Nidhin Sreekumar ,Ajit Haridas ,G.S.Godwin ,N.Selvaraju ,*

1 National Institute of Technology(NITC),Calicut,India

2 National Institute for Interdisciplinary Science and Technology(NIIST),Council of Scientific&Industrial Research(CSIR),Thiruvananthapuram,India

1.Introduction

Theneed for clean,biodegradableand carbon neutral alternativeenergy has led the R&D focus on microalgae as a biofuel source[1,2].Microalgae are considered a future source of biofuel,because it does not competewith agriculturalland and fresh water resources,and hasthepotential to be cultivated in quantities required for substituting mineral oil.However,at productivities achieved so far,algal oil production costs are several times that of mineral oil.Mass culturing methods of algae have to be optimised along with the cost optimisation of culturing methods so that the algal biofuelscan compete with the ever-depleting and polluting,fossil fuels[3].A proper optimisation of all the steps involved in the production process is vital for improving the economics of the whole process[4].Raceway ponds are shallow artificial ponds used in the cultivation of microalgae which is the most economical method for algal culture[5,6].The shortcomings of raceway ponds are that they are highly susceptible to environmental factors[7].A closed photobioreactor could be a better companion while studying the growth parameters of algal culture[8].The simultaneous studies of parameters of growth in a closed system along with a larger open system could reveal valuable insights into maximisation of lipid production and cost optimisation.

The preference of microalgae is due to its less complex structure and generation time also they are filled with protein,lipid and various other high value products such as nutraceuticals and fatty acids[9]Similar to any photoautotrophs microalgae also derive its energy from solar irradiance and process the energy through photosynthetic pathways to stored energy[10].Contemporary research efforts with microalgae are concentrated on finding high yielding species and have yielded lipid up to around 20%with a biomass yield of around 1000 mg·L−1.The selection of an algal species is a significant step in the commercialization of bio-oil as a source of fuel[11].There are many methods for selection of algae[12].The R&D effort on microalgae has followed two approaches:a)collecting,culturing and cultivating natural algal strains that have high lipid content or b)genetic engineering of algae to improve lipid content.Natural selection engineering is a new approach to the problem.In our novel method,a phototrophic consortium rather than specific strains is cultivated.The consortium is modified through natural selection by control of process conditions so that it has desired characteristics.To improve the photosynthetic rate and lipid production of microalgae,daytime cell division should be arrested and instead,night time division should occur since the lipid accumulation in microalgae cells happens during daytime[13].Daytime cell division can be arrested by facilitating a nutrient exhausted condition[14].The energy harnessed by photosynthesis during the day should be bypassed to storage instead of cell division.Microscopic studies revealed that it is possible by adjusting nutrient distribution pattern.

Besides CO2and light,algae require nutrient to grow,nitrogen(N)and phosphorous(P)being the most important ones[15].According to Red field ratio,the nutrient availability should be equal to or higher than 16:1(N:P)[16,17].The usage of indigenous,alternative sources of nutrients like sewage or seawater[18],apart from the vast diversity and availability,can significantly reduce the running cost of nutrient additions or water cost[19,20].The amount and kind of nutrients needed for algal growth depend very heavily on the species,but as an indication for the most important nutrients,about 5%(typically 7%–8%)of algal dry matter consists of nitrogen(N)and 1%of phosphorous(P)[13].Ammonium chloride(NH4Cl),Potassium dihydrogen orthophosphate(KH2PO4)and trace metal solution are the nutrients used.In this work,our aim is to get a consortium that has high lipid productivity;accordingly the nutrient concentration is changed.Ammonium chloride acts as the nitrogen source,and potassium dihydrogen orthophosphate acts as the phosphorous source.

2.Methodology

2.1.Experimental setup

2.1.1.Photobioreactor

A tubular photobioreactor(PBR)of volumetric capacity 10 L,made of transparent polycarbonate tubes was made to culture the algal consortium(Fig.1(a)).The irradiant area was 0.5 m2and was illuminated by two 40 W fluorescent tubes providing lighting equivalent to 418.92 μmol photons·m−2·s−1(31000 lx)on the tube surface.The temperature of the system was uncontrolled and closely monitored,(28 ± 3)°C.The circulation of the media was carried out using a peristaltic(Watson-Marlow)pump.Continuous monitoring of dissolved oxygen and p H was carried out using(Ponsel)probes.The aeration tank was provided with a constant mechanical stirring,at 20 r·min−1,just to prevent the settling of microalgae inside the aeration tank and not causing any shear damage to the cells.The CO2required was continuously supplied by compressed air sparged into the aeration/degassing tank.

