1-s2.0-S0960852414018288-main-2-2 - copia

woa

Brent M. Peyton , Ronald C. SimsaUtah State University, Department of Biological Engine

ducts anobiologyl Engineeical and

lm gructionphase

ynthesi

as an inexpensive source of nutrients, and as a biological wastewa-ter treatment alternative (Cai et al., 2013; Sturm and Lamer, 2011).Microalgae can utilize nutrients in wastewater for growth to gen-erate considerable amounts of biomass. However, recovery ofmicroalgae from the liquid medium is difcult and represents asubstantial capital cost in suspended cultivation systems

uently there is ams. Algalreactor (

eactor (RAAlgaewheel have been developed, and algal biolm growtonstrated in bench and pilot scale operations (ChristensoSims, 2011, 2012; Gross et al., 2013; Kesaano and Sims,Pizarro et al., 2006). However, there is still limited fundamentalinformation on algal biolm physiological processes and growthespecially in wastewater remediation.

Widespread application of algal biolm-based systems is alsolimited but can be promoted through integration of wastewatertreatment with the production of valuable bioproducts from theharvested algal biomass. Algal biomass composition (i.e., lipid,

Corresponding author at: 4105 Old Main Hill, Logan, UT 84322, United States.Tel.: +1 435 797 2785.

E-mail address: [email protected] (M. Kesaano).

Bioresource Technology 180 (2015) 715

Contents lists availab

Bioresource T

els1. Introduction

Cultivation of microalgae in wastewater streams has been pro-posed as a means of reducing competition for freshwater sources,

(Greenwell et al., 2010; Hoffmann, 1998), conseqgrowing interest in attached algal growth platforbased systems such as the rotating algal biolmalgal turf scrubber (ATS), revolving algal bior

http://dx.doi.org/10.1016/j.biortech.2014.12.0820960-8524/ 2015 Elsevier Ltd. All rights reserved.biolmRABR),B), andh dem-n and2014;Article history:Received 2 October 2014Received in revised form 22 December 2014Accepted 23 December 2014Available online 31 December 2014

Keywords:MicroalgaeBiolmsWastewaterDissolved inorganic carbonBiofuels

Microalgal biolms grown to evaluate potential nutrient removal options for wastewaters and feedstockfor biofuels production were studied to determine the inuence of bicarbonate amendment on theirgrowth, nutrient uptake capacity, and lipid accumulation after nitrogen starvation. No signicantdifferences in growth rates, nutrient removal, or lipid accumulation were observed in the algal biolmswith or without bicarbonate amendment. The biolms possibly did not experience carbon-limitedconditions because of the large reservoir of dissolved inorganic carbon in the medium. However, anincrease in photosynthetic rates was observed in algal biolms amended with bicarbonate. The inuenceof bicarbonate on photosynthetic and respiration rates was especially noticeable in biolms thatexperienced nitrogen stress. Medium nitrogen depletion was not a suitable stimulant for lipid productionin the algal biolms and as such, focus should be directed toward optimizing growth and biomassproductivities to compensate for the low lipid yields and increase nutrient uptake.

2015 Elsevier Ltd. All rights reserved.a r t i c l e i n f o a b s t r a c tbUniversity of Minnesota, Department of BioprocMontana State University, Department of MicrdMontana State University, Department of CivieMontana State University, Department of Chem

h i g h l i g h t s

Bicarbonate added to enhance algal bio Bicarbonate added to trigger lipid prod Nutrient removal inuenced by growth Bicarbonate addition enhanced photosering, Logan, UT 84322, United Statesd Biosystems Engineering and West Central Research and Outreach Center, St. Paul, MN 55108, United Statesand Center for Biolm Engineering, Bozeman, MT 59717, United Statesring and Center for Biolm Engineering, Bozeman, MT 59717, United StatesBiological Engineering and Center for Biolm Engineering, Bozeman, MT 59717, United States

owth and nutrient uptake.in nitrogen stressed algal biolms.of algal biolms.s in algal biolms.Maureen Kesaano a,, Robert D. Gardner b, Karen Moll c, Ellen Lauchnor d, Robin Gerlach e,e aDissolved inorganic carbon enhanced groaccumulation in wastewater grown micr

