Heterotrophic cultivation of T. obliquus under non-axenic conditions by uncoupled supply of nitrogen and glucose

Biochemical Engineering Journal 145 (2019) 127–136

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Regular article

Heterotrophic cultivation of T. obliquus under non-axenic conditions by uncoupled supply of nitrogen and glucose

T

Fabrizio Di Caprioa, , Pietro Altimaria, Gaetano Iaquaniellob, Luigi Torob, Francesca Pagnanellia ⁎

a b

Department of Chemistry, Sapienza University of Rome, P.le Aldo Moro 5, 00185, Rome, Italy Bio-P S.r.l., Via di Vannina 88, 00156, Rome, Italy

HIGHLIGHTS

strategy is demonstrated for heterotrophic non-axenic microalgae cultivation. • AUncoupled controlled supply of NO and glucose can reduce bacteria contamination. • Cell duplication occurred only under N-replete (C-deplete) conditions. • Biomass production occurred only under N-deplete (C-replete) conditions. • Under N-deplete microalgae biomass duplicated independently of x . • − 3

0

ARTICLE INFO

ABSTRACT

Keywords: Heterotrophic growth Non-axenic culture Microalgae Bacteria contamination Wastewaters Tetradesmus obliquus

A fed-batch strategy is proposed to produce microalgae biomass under non-axenic heterotrophic conditions. The strategy induces the alternation of N-deplete (Glucose-replete) and N-replete (Glucose-deplete) cultivation phases by the periodic and uncoupled supply of glucose and NO3− to the culture. Cultivation of the microalga T. obliquus with this strategy reduced the ratio of the bacteria to microalgae cell concentration from 1.6, attained by conventional photoautotrophic cultivation, to 0.03. During the N-deplete phase, microalgae duplication stopped and biomass concentration increased 1.9 times, while during the N-replete phase, microalgae duplicated halving their average size and losing about 25% of their weight. The process proved to be effective under several consecutive cycles. Biomass productivity until 6.1 g/Ld and biomass concentration until 26 g/L were achieved. The results demonstrate that the proposed strategy can effectively prevent bacterial contamination, paving the way to the large scale production of microalgae biomass under non-axenic heterotrophic conditions.

1. Introduction Microalgae can convert renewable and cheap resources to biomass containing lipids, carbohydrates, proteins and pigments [1,2]. These substances can be potentially employed for several applications including, for example, the production of biofuels, biopolymers, feed and food [3,4]. However, the lowest costs actually obtained for microalgae biomass production are currently around 3–10 euro/kg [5–7], which restrict the applications to the synthesis of high value products (e.g. nutraceuticals). The elevated costs of microalgae biomass are given by the application of expensive cultivation systems (open ponds, flat panel and tubular photobioreactors) with costs for reactors installation and for continuous power supply (for both cultivation and harvesting) that are not balanced by the biomass productivity [5,7]. ⁎

Some microalgae can also grow by using the heterotrophic and mixotrophic metabolism, thus employing organic substrates as sources of energy and/or carbon [8,9]. As compared to photoautotrophic cultivation, heterotrophic cultivation could reduce the production costs of microalgal biomass ensuring higher biomass productivity and allowing for the application of light-independent reactors characterized by considerably lower surface to volume ratio. However, it should be considered that while photoautotrophic cultivation uses only CO2 as carbon source, heterotrophy invariably requires organic molecules derived mainly from terrestrial plants. For this latter reason, economic and environmental sustainability of heterotrophic microalgae processes is promising mainly employing wastewaters as source of nutrients [10]. Microalgae heterotrophic cultivation rises the issue of culture contamination, mainly by bacteria and fungi [11,12], because these latter can compete with microalgae for the same substrates (organic carbon).

Corresponding author. E-mail address: [email protected] (F. Di Caprio).

https://doi.org/10.1016/j.bej.2019.02.020 Received 25 October 2018; Received in revised form 31 January 2019; Accepted 24 February 2019 Available online 25 February 2019 1369-703X/ © 2019 Elsevier B.V. All rights reserved.