2.1.2.Raceway pond reactor

Fig.1.(a)Schematic diagram of tubular photobioreactor.(b)Schematic diagram of raceway pond reactor.

An algal raceway pond reactor(RPR)made of opaque fibreglass was used to cultivate the microalgae consortium(Fig.1(b)).It had an irradiant area of 1 m2;liquid depth is 0.30 m and a volumetric capacity of 300 L.Agitated by a two bladed paddle system controlled by a three phase regulator.The reactor was fed with only fresh sea water,and the composed nutrientsare given overnight through a peristaltic(Watson-Marlow)pump.Harvesting is done on a daily basis of 50%to give a two-day cell retention time.The incident radiation was monitored using a pyranometer.A photosynthetically active radiation sensor(PAR)was provided outside and at the floor of the RPR.Continuous monitoring of dissolved oxygen and p H was carried out using outdoor probes(Mettler Toledo,InPro Series).Carbon dioxide was supplied to the RPR as the primary carbon source.The CO2addition was feedback controlled by p H at 8.0.The daily solar irradiance,on an average,ranged from 0.1 to 1.5 CCM(5000 μmol photons·m−2·s−1)over 830–1730 h.The temperature fluctuated diurnally,based on the solar irradiance and was controlled only by natural evaporation.The media temperature during the experiment was ranging from 24°C to 29°C.

2.2.Source of water and microorganism

The use of natural sources of nutrient would aid us in having a positive effect on the ergonomics of the developed system.The sources of water used in this work are(1)sewage sludge,collected from Sewage Treatment Plant,Sewage Farm,Valiyathura,Thiruvananthapuram,Kerala,India(8°27′34.2966″N,76°56′12.8076″E)and(2)sea water,collected from Vizhinjam Wharf,Thiruvananthapuram,Kerala,India(8°22′24″N,76°59′23″E).The naturally occurring algal consortium from each water source was enhanced and modified to yield lipid with nutrient stress and temporal phase separation protocol.The use of indigenous,naturally occurring consortia,consisting of Arthrospira sp.along with diatoms,Scenedesmus sp.,Chlorella sp.and Enteromorpha sp.,improves the chances of survival and will have the highest biomass yield asthe organismsare well acclimatized with the nutrient conditions[8,21].

2.3.Nutrients and its composition

Besides CO2and light,algae require nutrient to grow.The following formulation was used in RPR daily and was based on the water analysis.NH4Cl—2.8 g,KH2PO4—0.8 g,trace metal solution(16.7 ml)from a stock containing per litre:FeCl3—2000 mg,MnCl2·2H2O — 500 mg,ZnCl2— 50 mg,NH4Mo7O24·4H2O — 50 mg,CoCl2·6H2O — 50 mg,CuCl2·2H2O — 50 mg,EDTA — 500 mg,NaSeO3—100 mg,H3BO3—50 mg,AlCl3—50 mg,HCl—1 ml.The nutrients were made up to 1 L and pumped at a flow rate of 100 ml·h−1.In order to shift the cell division pattern,nutrient addition to the reactor was done during night time.In PBR sewage was used as primary nutrient.

2.4.Microscopic observation

Regular monitoring of samples with an epi fluorescence microscope(Leica DM2500)gives valuable responses[22].Photoactive algae in consortia were observed by auto fluorescence of the consortia under UV illumination.Accumulations of lipid in cells in the consortia were observed by staining with Nile Red dye,and fluorescence with Y3 filter[23].The gross morphology of the algal consortia was observed with a Nikon stereo microscope.Underwater attached growth in the raceway pond was observed with a borescope.