journal homepage: www.th, nutrient uptake, and lipidlgal biolms

le at ScienceDirect

echnology

evier .com/locate /bior tech

ce Tcarbohydrate, and protein content) is inuenced by the chemicalcomposition of the medium and the environmental growth condi-tions (e.g., temperature, pH, and light), which subsequently deter-mines the by-products that can be synthesized. Conventionally,microalgae grown as feedstock for biofuels require a two stage pro-cess where biomass accumulation occurs under nutrient-rich con-ditions followed by an environmental challenge to inducesecondary byproduct accumulation (e.g., tri-acylglycerols as energystorage compounds) (Su et al., 2011). Nutrient starvation is typi-cally employed as an environmental stress to stimulate lipid bio-synthesis in microalgae cultures (Devi et al., 2012; Rodol et al.,2009; Sharma et al., 2012). However, stimulation of lipid produc-tion in algal biolms as a result of nutrient starvation has not beenas successful as in suspended cultures (Bernstein et al., 2014;Schnurr et al., 2013).

Furthermore, information on the use of other lipid inducingtechniques such as chemical addition, pH stress, and temperatureeither independently evaluated or in combination with nutrientstarvation is limited in algal biolm studies. For example, additionof bicarbonate salts (HCO3) was reported as an effective trigger forlipid production in nutrient limited suspended microalgae cultures(Gardner et al., 2012, 2013; Peng et al., 2014; White et al., 2013).The bicarbonate salts not only induce lipid production, but alsoprovide a stable and readily available source of inorganic carbonessential for photosynthesis and microalgae growth (Chi et al.,2013; Mus et al., 2013; Wensel et al., 2014). In addition, Gludet al. (1992) observed an increase in photosynthetic rates and asimultaneous reduction in respiration rates (17%) in a diatom-dominated biolm community amended with bicarbonate.

The potential use of bicarbonate in minimizing photorespira-tion is especially of interest in algal biolms because of the highO2/CO2 ratios due to localized supersaturated oxygen concentra-tion from active oxygen photosynthesis (Bernstein et al., 2014;Glud et al., 1992). Photorespiration is a competing process to car-boxylation, where ribulose-1,5-biphosphate carboxylase oxygen-ase (RuBisCO) acts as an oxygenase, thereby inhibiting carbondioxide xation and subsequently reducing photosynthetic ef-ciency. The study presented here evaluated the effects of addingdissolved inorganic carbon in the form of 2 mM HCO3 to syntheticwastewater medium to grow algal biolms in order to:

(1) Enhance algal biolm growth, nutrient uptake, and lipidaccumulation during nutrient deplete culturing.

(2) Increase photosynthetic rates with biolm depth within thephotic zone.

2. Methods

2.1. Microalgal biolm culturing and sampling

The chlorophyte isolate Botryococcus sp. strain WC-2B, previ-ously described in Bernstein et al. (2014), was cultured in 8 L lab-oratory scale rotating algal biolm reactors (RABRs) operated at12 rpm and 25 C. Each reactor was comprised of two plastic cylin-drical wheels (10 cm diameter) onto which 3/16 inch (diameter)untreated cotton cord was attached as the biolm substratum.Synthetic wastewater was made to simulate typical mediumstrength domestic wastewater for total nitrogen (TN) and totalphosphorus (TP) concentrations without a carbon source (Metcalfand Eddy, 2003). The medium consisted of 60 mg L1 NH4Cl,150 mg L1 NaNO3, 16 mg L1 Na2HPO4, 15 mg L1 K2HPO4,4 mg L1 KH2PO4, 75 mg L1 MgSO47H2O, 25 mg L1 CaCl2H2O,

1 1

8 M. Kesaano et al. / Bioresourand micronutrients (8.82 mg L ZnSO47H2O, 1.44 mg L MnCl2-4H2O, 0.71 mg L1 MoO3, 1.57 mg L1 CuSO45H2O, 0.49 mg L1Co(NO3)26H2O and 4.98 mg L1 FeSO4).The experimental set up consisted of four laboratory RABRsunder uorescent lights with a photosynthetically active radiation(PAR) of 227 65 lmol m2 s1 on a 14:10 L/D cycle. Duplicatereactors were amended with 2 mM HCO3 in the form of NaHCO3and another duplicate set without HCO3 amendment was culturedfor comparison. The reactors were operated in sequenced batchmode with a 5 day hydraulic retention time (HRT) for a period of18 days, after which nitrogen stress was induced for an additional5 days by replacing all liquid medium with synthetic wastewaterwithout a nitrogen source. For each cycle of hydraulic retentiontime, the reactors were drained, cleaned, and lled with fresh med-ium. Prior to the start of the experiment, the medium was inocu-lated with microalgae and the RABRs operated for 3 days(seeding period) to allow the microalgae to attach to the ropestrands. As shown in Fig. S1, after the seeding period, the RABRswith the exception of the substratum (rope strands) were coveredwith black polyethylene sheet to minimize microalgae growth inthe liquid medium. Culturing and sampling was performed undernon-aseptic conditions (open air).