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Nomenclature x0 xf x(t) xG,f xN,f ΔxR xmax Yx / NO3 Yx / glu Px Px,max glu0 glu(t) q0 qmax

q(t) N0 N(t) η tf ΔN

Initial biomass concentration (as dry weight) of the cultivation phase, g/L Final biomass concentration (as dry weight) of the cultivation phase, g/L Biomass concentration (as dry weight) measured at time t, g/L Biomass concentration at the end of the C-replete/N-deplete phase, g/L Biomass concentration at the end of the C-deplete/N-replete phase, g/L Biomass consumed during the C-deplete/N-replete phase, g/L Maximum biomass concentration (as dry weight), g/L Biomass to NO3− yield factor, g/g Biomass to glucose yield factor, g/g Average biomass productivity, g/Ld Maximum biomass productivity, g/Ld Initial glucose concentration, g/L Glucose concentration measured at time t, g/L Minimum nitrogen quota, g/g Maximum nitrogen quota, g/g

fc mc cell(t) cell0 cellf cellmax Pcell Pcell,max Ycell / NO3 Ycell / glu UHS CS ETW

Nitrogen quota measured at time t, g/g Initial nitrogen concentration, g/L Nitrogen concentration measured at time t, g/L Fattening factor Duration time of the cultivation phase, d Nitrogen assimilated during the C-deplete/N-replete phase, g/L Amount of biomass consumed for every gram of nitrogen assimilated during C-deplete/N-replete phase, g/g Mean microalgae cell weight, pg/cell Microalgae cell concentration measured at time t, 106/mL Initial microalgae cell concentration of the cultivation phase, 106/mL Final microalgae cell concentration of the cultivation phase, 106/mL Maximum microalgae cell concentration, 106/mL Average microalgae cell productivity, 106/mLd Maximum microalgae cell productivity, 106/mLd Microalgae cell to NO3− yield factor, 106/g Microalgae cell to glucose yield factor, 106/g Uncoupled heterotrophic strategy Coupled heterotrophic strategy Enriched tap water

main idea behind the implemented cultivation strategy was that microalgae can grow when either nitrogen or organic carbon are not present in the culture medium by consuming the internal quota of nitrogen and by photosynthesis, respectively, whereas most bacteria can grow only if all nutrients are simultaneously present in the culture medium. These behaviours are generally described by Droop model and Monod model [13,21]. However, the study carried out by Deschênes et al. [20] was restricted to the analysis of mixotrophic conditions, and no study has previously investigated the implementation of the uncoupled supply strategy under heterotrophic conditions. In addition, no guidelines have been provided on how to determine the parameters of the uncoupled supply strategy (e.g. time between two successive supplies, amount of nitrogen and carbon to be supplied). In this article, we describe how the prevention of simultaneous presence of organic carbon and nitrogen in the culture medium by the uncoupled supply strategy imposes, under heterotrophic conditions, to overcome severe difficulties which are not encountered under mixotrophic conditions (Section 3). In order to overcome these difficulties, guidelines are developed and demonstrated to orchestrate the supplies of nitrogen and organic carbon. This developed supply strategy is hereafter referred to as uncoupled heterotrophic supply strategy (UHS). In Section 4, results and discussion are reported including the description and demonstration of the proposed UHS strategy.