2.5.Total biomass quantification

Total suspended solids(TSS)are solid materials including organic and inorganic that are suspended in the water i.e.neither dissolved nor settled[24].A known amount of sample is filtered carefully using the filtration unit employing a pre-weighed Whatman No.1 filter paper pre-dried at 105°C.The residue collected along with the filter paper is then transferred to a pre-dried and weighed silica crucible and heated at 105°C for 1 h.After cooling,the crucible and its contents are weighed again,and TSS is calculated from the difference in mass.The formula used:

2.6.Quantitative lipid content estimation:(chloroform–methanol 1:1)

Standard isation of extraction procedure has been carried out with various ratios of chloroform–methanol mixture.Maximum lipid extracted with 1:1 ratio of chloroform–methanol solvent.100 mg of lyophilized sample is weighed accurately to which 4 ml methanol,4 ml chloroform,2 ml water and mix well.Centrifuge at 2000 r·min−1for 10 min,at 5 °C to 25 °C.Remove the upper layer(methanol/water)using a Pasteur pipette.Transfer the lower layer into a transparent tube with a Pasteur pipette.Leave the solids behind,if present.Re-extract the solids left at the bottom using 2 ml of 1:1(v/v)chloroform/methanol.Pass the combined layers through anhydrous sodium sulphate using Whatman No.1 filter paper in a funnel,into a pre-weighed container suitable for a rotary evaporator.Remove the solvents using a rotary evaporator under reduced pressure,at 40°C.Calculate the mass of the lipid:

2.7.Physico-chemical analysis

The p H and dissolved oxygen were determined by portable probes(Ponsel).Nitrate and phosphate were measured using a UV–visible spectrophotometer[25].EDTA titrimetry was followed for calcium and magnesium estimation[25].Estimation of silicate was done by molybdosilicate method[25].BODand COD were estimated by standard methods[24].

2.8.Statistical analysis

For batch studies as well as pilot plant studies,duplicates were carried out to confirm the findings.For PBR studies,all the nutrient loads were carried out twice to confirm the results.For DNA studies in RPR,the results were confirmed using replicates.The data acquired were subjected to statistical analysis for variability check and standard errors.The level of replication was subjected to T-Test for assuring the acceptance of the results.

2.9.Total genomic DNA estimation

Optimised DNA extraction procedure was used on RPR samples collected at various time intervals in a day.Samples were collected at every 10 am and 5 pm to investigate the cell division pattern.Collected samples were immediately subjected to further DNA extraction procedure.

1 L of the microalgal sample was collected,settled,concentrated and fed into amechanicalattritor for cell disruption.The cell suspension was collected and stored in a clean 2 ml Eppendorf tube.Organic solvents such as phenol and chloroform interact with hydrophobic components of protein and lipoprotein,causing denaturation.The precipitate of denatured cellular material remains within the organic phase or collect at the interface between the two phases which is separated by centrifugation.Lipids will partition effectively into the organic layer.Chloroform was used to separate aqueous and organic phase and also help phenol to remove proteins.Isoamyl alcohol was used as an antifoaming agent during extraction.The ratio of phenol:chloroform:isoamyl alcohol was 25:24:1.

The DNA from cell lysates and the suspension was recovered by the use of isopropanol.With the aid of isopropanol,reversible denaturation(i.e.,the helical structure is extensively destroyed)and subsequent precipitation of DNA occur.Ethanol(70%)was used for washing the DNA because alcohol soluble salts are removed in this step and making final DNA clearer.Absolute alcohol could not be used due to its high evaporation rate.The DNA pellets were resuspended in TE buffer or Milli-Q water(if stored).Suspension was analysed in a Biophotometer.

2.10.Growth modelling

The growth dynamics of microalgae in consortia is significantly different than that in a pure culture.But the growth rate of consortia is still a function of irradiance and follows equation μ=f(Iavg)where μ is the specific growth rate(d−1)and Iavgis the average irradiance received by the culture(μmol·m−2·s−1).The biomass concentration was determined daily by measuring absorbance at 750 nm with a spectrophotometer(UV/Vis Spectrophotometer,Thermo Fisher Scientific Inc.,USA).Incident irradiance was measured by a PAR sensor.Assuming monod kinetics,specific growth rate,μ follows as:

Here μ is the specific growth rate(d−1),μmaxis maximum specific growth rate(d−1),Iavgis the average irradiance(μmol·m−2·s−1),and Ikis irradiance saturation constant(μmol·m−2·s−1).The average irradiance was calculated from the expression:

Here,Kais the extinction coefficient of the biomass,I0is the incident irradiance on the culture surface,and φeqis the length of the light path from the surface to any point in the bioreactor.For RPR system,ideally,φeq=d,the depth of RPR.If φeq＜ d,then light does not reach the bottom of the RPR.For tubular photobioreactor systems,φeqis a function of tube diameter φ and the angle of declination(θ)of the light source from the vertical.In our case,θ is 0°.φeq=φ/cosθ.