Rope samples with attached microalgae were excised foroxygen microsensor measurements, microscopy characterization,biomass dry weight measurements, and lipid analysis. Biomass celldry weights (CDW, gcdwm2) were obtained by removing the bio-lm from a known length of cord into a pre-weighed aluminumweigh boat using a at end spatula. The biomass was dried at70 C for 18 h until the biomass weight was constant. BiomassCDWs were calculated by subtracting the dry weight of the ovendried boat with biomass and normalizing by the total cylindricalsurface area for the length of cotton cord substratum excised.

2.2. Water quality monitoring

Nitrate (NO3), nitrite (NO2), and orthophosphate (PO43) con-centrations were monitored in the bulk medium and measuredby ion chromatography (IC) using a Dionex IonPac AS22 carbonateeluent anion-exchange column set at a ow rate of 1.2 mL min1.IC data was analyzed by Chromeleon 7 Chromatography Data sys-tem (CDS) software. Ammonium (NH4+-N) concentrations weredetermined according to the 2-phenylphenol method (Rhineet al., 1998) with a BioTek PowerWave XS microplate reader (Ver-mont, USA) at an absorbance of 660 nm. The dissolved inorganiccarbon (DIC) was measured on 8 mL ltered (0.2 lm pore size l-ters) medium samples using a Skalar FormacsHT/TN TOC/TNanalyzer (model CA16, Netherlands) and Skalar LAS-160 autosam-pler. DIC was quantied using peak area correlation against a stan-dard curve from a bicarbonate-carbonate mixture (Sigma Aldrich).Culture pH and optical density (OD) measurements were takenusing a standard laboratory Accumet pH electrode (FisherScientic) and Genesys 10 UV-Model 10-S spectrophotometer(Thermo Electron Corporation), respectively.

2.3. Oxygen microsensor analysis

Clark-type oxygen microelectrodes (10 lm tip diameter; OX-10Unisense) and specialized computer controlled hardware (Uni-sense) were used to analyze the reactive transport of dissolvedoxygen with biolm depth under steady-state diffusive conditionscorresponding to light and dark conditions. Photosynthetic rates(coupled with photo-respiration) were estimated using the light/dark shift technique (Khl et al., 1996; Revsbech and Jrgensen,1986). The light/dark shift measurements are valid under the fol-lowing assumptions: (1) initial steady state oxygen distributionis achieved before darkening, (2) oxygen consumption rates before

echnology 180 (2015) 715and after dark incubation are identical, and (3) identical diffusiveuxes are maintained during the measurement time at each posi-tion. Two point calibrations were performed for the oxic conditions

(medium saturated with air) and anoxic conditions (mediumsparged with nitrogen gas).

2.4. Biodiesel analysis

Biodiesel precursors i.e., free fatty acids (FFAs), mono-acylglyce-rols (MAGs), di-acylglycerols (DAGs), and tri-acylglycerols (TAGs)were extracted from dried biomass by bead beating extractionand the biodiesel potential (total FAMEs) was determined by directin situ transesterication according to protocols published byLohman et al. (2013). The total FAMEs and the fatty acid composi-tions of these FAMEs were quantied using gas chromatography-mass spectroscopy (GCMS; Agilent 6890N and 5973 NetworkMS). The FFAs, MAGs, DAGs, and TAGs were analyzed using gaschromatography ame ionization detection (GC-FID; Agilent6890N).

3. Results and discussion

1.79 g m2 day1 for biolms with bicarbonate and biolm with-out bicarbonate added, respectively.

Growth curves for the algal biolms (attached to rope) and mic-roalgae growth in suspension are shown in Fig. 2A. Microalgaegrowth in the bulk medium was negligible over the study periodindicating that covering the reactors with black plastic effectivelyprevented light penetration and minimized growth in suspension.There was no statistical difference observed in growth characteris-tics for algal biolms amended with bicarbonate and biolms thatdid not receive bicarbonate (p value of 0.4517 from t test).Although it was hypothesized that the addition of bicarbonatewould increase the algal biolm growth, this was not observed.With the 8 L medium reservoirs, even the unamended algal bio-lms were not carbon limited, such that bicarbonate addition didnot enhance growth in this reactor system. DIC measurementsremained relatively constant for each 5-day retention time withslight differences observed in the medium concentrations, withthe exception of the rst 3 days, (Fig. 2B).