Since bacteria have growth rates about one order of magnitude larger as compared to microalgae, the extinction of microalgae can be easily attained [13]. The strategy typically adopted to prevent contamination of heterotrophic microalgae cultures is the accurate sterilization of everything coming into contact with microalgae: the reactor, the culture medium and the supplied gases [14]. For this purpose, conventional sterilization methods including, for example, filtration, chemical and thermal treatment, are implemented [15]. The application of axenic inocula is also required making necessary the preliminary separation of bacteria that generally live in association with microalgae [16]. Antibiotics are typically used to achieve this objective [17]. However, obtaining axenic microalgae cultures is difficult because bacteria often live in close association with microalgae in symbiotic relationships [16]. Contamination becomes an even more severe concern if wastewaters are employed as source of substrates. Wastewaters invariably contain a large variety of suspended solids and compounds, which makes difficult to achieve sterilization by conventional methods [11,18]. Overall, the costs that must be sustained to maintain axenic conditions limit the recourse to axenic heterotrophic cultures only to few industrial applications aimed at the production of high value compounds [14]. Remarkably, in most of the previous studies on mixotrophic and heterotrophic microalgae cultivation, researchers have mitigated or solved the contamination issue by performing experiments with very low concentrations of organic carbon (COD between 0.1–1 g/L) or under fully sterilized conditions [19]. However, working at low COD concentrations limits improved growth due to heterotrophic metabolism, while fully sterilized conditions are hardly sustainable in industrial practice (owing to the sterilization costs). Based on the illustrated analysis, the development of strategies to control bacterial contamination of non-axenic microalgae cultures is essential to drive the large scale production of microalgae biomass under heterotrophic cultivation conditions. In a previous work, Deschênes et al. [20] demonstrated the possibility to control bacterial contamination under mixotrophic conditions by preventing the simultaneous presence of nitrogen and organic carbon in the culture medium. This condition was achieved by the “uncoupled” periodic supply of organic carbon and nitrogen. The term “uncoupled” is here employed to indicate that organic carbon and nitrogen were always supplied at different times. The

2. Materials and methods 2.1. Microalgal strain A strain of Tetradesmus obliquus was previously selected in Siracusa (Italy) and identified by IGA Technology Services by means of a genetic analysis based on ITS region [22]. T. obliquus is generally known as Scenedesmus obliquus, but it has been recently reclassified by Wynne and Hallan [23]. This strain was cultivated by Bio-P s.r.l. in an outdoor pilot plant located in Rome (Italy), as described in a previous work [24]. The pilot plant is made of 10 column photobioreactors, 21 L each one (Ø = 14 cm, h = 150 cm). In this plant, the cultivation medium was obtained by enriching tap water [25] with pure salts, obtaining the composition reported in Table 1. The reactors were continuously fed 128

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An aliquot of the harvested pellet (Section 2.1) was diluted with BG11 medium (Table 1) without NaNO3 in 500 mL conical flasks. Three biological replicates were tested at initial biomass concentrations equal to 0.3, 0.5, 0.6 and 1 g/L and at an initial glucose concentration equal to 10 g/L. Biomass and glucose concentrations were measured at different times of the batch and the yield factor was determined as follows:

Table 1 Average chemical composition of the BG11, of the cultivation medium used in the pilot plant and of the medium used for laboratory tests in heterotrophy (ETW). Pilot plant and ETW medium were obtained by mixing tap water and pure mineral salts. Values marked with an asterisk were varied in the different tested conditions. Parameter

BG11

Pilot Plant

ETW

HCO3− (mg/L) Ca (mg/L) Mg (mg/L) NO3 (mg/L) F (mg/L) K (mg/L) Na (mg/L) SO42− (mg/L) Fe (mg/L) Mn (μg/L) PO43− (mg/L) EDTA (mg/L) H3BO3 (μg/L) Zn (μg/L) Mo (μg/L) Cu (μg/L) Glucose (g/L)

14.5 9.8 7.4 0 – 1090* – 13.7 5 – 410* 29.6 1.3 0.2 16.6 1 60 65 7 0.64 –

405 112 25 260 0.12 15 105 46 1.3 0.5 17 1 60 65 7 0.64 –

405 112 30 0 – 750* 0.12 134 105 – 1500* 120 8.5 0.5 163 1 60 65 7 0.64 0 – 20*

Yx / glu =

2.4. Determination of the biomass increase under heterotrophic N-deplete conditions The microalgae biomass harvested from the pilot plant (Section 2.1) was diluted with a medium containing the same nutrients of the BG11 medium but without NaNO3. The concentration of every nutrient was fixed by the following equation:

[S ]i, HM = n [S ]i, BG11

(3)

where [S]i,BG11 and [S]i,HM are the concentrations of the nutrient i in the BG11 and in the employed medium, respectively, while n is the ratio between the targeted amount of produced biomass and the biomass that can be produced by supplying the BG11 medium. In addition to the BG11 nutrients, 2.5 g of glucose were supplied to the culture per gram of the targeted amount of produced biomass. Eq. (3) and the amount of glucose supplied per gram of produced biomass were derived by assuming that the concentrations of nutrients in the BG11 and 2.5 g/L of glucose are required to produce 1 g/L of microalgae biomass. Heterotrophic cultivation tests were carried out in 100 mL bottles with x0 ranging from 0.3 g/L to 13.5 g/L. The duration of any batch was 2 days. All the heterotrophic tests were conducted using flasks covered with aluminum foils to exclude light penetration into the culture, continuously stirred by magnetic agitation and continuously fed with 0.15 L/min of air. The pH was adjusted daily at 7.0 ± 0.5 during the cultivation by NaOH or HCl addition.

2.2. Determination of biomass yield factor on NO3− in photoautotrophic batch cultivation The biomass to NO3− yield factor (Yx / NO3 ) was determined under photoautotrophic conditions by cultivating T. obliquus harvested from the pilot plant (see Section 2.1) with the BG11 medium (Table 1) in 300 mL conical flasks, under constant light exposition (24 h/24 at 80 μmol·s-1 m-2), continuous mechanical stirring, constant temperature (30 ± 2 °C) and continuous air feeding (0.1 L/min). Biomass and NO3concentrations were measured at the beginning and at the end of the batch, and the yield factor was determined as follows:

x (t ) x0 NO3 0 NO3 (t )

(2)

where glu(t) and glu0 are the glucose concentrations at time t and at the beginning of cultivation, respectively. All the heterotrophic tests were conducted using flasks covered with aluminum foils to exclude light penetration into the culture, continuously stirred by magnetic agitation and continuously fed with 0.15 L/min of air. The pH was adjusted daily at 7.0 ± 0.5 during the cultivation by NaOH or HCl addition.

with air (5 L/min), while additional pure CO2 was furnished on demand to maintain the pH at 8. The experiments reported in this work have been carried out by using as inoculum T. obliquus taken from the described pilot plant. T. obliquus microalgae were harvested from the pilot plant (at a concentration between 1–1.5 g/L), transferred to laboratory 1 L conical flasks, replenished with NaNO3 1.5 g/L and maintained 24 h under constant illumination (100 μmol·s−1 m-2), constant agitation and air feeding (0.1 L/min). This latter phase was conducted to assure that microalgae could reach the maximum internal nitrogen quota (qmax) [26]. Afterwards, a microalgae pellet was harvested by 5 min centrifugation at 3500 rpm and washed two times with distilled water. Biological replicates are considered as inocula taken from different reactors of the pilot plant. All the cultivations were performed under non-axenic conditions.

Yx / NO3 =

x (t ) x 0 glu0 glu (t )

2.5. Heterotrophic fed-batch cultivation T. obliquus microalgae harvested from the pilot plant (see Section 2.1) were resuspended in 500 mL conical flasks diluted to x0 = 1 g/L in 300 mL of the ETW medium (Table 1). The two following fed-batch strategies were implemented to supply glucose and NO3− to the culture:

(1)

- Heterotrophic Coupled Supply (CS): glucose (10 g/L) and NaNO3 (1 g/ L) were simultaneously supplied at the beginning of the test, and they were simultaneously replenished at different successive times when they were exhausted (Fig. 3F). - Heterotrophic Uncoupled Supply (UHS): glucose was supplied at the beginning of the test, while NaNO3 was supplied only after glucose was exhausted. Glucose was supplied once again to the culture after NO3− depletion was attained. This cycle was replicated until the end of the experiment. The amounts of glucose and NO3− supplied to the culture during the experiment are reported in Fig. 3E. These nutrients were increased proportionally to the achieved biomass concentration as detailed in results Section (4.3). For all the conditions, two biological replicates were tested.