For industrially viable large scale production of microalgae,volumetric productivity(Pv)and areal productivity(Pa)of lipid rich biomass must be maximised.The volumetric productivity,Pvis represented as Pv=μX.Here,Pvis the volumetric biomass productivity(mg·L−1·d−1),μ is the specific growth rate(d−1)and X is the biomass concentration of culture(mg·L−1),which was measured regularly.Consecutively,areal productivity(Pa)needs to be calculated.The volumetric productivity calculation is common for both open pond system and tubular PBR system,but areal productivity varies as the incident area is different.For tubular PBR,areal productivity is calculated as:

Here,Pais areal biomass productivity(mg·L−1·d−1),Pvis the volumetric biomass productivity(mg·L−1·d−1), φ is the tube diameter(m)and N is the number tubes in the array.

Similarly,in an open pond system,areal productivity depends on the area of the pond exposed.

Here,A is the exposed area(m2)and d being the depth of the pond(m).

3.Results

3.1.Chemical analysis of nutrient sources

Table 1 depicts the physicochemical estimation of the seawater collected for the use as culture media.The supplementary nutrients were decided based on this evaluation.Table 2 illustrates the physicochemical values of the sewage sludge collected from the sewage farm.A 50%diluted sludge has the nutrient composition as represented in Table 3.

Table 1 Physico-chemical parameters of collected seawater

Table 2 Physico-chemical parameters of collected sludge

Table 3 Chemical estimation of 1:1 diluted sludge(used as media)

3.2.Cell disruption

The samples were subjected to cell disruption by different methods like physical grinding and attrition with zirconium beads.The time duration of beating,weight of zirconium beads and speed of rotor rotation were optimised.The attritor was calibrated at 1500 r·min−1for 20 min with 150 g of zirconium beads.With this calibration,bead beating seems to be very effective for the better quantity,but purity was not much improved.Total gDNA extraction via this method results in the improved quantity of total genomic DNA than extraction but purity obtained was reduced.Another method adopted for cell disintegration was a cell beater that has the same principle as that of an attritor the difference being the beads used and the speed of operation.The beads used were glass beads of 0.1 mm to 1 mm,and the speed of operation was 5000 r·min−1.This method gave sufficient purity and greater quantity.The results are as depicted in Table 4.

Table 4 Optimisation of cell disruption

3.3.Biomass and lipid pro files

Fig.2(a)displays the biomass pro file of culture in PBR with sewage under various lighting cycles.Fig.2(b)depicts the Biomass,Dissolved oxygen and p Hpro file of microalgal culturein a PBRat different nutrient loadings.The light exposure was16 h of light and 8 h dark.The nutrient load provided was NH4CL— 52.22 mg·L−1;KH2PO4— 3.75 mg·L−1;Trace nutrients— 1.2 mg·L−1and a one-time addition of 1 g NaSiO2for enhancing the growth.The nutrient levels were set in accordance with the Red field ratio,an atomic ratio of nutrients found in phytoplankton,N:P=16:1[8].

Fig.2.(a)Biomass pro file of culture in PBR with sewage under various lighting cycles(light hours:dark hours).(b)Biomass,p H and DO pro file of seawater culture at various nutrient concentrations in PBR.(c)Batch growth curve of consortia with sewage.

The continuous analysis of the microalgal sample collected from the raceway pond reactor,under seawater as nutrient source,twice daily over a period of 10 days was carried out in triplicate and the datas obtained is as shown in Fig.3.The biomass pro files of the system during the period of study are presented in Fig.3.The quantification of Total gDNA of the sample is done by the optimised CTAB protocol for microalgal species.The expected DNA quantity plotted gives an idea of how the genetic matter is increasing overnight.The Total gDNA is an indication of the microalgal cell division[26].The process of daily 50%harvesting halves the genetic matter of the whole system,which is readjusted by the overnight cell division.