3.2. Removal of nitrogen and phosphorus from synthetic wastewater

M. Kesaano et al. / Bioresource Technology 180 (2015) 715 93.1. Microalgae growth rate and productivity

Microalgae successfully attached to the cotton cord and grew asa biolm for the entire study period (Fig. S1). The lag phase wasminimized by the 3-day seeding period. The microalgal biolmswere in exponential growth from days 3 to 10 as determined fromlinear the portion of the natural log transformed growth data andthe stationary phase occurred after 10 days of growth (Fig. 1).Curve tting of the growth data also showed that the 1st orderequation provided a better description of the microalgal growthbetween days 3 and 10 compared to the zero order equation withR2 values of 0.946 and 0.999 for biolm amended with bicarbonateand those without bicarbonate respectively (Fig. S2). The maxi-mum specic growth rates measured during the exponential phasewere 0.18|0.07 (mean|range) and 0.20|0.07 day1 for algal biolmsamended with bicarbonate and the unamended control, respec-tively. The maximum areal biomass density measured during thestationary phase was 20.95 and 25.98 g m2 for biolms withbicarbonate and biolm samples without bicarbonate, respec-tively. Additionally, the biolm production rates, calculated asthe total biomass accumulated per rope surface area divided bythe time taken to reach stationary phase, were 1.45 andFig. 1. Algal biolm growth curves from natural log transformed data showing the exponvalues describing the exponential phase of biolms with and without bicarbonate amenusing algal biolms

A basic requirement of wastewater treatment is the removal ofnutrients (i.e., nitrogen and phosphorus) to acceptable limits priorto discharge. Microalgae based systems promote nutrient removalthrough plant uptake and subsequent harvesting of the nutrient-rich biomass from the efuent. In addition, microalgae increasethe medium pH via photosynthesis thereby promoting volatiliza-tion of ammonia and possible precipitation of phosphate ions(Boelee et al., 2012). It should be noted that all the RABRs werecovered in black polyethylene, cleaned, and had the bulk mediumreplaced every 5 days to minimize algal growth in the bulk med-ium, which also minimized the pH increase of the medium result-ing from photosynthesis. Therefore, at the measured pH of8.5 0.15 for medium amended with bicarbonate and 7.97 0.22for medium without bicarbonate respectively, nutrient removalwas attributed to the activity of the biolms.

The synthetic wastewater was prepared with ammonia andnitrate salts as the only nitrogen sources. Initial concentrations oftotal nitrogen and phosphorus in the medium were approximately40 mg-N L1 and 7 mg-P L1, respectively, giving a molar N:P ratioof approximately 13:1. The measured residual total nitrogenential phase (days 310) and stationary phase (days 1118). Insert: equations and R2

dment, respectively.

ce T10 M. Kesaano et al. / Bioresourconcentrations (including NO2-N) ranged from 7.95 to 19.66 and8.20 to 19.72 mg-N L1 for RABRs with and without bicarbonateamendment, respectively. Similarly, nal total phosphorus concen-trations ranged from 3.39 to 3.57 and 3.35 to 3.55 mg-P L1 forRABRs with and without bicarbonate amendment, respectively.The lowest N and P residual concentrations were obtained duringthe retention time cycles corresponding to the exponential growthphase of the biolms (Fig. 3). Therefore, as expected, nutrientremoval from the wastewater was closely linked to algal biolmgrowth i.e., higher removal efciencies were obtained during theexponential growth phase of the biolm compared to the onsetof the stationary phase.

The N and P removal efciency ranged from 27% to 74%(NO3-N), 89% to 100% (NH4+-N), and 19% to 41% (PO43-P) duringthe experiments, with no signicant difference observed betweenliquid samples from reactors amended with bicarbonate and thosethat did not receive additional dissolved inorganic carbon.Similarly, for the entire duration residual N and P concentrationsfollowed the same trend in cultures amended with bicarbonateand those that did not receive bicarbonate (Fig. 3). Completeuptake of ammonium ions was observed unlike nitrate ions inthis study, probably due to preferential uptake of ammonia by

Fig. 2. Growth curves for attached (solid lines) and suspended (dotted lines) microalgaamended with bicarbonate and without bicarbonate addition. Error bars for algal biolmbars for suspended growth represent the range (n = 2). Vertical dotted lines represent eechnology 180 (2015) 715microalgae compared to nitrate (Eustance et al., 2013). Microalgalcultures supplied with mixed nitrate and ammonium sources mayrepress NO3-N uptake due to feedback inhibition, since ammo-nium is an end product of assimilatory nitrate reduction(Crofcheck et al., 2012). Similar to the algal biolm growth results,phosphate and nitrogen removal rates were not inuenced by theaddition of bicarbonate to the medium. Maximum nutrientremoval from wastewater with algal biolms can be attained viaharvesting at the end of the exponential growth phase preferablyafter 810 days of growth using this RABR system.

