 days

Avg. 27.5 °CDaily feed (Avg)

7.227.194.831.140.633.666.710.09

10 days (Avg)9.937.399.680.150.021.101.260.05

7 days (Avg)9.807.657.730.350.071.051.640.08

4 days (Avg)9.777.368.080.550.181.472.410.06

Table 3. Nutrition removal efficiency and carbon, nitrogen and phosphorous ratio.

3.2 Total Suspended Solids (TSS)The three rectors (P1, P2 and P3) of

Total Suspended Solids (TSS) results werepresented in (Figure 3A). The average of P1was 0.15 g/L with the range from 0.12 to0.18 g/L. The average of P2 was 0.14 g/Lwith the range from 0.07 to 0.22 g/L. Theaverage of P3 was 0.11 g/L with the range

from 0.03 to 0.16 g/L. TSS represented thetotal amount of suspended solids in thealgae growth units. TSS was referred to thetotal solids retained by 0.45�m filter paperand could be used as a measurement of algalbiomass after drying [15]. Brune [16] statedtheoretically, the longer detention time atincreasing depth to achieve stable

Parameter

nitrogen nutritionremoval efficiencyphosphorus nutritionremoval efficiencyRedfield ratioCNP ratio (this study)

Feed (medium)

--

106:16:120:75:1

Reactor10 days (avg)

81%

44%

-73:25:1

7 days(avg)76%

11%

-36:51:1

4 days(avg)64%

33%

-21:40:1

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3.3 Volatile Suspended Solids (VSS)The volatile suspended solids (VSS)

results were illustrated in (Figure 3B). VSS isone of the methods to estimate the biomassconcentration of algal sample [17]; Barthelet al. [18]. VSS was measured of organicportion of algae. The average algae biomass(VSS) in P1 was 0.06 g/L with the range from0.02 to 0.09 g/L. The average of P2 was0.07 g/L with the range from 0.02 to0.12 g/L. The average of P3 was 0.06 g/Lwith the range from 0.02 to 0.10 g/L. Goingthrough the available literature of the algalbiomass VSS, all the studies focused on thewastewater or artificial medium and there isno any relevant paper on natural watermedium at all.

3.4 Fixed Suspended SolidsThe fixed suspended solids (FSS) results

were shown in (Figure 3C). The average FSSwas 0.06 g/L and ranged 0.03-0.08 g/L.The average of P2 was 0.07 g/L with therange from 0.02 to 0.12 g/L. The average ofP3 was 0.06 g/L with the range from 0.01 to0.10 g/L. There is no any paper used FSS

as biomass index but FSS is importantmeasurement of inorganic portion of algae.We measured FSS for accuracy check ofTSS with VSS, since TSS is the sum of VSSand FSS [19]. On the other hand, potentiallyFSS could be a good index for some specieswhich contained a lot of inorganic ingredientsuch as diatom [21, 22]. Because of the largeinorganic portion of diatom, FSS might be agood index for them. But TSS and VSS arestill the most popular indexes of algal biomass.

3.5 The Potential of Natural Mediumfor Algal Biomass Production

Microalgae can get essential nutrition fromnatural water body [23-25]. Utilizing thisgrowth uptake function we could apply thenatural medium in controlled environmentssuch as lab or field scale growth units(ex. HRP and photo-bioreactors) or evenfurther applied in natural environment withwatershed management, but nowadays mostof researchers and algae manufactures areusing artificial medium which is expensiveto produce algal biomass. Our study could takeadvantage of nutrients available in natural

population. The total algal biomass waspossibly responded by increasing densitywith detention time.

The statistical t-test results were shownin (Table 4), algae biomass growth in reactorsP1 and P2 was same and reactor P3 wasdifferent from those rectors. There were lotsof production units in literature, such ascontinuous-flow stirred-tank reactors [17],high rate algal pond [18], Integrated

Duckweed and Stabilization Pond System[19], etc. The only literature of naturalmedium was Aquaculture Facility USA whichwas used neighboring stream water,supplemented with inorganic nutrientsand achieved the algae biomass productionof 0.03 to 0.11 g/L [12]. Consequently, theresults showed our reactor designand detention time setup could get higherbiomass.

Table 4. t-tests among reactors P1, P2 and P3.

Biomass TSSP1 & P2P1 & P3P2 & P3

t value-0.115692.885552.54853

p-value0.908950.009470.00947

test resultacceptrejectreject

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water to reduce the total cost.Microalgae play an important role in

self-purification of contaminated naturalwaters and offer an alternative for advancenutrition removal in water or wastewater.The idea to incorporate microalgae as anagent of bioremediation was firstlyproposed by Oswald and Gotaas in 1957[2] and microalgae have been widely studiedcurrently. The cultures with natural watermedium would offer several distinctive

advantages: (1) with no artificial medium,algae growth won’t generate additionalcontamination for environment. (2) Aftersecondary or tertiary treatment discharge,we could take advantage of the remainingnutritious in the water body and to reducethe nutrition furthermore. This environmentalfriendly process offers a substantial potentialsource of algae biomass to provide bioenergyand to reduce the greenhouse gas, carbondioxide.

Figure 3. Algae biomass in the reactors: total suspended solids (A), volatile suspendedsolids (B), fixed suspended solid (C).