Fig.3.Biomass pro file and Total gDNA quantity over the days and the Expected morning values.

A plot of the Total genomic DNA values over the days along with the calculated Biomass values during the morning will give an overview of how the biomass growth responds.The difference of the TgDNA values between morning and the evening samples describes the cell division during daytime and the difference of the morning value to the previous day evening value represents the overnight multiplication of genetic matter.The comparison of lipid pro file and the DNA difference values over the period of study are given in Fig.4.In Fig.4,Day difference(E–M)denotes the difference between Total gDNA quantities of evening samples and morning samples of the same day(cell division during day),Night difference(M2–E1)denotes the difference between Total gDNA quantities of next day morning samples and evening samples of the previous day(cell division during night).

Fig.4.Total lipid pro file versus day time and night time DNA division.

A statistical analysis of the results shows ample replicability,and it was also observed that the replication was in accordance and supports the hypothesis.

Fig.5 depicts the microscopic observations of lipid yielding consortia present.

Fig.5.Self-settling flocs of lipid producing microalgae consortium.

3.4.Effect of pH

At p H 6.5 condition biomass production was 140–150 mg·L−1·d−1.Whereas at p H 8.1 it was 90–100 mg·L−1·d−1(Fig.6).At p H 6.5,total lipid production of biomass was up to 14%.But at p H 8.1,total lipid production of biomass improved up to 20%.Under N/L 2 condition with p H 6.5 total lipid production of biomass was up to 14%while with p H 8.1 it was20%(Fig.6).A 30%higher total lipid production was obtained under p H 8.1 and a higher biomass was obtained at a p H of 6.5.

Fig.6.Biomass and lipid pro file at different p H.

4.Discussions

The objective of this study was to check the hypothesis of improving the lipid yield of microalgal consortium by providing temporal phase separation of cell division.By nature,cell division is an energyintensive primary process of any cell[27,28].The energy harnessed by a cell from photosynthesis will be primarily diverted to cell division,under nutrient-rich conditions,followed by biomass production and the leftover energy will beconverted to storage food,protein or lipid depending on the ambient conditions[29].To usealgaeasalipid producer,it is required to bypass this energy consumption pattern,and to harvest the lipid,as and when it is produced,before it is lost to cellular respiration.To achieve this,temporal phase separation of energy harvesting phase and energy expending phase,nutrient loading pattern was altered.To arrest cell division(energy expending phase)during daytime(energy harvesting phase),a nutrient deplete condition was provided.By providing nutrient deplete condition during photosynthesis period has a two-fold effect,the consortia will produce maximum storage food as well as the production cycle will be leaning towards lipid production rather than protein.On the other hand during night time,to facilitate cell division,nutrient loading was performed.Biomass harvest was aptly placed between these two periods,to extract maximum amount of lipid.

The use of indigenous sources of water other than fresh water reduces the load on the overall economics of the system[30].In order to accomplish this mission,non-potable and potentially nutrient rich sources like municipal sewage and seawater were considered.The physicochemical analysis of the sources was carried out in order to fix an appropriate supplementary nutrient.Tables 1,2 and 3 depict the analysis results.The nutrient concentration should be adjusted such that the availability of phosphate and nitrogen follows the Red field ratio[31].The phosphates are essential for proper cell division whereas nitrogen is one of the major building blocks in cellular mass development.

In order to make algal biofuel economically viable at practical biomass yields,the phototrophic biomass should have the following characteristics:

a)should be stable and perpetual

b)should have inexpensive and complete separability from water either by coarse straining or rapid gravity settling for harvesting

c)should be neutrally buoyant during daytime for resuspension in raceway ponds

d)should have high lipid content

e)there should be only limited grazing activity

f)should be capable of cultivation in clay lined open raceway ponds.

By employing the natural selection engineering protocol in conjecture with temporal phase separation we produced biomass which are automatically stable and perpetual,provided the process conditions are held stable.Inherent variability of seasonal temperatures,irradiances and the length of daylight,in case of RPR,cannot be controlled.Therefore,there will be variation in biomass.The day to day changes in irradiance leads to changes in yield rather than in diversity.When the culture is perpetual,there is no need to raise seed cultures to inoculate the cultivation ponds.Hydrodynamic selection pressures are applied to selected algae that are easily harvested.Even though different water sources were used the consortia was dominated by Arthrospira sp.along with diatoms,Scenedesmus sp.,Chlorella sp.and Enteromorpha sp.The Arthrospira is a species which can survive in seawater,brackish as well as nutrient rich conditions[32].