The nitrite concentrations observed in solution were probably aresult of incomplete nitrication of ammonia since the algal bio-lms were grown in a non-aseptic oxygenated environment(Fig. 3). An abiotic control was used to verify that the presence ofNO2-N ions was due to biological processes (Table S1). The chemo-autotrophic bacteria involved in nitrication require a carbonsource such as CO2 or HCO3, therefore the reactor with bicarbonatetreatment possibly had more favorable initial conditions for thebacteria to grow, thus the higher nitrite concentrations observed(Fig. 3). However, quasi-steady state concentrations of nitrite wereeventually attained and the difference ceased to be signicant laterin the experiment.

e (A) and dissolved inorganic carbon (DIC) concentrations (B) in laboratory-RABRsareal density and DIC measurements represent the standard deviation (n = 4). Errornd of 5 day hydraulic retention time.

3-

ende

ce Te3.3. Microalgal biolm photosynthesis and coupled respiration

0.0

0.5

1.0

1.5

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1 3 5 6 8 10 11 13 15 16 18

NO

3--N

, mM

Days

1 3 5 6 8 10 11 13 15 16 180.0

0.5

1.0

1.5

2.0

2.5

NH

4+-N

, mM

Fig. 3. Ammonium, nitrate, nitrite, and phosphate ion concentrations in medium am(n = 2). Vertical dotted lines represent end of 5 day hydraulic retention time.

M. Kesaano et al. / Bioresour3.3.1. Oxygen microproles under illuminationOxygen microproles were taken before and after N-depriva-

tion was initiated, at 18 and 23 days of RABR operations. Steadystate oxygen microproles for biolm samples under light showedan initial increase in oxygen concentrations (compared to equilib-rium with saturated air 260 lM oxygen), which peaked at adepth of 200 25 lm from the biolm surface (biolm/air inter-face) for both N-replete and N-deprived biolms (Fig. 4A and B).Oxygen production in illuminated algal biolms is a result of pho-tosynthesis, and spatial gradients of light are known to affect therate of oxygenic photosynthesis and corresponding oxygen con-centrations in algal biolms (Wieland and Khl, 2000). Photosyn-thetic activity was highest in the upper layers of the biolm anddecreased with biolm depth, possibly due to light attenuationand/or substrate diffusion limitations. Biolms cultured under N-replete conditions had peak oxygen concentrations that were twicethat of N-deprived biolms (Fig. 4A and B). Furthermore, underillumination there were no anoxic zones observed in N-replete bio-lms, an indication that the oxic zone (oxygen penetration depth)extended into the cotton cord substratum (Fig. 4A). In nitrogenreplete systems, the steady state oxygen microproles showedno signicant differences under either light or dark conditions forbiolms with or without bicarbonate (Fig. 4A and C).

On the contrary, differences in steady state oxygen micropro-les were revealed between N-deprived algal biolms with andwithout bicarbonate amendment (Fig. 4B). For example, bicarbon-ate amended biolms had higher oxygen concentrations comparedto biolms that did not receive bicarbonate. This is an indication ofeither higher photosynthetic rates and/or reduced oxygen con-sumption rates due to respiration. Indeed, higher photosyntheticrates and lower areal respiration rates (in the light) were calcu-lated for bicarbonate amended biolm samples under N-stress(Table 1). Additionally, anoxic zones were observed in N-deprivedalgal biolms and the depth of oxygen penetration for the bicar-0.00

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1 3 5 6 8 10 11 13 15 16 18

PO4

-P, m

M

Bicarbonate addedNo bicarbonate added

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0.10

0.15

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1 3 5 6 8 10 11 13 15 16 18

NO

2--N

, mM

Days

d with bicarbonate and without bicarbonate addition. Error bars represent range for

chnology 180 (2015) 715 11bonate amended biolms was 1500 lm compared to 850 lm forbiolms without bicarbonate addition (Table 1).