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3.6 Ecological SustainabilityThe symbiotic life style involves mutual

ecological, physiological, morphological andmolecular adaptations between the partners.An alga has that symbiotic nature; for anexample, the bacteria provides algae withbioavailable forms of inorganic for algaegrowth; at the same time, the algae biomasswould be available for bacteria to utilizeand this mutual relationship was naturallyoccurring in any aquatic ecosystem on earth.Another example: algae metabolism acquirevitamin B12 (cobalamin) primarily as acofactor through a symbiotic relationshipwith bacteria, owing to algae’s loss of thevitamin B12-independent producing enzyme[4]. Our growth system is one of the simplestalgal-bacterial symbiotic units; the naturalwater medium is a practical resource for algaegrowth in lab or plants as well as in nature.

The symbiosis design of natural mediumpractice has ecological perception and wouldbe competitive for algae biomass applicationsin near future.

Even more if we could apply thisknowledge to any water body such asreservoirs with harvesting technology i.e.micro/macro filtration, the massive algalbiomass in water body has gigantic potentialfor the production of bioenergy, whichcame early from Prof. Oswald’s idea [3];Vilchez et al. [2]. The energy conversiontechnologies for utilizing microalgae biomasscan be separated into two basic categories ofthermochemical and biochemical conversion.Figure 4 depicted the outline of conversionprocesses; these details were adopted fromliterature [1, 7-9, 25-28]. Consequently algalbiomass has a tremendously prospectivepotential.

Figure 4. Option for algae biomass to bio-energy.

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Climatic change will potentially bringunprecedented challenges of mankind fromthe combined effects of the uncertainty inthe climate system to increase temperature,to change water and carbon cycle [1, 25, 26]etc. Addressing these challenges will require asustainable strategy to face the impacts ofclimate change and to reduce CO2 which wasdocumented as a primary greenhouse gas[26]. Algae biomass production could beapplicable to the utilization of greenhousegas. Carbon dioxide, the most notoriousfugitive of the greenhouse gas [23-25] issteadily increased due to anthropogenicactivities. Currently, algal bio-fixation isbroadly recognized as one of the bestalternatives to reduce CO2. There are severalunique advantages for this approach: (1)the best growth rate among the plants, (2)low impacts on world’s food supply, (3)specificity for CO2 sequestration withoutgas separation to save over 70% of total cost,(4) excellent treatment for combustion gasexhausted with NOx and SOx, (5) high valueof algae biomass including of food, nutrition,pharmaceutical material, fertilizer, aquaculture,biofuel, etc. [1, 26]. Consequently, algaebio-fixation technology could be the beststrategy for greenhouse gas reduction.

Although algal CO2 sequestration offersthe opportunities to reduce the concentrationfrom air, all the recent research and scientificevidences are done on extra manmadeaddition. Those technologies didn’t absorband captivate the atmospheric CO2 directly.

This study explored natural watermedium to grow algae with no additionof artificial medium and any extramanmade CO2 source; the significant biomassproduction was achieved as aforementioned.Since, our system tried to mimic the naturalwater environment. Compared to the generalalgae studies of CO2 fixation, this studycould possibly develop to the most ecological

way to absorb CO2 which completelycame from atmosphere directly. If we couldadapt this concept to the water-bodies i.e.ponds, lakes, reservoirs and oceans, it wouldbe an applicable method to reduce CO2

concentration in nature.Ramaraj et al. [25] demonstrated that the

effectiveness of the niche of freshwater algaeas a remover of CO2 from the ecosystem,and its potential to perpetuate carbon cycleare to be systematically. However microalgaeare photosynthetic and hence capable ofleading independent life, they are symbioticallyclosely associated with non-photosyntheticprokaryotes and protists in a complexmanner. This ecological situation mayprovide sustenance to microalgal life andmetabolic processes, including CO2 fixation,though it can limit the net microalgal CO2

fixation efficiency at any instant [24, 25].Furthermore, this biotech application was inthe most natural and the most ecologicalway to accomplish the “self-design” and“self-function” of the nature. Therefore,algae growth had great potential for CO2

bio-fixation and deserved a close look, andthis study might develop to be one of themost promising and environmental-friendly technologies for CO2 capture fromair.

4. CONCLUSIONSThis study was undertaken as an effort

to develop a technique which couldprovide information regarding algalbiomass production in a natural environment.The mixed algal growth units were imitatedthe ecosystem in lab. TSS and VSS techniqueswere adopted as the indexes to estimatealgal biomass. The algal bacterial symbiosissystem showed that the biomass yield wasslightly in 10 days (reactor P1) and 7 days(reactor P2) detention time. The 10 daysdetention time was consumed less amount

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of water medium to reduce the cost. Theshort detention time of 4 days (reactor P3)was produced less biomass. The resultsconfirmed that the detention time hassignificant role in algae growth.Furthermore this paper briefly heightedthe potential of natural medium foralgae biomass production which canbe applicable difference aspects suchas economic benefits, environmentaladvantages and ecological sustainabilityincluding symbiosis, green-tech of bioenergyand CO2 sequestration. Consequently, weshowed the production of algae on naturalwater medium is possible and feasible inthe ecological sense.

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