For seawater culture with 16:8 light–dark cycles,the biomass,p Hand DO pro file at different nutrient concentrations in PBR are as depicted in Fig.2(b).Ahigher nutrient load would generally result in higher biomass,but there is a higher limit to the biomass increase based on light availability and p H.The consecutive batch experiments revealed that the biomass tends to fall at higher p H.There is aneed to adjust the p Hat around 8.8 so as to get maximum biomass[33].The batch studies reveal the effect of pH over biomass growth and lipid production(Fig.6).It was evident that the levels of p H need to be adjusted to optimise the growth in the system.Generally lower p H favours an increase in biomass but lipid accumulation is favoured at higher p H.The p H can be adjusted by a feedback control over the CO2pumped into the system.From the batch experiment,it was also observed that the culture was saturated at 5.625 mg·L−1of phosphate concentr ation and did not respond to further increase.The culture may be considered to be light limited inside the PBR.

A semi-continuous process on RPR was devised to facilitate maximum lipid accumulation in algae.In order to promote maximum lipid recovery,biomass harvest was introduced before the cells are allowed to utilise the energy for cellular respiration.Also,the cell division was restricted to night time by a time controlled addition of nutrients since the lipid accumulation in microalgae cells happens during daytime through photosynthesis under stress conditions.In order to shift the cell division pattern,nutrient addition to the reactor was done during night time and nutrient consumption pattern in there actor shows added nutrients were depleted within 12 h of nutrient addition.Figs.3 and 4 shows the effect of night time nutrient loading.In-spite the 50%harvest of biomass,the cell number was auto adjusted at par with the pre-harvest consortia.However,the biomass of the consortia is not drastically improved as biomass increase requires energy from photosynthesis(Fig.3).

The nutrient rich night conditions favour cell division and the nutrient deplete daytime arrests the cell division.This condition combined with evening time biomass harvest ensures minimum loss of lipid to cellular respiration.Fig.4 gives the effect on lipid.It can be observed that whenever the cell division is less during the day,the harvested lipid levels are high,which confirms our hypothesis.

Table 5 Statistics of Biomass pro file of seawater culture at various nutrient concentrations in PBR,corresponding to Fig.2(b)

Table 6 Statistics of Biomass pro file and Total gDNA quantity over the days in RPR,corresponding to Fig.3

Statistical analysis(Tables 5–7)reveals that the replication of experiment supports the conclusion as well proves the replicability of the experiments under similar conditions.

Table 7 Statistics of total lipid pro file versus night time DNAdivision in RPR,corresponding to Fig.4

An inverse association can be formed with cell division during day time and the lipid produced.Fig.7 depicts the relationship between lipid production and daytime cell division.

Fig.7.Relation between lipid and cell division.

5.Conclusions

In the quest for sustainable alternate energy,microalgal biodiesel is the most promising outcome which could connect the old technologies of internal combustion engines to new age pollution freetechnologies.A novel approach of naturals election of consortia for lipid production was established in this work.The use of consortia would bring down the production cost of biodiesel as they do not require sterile conditions.The lipid maxim is ation of the consortia was successfully carried out with novel temporal phase separation protocol in combination with p H control.The consortia yielded up to 22%lipid with biomass of around 800 mg·L−1in PBR.In outdoor open pond the yield was around 12%–15%lipid and around 600 mg·L−1biomass.From the work it can be confirmed that the temporal phase separation has a positive lipid accumulation capability.Moreover approximately 40%increase in lipid levels could be achieved,in both modes of cultivation,without much loss in biomass.

Acknowledgements

The raceway pond was set up aspart of CSIR-NMITLIproject“Biofuel from marine microalgae”,at NIIST by Dr.Ajit Haridas.Authors are thankfulto thefollowing for their contributions,Dr.Aneesh C.,Mr.Faisal M.,Mr.Rajendra Prasad,Ms.Farza Naushad,Ms.Sayana C.R.,Ms.Anie Mariya,Mrs.Priya P.,Dr.Anju S.,Dr.Anupama P.and Dr.Pradeep S.

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