3.3.2. Oxygen microproles in the darkOxygen is consumed by algal biolms in the dark as a result of

respiration. Assuming oxygen diffusivity is constant, the rate atwhich oxygen decreases (slope) is an indication of the consump-tion rate i.e., a steeper decline in oxygen concentration indicatesgreater consumption and a smaller depth of oxygen penetrationcan be assumed to occur as a result of high heterotrophic activity(Glud, 2008). Steady state oxygen concentrations for biolms inthe dark decreased with depth to anoxic conditions for bothN-replete and N-deprived biolms (Fig. 4C and D). Biolms underN-replete culturing showed a more gradual decline in oxygen con-centration compared to N-deprived biolms, where steeper slopesand shorter oxygen penetration depths were observed. This was anindication of greater potential for heterotrophic oxygen consump-tion in N-deprived biolms compared to N-replete biolms, anobservation that is contrary to what was reported in Bernsteinet al. (2014). The current study provided a longer N-starvation per-iod of 120 h compared to 60 h in the study by Bernstein et al.(2014), which may have promoted greater heterotrophic activityin the N-deprived biolms.

Both before and after N-deprivation, biolm samples amendedwith bicarbonate had greater oxygen penetration depths underdark conditions compared to biolms that did not receive bicar-bonate. Oxic zones of 1100 25 lm and 950 25 lm in depthwere estimated for biolms amended with bicarbonate and with-out added bicarbonate under N-replete culturing. Similarly, oxygenpenetration depths of 650 25 lm and 300 25 lm for biolmsamended with bicarbonate and without added bicarbonate duringN-deprivation were observed (Table 1). This showed that the bicar-bonate amended biolms had lower oxygen consumption in the

Fig. 4. Steady state oxygen microproles for illuminated algal biolms under nitrogen replete (A) and nitrogen deprived (B) conditions. Error bars represent standarddeviation of replicate proles (n = 3); steady state oxygen microproles in the dark for algal biolms under nitrogen replete (C) and nitrogen deprived (D) conditions. Errorbars represent the standard deviation of replicate proles (n = 3); and representative photosynthesis proles for algal biolms under nitrogen replete (E) and nitrogendeprived (F) conditions. Zero depth (surface) is at the algal biolm/air interface.

Table 1Measurements of photosynthetic rates, respiration rates, and relevant depth parameters for laboratory grown microalgal biolms with and without bicarbonate amendment.

Parameter (lmol O2 cm2 s1) Bicarbonate No bicarbonate

N-replete N-deprived N-replete N-deprived

Gross photosynthesis, Pg 6.27E04 2.26E04 3.08E04 2.02E04Net areal rate of biolm photosynthesis, Pn (% Pg) 2.43E04

(38.74%)9.2E05(40.76%)

2.21E04(71.59%)

7.43E06(3.68%)

Net areal rate of photic zone photosynthesis Pn,phot (% Pg) 2.99E04(47.72%)

1.36E04(60.26%)

2.61E04(84.87%)

5.52E05(27.33)

Areal respiration of the biolm, Rlight (% Pg) 3.84E04(61.26%)

1.34E04(59.24%)

8.75E05(28.4%)

1.94E04(96.32%)

Areal respiration of the photic zone, Rphot (% Pg) 3.28E04(52.28%)

8.97E05(39.74%)

4.66E05(15.13%)

1.47E04(72.67%)

Respiration in the dark, Rdark 0.59E04 1.49E04 0.74E04 0.98E04Depth of photic zone, Lphot (lm) 1000 100 600 100 700 100 600 100Depth of oxic zone in light (lm) >1950 1500 >1950 850Depth of oxic zone in the dark (lm) 1100 25 650 25 950 25 300 25

12 M. Kesaano et al. / Bioresource Technology 180 (2015) 715

dark compared to the biolms without bicarbonate amendment forboth nutrient conditions.

3.3.3. Spatial rates of photosynthesis and respirationThe gross photosynthesis proles were generated at a spatial

resolution of 100 lm vertical depth using the volumetric photo-synthetic rates (i.e., the rate of oxygen depletion within 3 s of darkincubation) determined from the light/dark shift technique. Photo-synthesis occurred within a depth of 500 lm from the biolm sur-face (Fig. 4E and F). Similarly, increasing rates of areal grossphotosynthesis (Pg) resulted in higher areal net biolm photosyn-thesis (Pn) and photic zone photosynthesis (Pn,phot), whichcorresponded to deeper oxic zones (Table 1).