x(t) and NO3 (t) are the biomass and NO3− while NO3 0 and x0 are the biomass and NO3−

where concentrations at time t concentrations at the beginning of cultivation. N-replete conditions (NO3 > 50 mg/L) were maintained over the entire duration of the batch (5–10 days). This test was repeated using 3 biological replicates for different initial biomass concentrations: 0.01, 0.05, 0.1 and 0.3 g/L. 2.3. Determination of biomass yield factor on glucose in heterotrophic batch cultivation In order to determine biomass to glucose yield factor (Yx/glu), heterotrophic batch cultivation tests were performed using T. obliquus suspensions harvested from the pilot plant (Section 2.1) as inoculum. 129

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The initial pH was corrected to 7.0 and it was adjusted every day to this value by adding NaOH 1 M and/or HCl 1 M.

energy source, i.e., by using the stored starch and/or lipids as the exclusive sources of energy. If this is not the case, it would be impossible to recover N-deplete conditions after the supply of NO3− during the Cdeplete phase. Notice that, under mixotrophic conditions, NO3− can be in contrast consumed through the photosynthetic metabolism, making it always possible to recover N-deplete conditions. If microalgae can use NO3− under heterotrophic conditions without an external energy source, guidelines for the implementation of the UHS strategy should be developed addressing the two following issues: Firstly - during N-replete/C-deplete phase, the maximum amount of NO3− that can be assimilated by microalgae under heterotrophic conditions without an external organic carbon source, is that required to shift from the initial (q0) to the maximum nitrogen quota (qmax). If the NO3− supplied at the beginning of the C-deplete phase exceeds this amount, NO3− will not be entirely consumed by microalgae, excluding the possibility to restore N-deplete condition. Differently, under mixotrophic conditions, the photosynthetic metabolism allows to restore Ndeplete condition irrespective of the supplied amount of NO3−, providing that no limitation to photosynthetic growth (depletion of other nutrients) arises. Secondly - during N-deplete/C-replete phase, the maximum amount of organic carbon that can be consumed by microalgae is that required to bring microalgae from the maximum (qmax) to the minimum nitrogen quota (q0). Initial nitrogen quota is equal to qmax because an inoculum harvested from cultures maintained under N-replete condition is used. The organic carbon supplied at the beginning of the N-deplete phase exceeding this amount will not be consumed by microalgae, preventing the possibility to reach C-deplete condition. Therefore, taking into account the uncertainty about the estimation of qmax and q0, the supplied organic carbon amount should be lower than the organic carbon amount required to attain the estimated qmax, thus decreasing the biomass produced by organic carbon degradation. While this decrease can be compensated under mixotrophic conditions by the photosynthetic microalgae growth, it could determine under heterotrophic conditions an unacceptable decrease in the biomass productivity. Otherwise, if organic carbon is added in excess to avoid reduced biomass growth, unacceptable bacteria contaminations can arise. In accordance with the above illustrated analysis, major difficulty in the application of the UHS strategy is to obtain reliable estimates for the amounts of glucose and NO3− to be supplied when N-deplete or C-deplete conditions are attained, respectively. In this work, a procedure is illustrated to quantify the amounts of glucose and NO3− to be supplied. The procedure relies on the preliminary estimation of the yield factors, Yx/glu and Yx / NO3 , and of the minimum and maximum nitrogen quotas, q0 and qmax. The preliminary experimental analysis performed to estimate Yx/glu, Yx / NO3 , q0 and qmax is illustrated in Sections 4.1 and 4.2, while the application of these parameters to drive the implementation of the UHS strategy is analysed in Section 4.3.