However, photosynthetic rates signicantly varied with bothnutrient conditions and presence/absence of bicarbonate in med-ium. Biolm samples under nutrient replete culturing had higherphotosynthetic rates (P , P , and P , ) compared to N-deprived

in oxygen concentration in the biolms amended with bicarbonateduring N-replete culturing (Fig. 4). For algal biolms culturedunder N-deprived conditions, lower Rlight and Rphot were observedwith added bicarbonate compared to biolm samples withoutbicarbonate (Table 1). This indicated that addition of bicarbonatereduced light respiration in N-deprived biolms possibly due toan increased DIC supply.

Dark respiration measurements were greater for N-deprivedbiolms indicating a higher capacity for heterotrophic (or lightindependent) respiration. The inuence of bicarbonate additionon Rdark values varied with nutrient condition. For example,N-replete cultures had higher Rdark in biolms that did not receivebicarbonate, whereas higher Rdark were observed in biolmsamended with bicarbonate for N-deprived cultures (Table 1).

3.4. Biofuel precursor production

ry gr

M. Kesaano et al. / Bioresource Technology 180 (2015) 715 13g n n phot

algal biolms indicating a greater potential for photo-productivitywhen nutrient replete (Fig. 4 and Table 1). Biolms amended withbicarbonate also had higher photosynthetic rates (Pg, Pn, andPn,phot) compared to the biolms that did not receive bicarbonatefor both N-replete and deprived conditions (Table 1). The distribu-tion of Pn and Pn,phot as a fraction of the gross photosynthesis in thebicarbonate amended biolms was different from that of biolmsthat did not receive bicarbonate. Pn and Pn,phot represented agreater proportion of gross photosynthesis under N-deprived con-ditions for bicarbonate amended biolms, whereas for biolmsamples that did not receive bicarbonate the reverse was observedi.e., Pn and Pn,phot represented a greater proportion of gross photo-synthesis under nutrient replete conditions.

Dark respiration and photorespiration are the two basic types ofrespiration that occur in photosynthesizing microalgae. Dark respi-ration is assumed to be constant and occurs both in the light anddark whereas photorespiration is mostly active in the light and afew seconds after dark incubation (Wieland and Khl, 2000). Thedark respiration term (Rdark) was obtained as the slope of the initialportion of the O2 microproles (linear part) in the dark. The lightrespiration terms (Rlight and Rphot) were determined as the differ-ence between Pg, and Pn and Pn,phot, respectively. Although, therewas no clear trend observed for respiration rates (Rlight and Rphot)across nutrient conditions, addition of bicarbonate to the biolmsrevealed some differences. For biolms cultured under N-repleteconditions, higher areal respiration rates (Rlight and Rphot) wereobserved in bicarbonate amended biolms compared to biolmsthat did not receive bicarbonate (Table 1). This may have beendue to the higher photosynthetic rates and subsequent increase

Table 2Total and percent composition of extractable biofuel precursor weight (%) in laborato

Precursor molecules, weight % (w/w) Condition

Nutrient replete

aBicarbonate

C14 FFA 1.44|0.01C16 FFA 1.11|0.26C18 FFA 0.73|0.21C16 MAG 0.11|0.05C18 MAG 0.11|0.01C16 DAG 0.09|0.03C18 DAG 0.21|0.06C16 TAG 0.49|0.41C18 TAG 1.32|1.30

Sum of extractablesWeight % (w/w) 5.62|1.08

Areal concentration (gm2) 1.03

a Mean and range (|) for n = 2.Extractable biofuel precursor molecules (FFAs, MAGs, DAGs andTAGs) and total biofuel potential (as FAMEs, i.e., extractable andnon-extractable molecules) for each biolm type, both before andafter N-starvation, were measured and are presented in Table 2and Fig. 5. An increase in total extractable precursor concentrationswas observed in the biolms after the 120 h N-starvation period(Table 2). Stressed microalgae have been reported to accumulateTAG as a carbon and energy storage material (Mus et al., 2013).The sum of extractable precursors increased from 5.62% to 7.13%(w/w) for biolms amended with bicarbonate and 4.84% to 5.18%(w/w) for the biolms that did not receive bicarbonate, respec-tively (Table 2). Although the FFA, MAG, and DAG concentrationsremained relatively constant, twice as much TAGs accumulatedin the biolms after N-starvation leading to the overall increasein total biofuel precursor molecules (Table 2). Bicarbonateamended algal biolms had higher weight percentage of extract-able molecules.

The total FAME-weight percent and yield for N-replete and N-starved biolms with or without bicarbonate amendment weresimilar (Fig. 5A and C). Although, the total FAME potential rangedfrom 12% to 20% (w/w) of the biomass (Fig. 5B), the total extract-able lipids were less than 10% (w/w) (Table 2). As previouslyreported by Bernstein et al. (2014), the most notable differenceregarding lipid production in the RABR-grown algal biolms wasthe difference in the total extractable weight percent of lipidsbetween the N-replete and deplete conditions (Table 2). Depletionof nitrogen and addition of dissolved inorganic carbon in the med-ium were not effective in stimulating substantial lipid productionin the microalgal biolms. Qualitative analysis of lipid proles

own microalgal biolms with and without bicarbonate amendment.

Nutrient deplete

aNo bicarbonate aBicarbonate aNo bicarbonate

0.67|0.14 1.07|0.18 1.23|0.151.49|0.03 0.86|0.58 1.45|0.211.53|0.05 0.48|0.35 0.79|0.130.18|0.01 0.11|0.09 0.13|0.030.16|0.01 0.09|0.04 0.13|0.020.10|0.02 0.09|0.05 0.10|0.000.19|0.06 0.19|0.06 0.17|0.010.17|0.08 0.58|0.41 0.35|0.150.36|0.37 3.65|1.51 0.84|0.40

4.84|0.71 7.13|0.57 5.18|0.42

1.22 1.30 1.30

E peand

ce TFig. 5. Total FAMEs and free fatty acid composition of the FAMEs. (A) Percent FAM(g m2). Error bars represent range (n = 2). ND and NR represent nitrogen deprived14 M. Kesaano et al. / Bioresourusing images from CLSM showed the same result, the microalgalbiolms only showed a slight increase in lipids after N-starvation(Fig. S3). Previous studies have attributed the inability ofN-depletion in the growth medium to induce lipid production inalgal biolms to possible nutrient re-cycling within the biolmsand resilience of algal biolms to environmental stress (Bernsteinet al., 2014; Schnurr et al., 2013).

4. Conclusions

For this study, there was no signicant difference in algal bio-lm growth, nutrient removal, and lipid accumulation betweenalgal biolms amended with bicarbonate and those that did notreceive bicarbonate. However, an increase in photosynthesis rateswas observed in algal biolms amended with bicarbonate. Theinuence of bicarbonate on photosynthetic and respiration rateswas especially noticeable in biolms that experienced nitrogenstress, as compared to biolms in nutrient replete conditions.

Medium N-depletion may not be a suitable stimulant for lipidproduction in algal biolms; rather focusing on optimizing growth,nutrient removal rates, and/or biomass productivities may be morebenecial.

Acknowledgements

The authors would like to acknowledge the Utah ScienceTechnology and Research Initiative (USTAR), Utah Water ResearchLaboratory (UWRL), Church and Dwight Co., Inc., National ScienceFoundation (NSF) CHE-1230632, and the U.S. Department ofEnergy (DOE) Ofce of Energy Efciency and Renewable Energy(EERE) Biomass Program under Contract No. DE-EE0005993 fornancial support, as well as Montana State University, Center forBiolm Engineering (CBE) for technical support. Also special thanksr total FAME (w/w), (B) percent FAME per biomass (w/w), (C) areal concentrationreplete algal biolms, respectively.

echnology 180 (2015) 715to Katie Davis, Luke Halverson, and Todd Pedersen for help andassistance in the CBE laboratories.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2014.12.082.

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M. Kesaano et al. / Bioresource Technology 180 (2015) 715 15

Dissolved inorganic carbon enhanced growth, nutrient uptake, and lipid accumulation in wastewater grown microalgal biofilms1 Introduction2 Methods2.1 Microalgal biofilm culturing and sampling2.2 Water quality monitoring2.3 Oxygen microsensor analysis2.4 Biodiesel analysis

3 Results and discussion3.1 Microalgae growth rate and productivity3.2 Removal of nitrogen and phosphorus from synthetic wastewater using algal biofilms3.3 Microalgal biofilm photosynthesis and coupled respiration3.3.1 Oxygen microprofiles under illumination3.3.2 Oxygen microprofiles in the dark3.3.3 Spatial rates of photosynthesis and respiration

3.4 Biofuel precursor production

4 ConclusionsAcknowledgementsAppendix A Supplementary dataReferences