2.6. Chemical and biochemical analyses Biomass dry weight was determined by filtering a prescribed volume of microalgal suspension through 0.7 μm glass microfiber filters (VWR), which were then dried at 105 °C. Microalgae cell concentration was measured by manual count in a 10−4 mL Thoma chamber carried out by a Leitz Laborlux 12 optical microscope. NO3- concentration was measured by using an ion-selective electrode (perfectION™, Mettler Toledo). Glucose concentration was measured by Dubois method [27]. Bacteria concentration was determined as unit forming colonies (CFU) by plating 20 μL of suspension obtained through serial dilutions on the LB solid medium in Petri dishes, which were incubated at 30 °C for 3 days. In order to determine the total carbohydrate content, the biomass was harvested by 5 min centrifugation at 8000 rpm, washed twice with distilled water and then stored at −20 °C until analysis. The biomass carbohydrate content was determined by acid saccharification followed by Dubois analysis, as described in a previous work [28]. 2.7. Statistical treatment of data The growth tests were carried out by using two or more biological replicates. The significance of the differences between samples was evaluated by using t-test and ANOVA (α = 0.05). SD and SE indicate standard deviation and standard error respectively. 3. Uncoupled supply under heterotrophic conditions: main ideas and difficulties In this work, we develop a heterotrophic uncoupled strategy (UHS) to prevent bacterial contamination under heterotrophic cultivation conditions. The objective is to optimize the uncoupled strategy to enforce the cyclic alternation of the following cultivation phases: - N-depleted/C-replete phase: glucose is supplied to microalgae suspended in a medium without NO3−. If the microalgae used to prepare the inoculum are harvested from cultures previously maintained under N-replete conditions, they will employ the supplied glucose to accumulate lipids and carbohydrates. - N-replete/C-deplete phase: after glucose deplete conditions are attained, NO3− is supplied to the culture. During this second phase, microalgae accumulate nitrogen and undergo cell division. The internal carbon reservoirs (lipids and carbohydrates) are consumed to obtain the energy needed to perform these metabolic activities. Once N-deplete condition is attained, glucose can be supplied once again to the culture to induce the intracellular accumulation of carbon.

4. Results and discussion

It is now important to notice that the application of the UHS strategy introduces difficulties that do not arise under mixotrophic conditions, which were investigated by Deschênes et al. [20]. Unlike mixotrophic cultivation, heterotrophic cultivation excludes the photosynthetic metabolism, implying that microalgae cannot use light and inorganic carbon (dissolved CO2) as additional sources of energy and carbon, respectively. This introduces critical issues that must be solved in order to ensure that the UHS strategy could effectively enforce the alternation of N-deplete/C-replete and N-replete/C-deplete phases. The first question that should be answered to evaluate the technical feasibility of the proposed strategy is whether microalgae metabolism can accommodate the transition from N-deplete/C-replete to N-replete/ C-deplete cultivation conditions. To the best of our knowledge, there is no evidence that microalgae can use NO3− under heterotrophic conditions without an external

4.1. Determination of the biomass to NO3− yield factor In order to determine the biomass to NO3− yield factor Yx / NO3 , batch photoautotrophic cultivation tests were performed with different initial biomass concentrations (between 0.01 and 0.3 g/L). No statistically significant difference was found between the Yx / NO3 values (Eq. (1)) attained with different initial biomass concentrations x0, which can be attributed to the application of a medium replete in every nutrient during the duration of the batch. Under these conditions, a balanced growth, with the production of a biomass with almost constant biochemical composition can be expected. The mean Yx / NO3 value was 3.3 g/g (SE = 0.6, SD = 1.6). This value is comparable to values previously reported for Desmodesmus sp. and H. pluvialis, which are 3.3 and 3.2 g/g respectively [29,30]. 130

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Since cultivation experiments were performed under N-replete conditions with inocula harvested from cultures previously maintained under N-replete condition, the biomass nitrogen quota can be assumed to be invariably equal to qmax over the cultivation period. A reliable estimate for qmax is then given by (N0 – N(t))/(x(t) – x0) irrespective of the cultivation time t. This gave a mean value for qmax equal to 0.07 ± 0.01 g N/gx. Notice that, since no statistically significant effect was induced by variations in x0, the reported Yx / NO3 and qmax values were determined by averaging the results of tests carried out with different x0. As suggested by Laurens et al. [31], the microalgae protein content attained under N-replete phase was computed by multiplying qmax times 4.78, which is lower than the factor 6.25 conventionally adopted. This gave a mean protein content equal to 33 ± 5%, which is lower than the values (50–60 %) previously reported for strains of the same species [32,33], but in agreement with results of a previous work with the same strain [22].

q0 =

qmax 1.87

(5)

With the qmax value estimated based on photoautotrophic cultivation tests (see previous section), Eq. (5) gives q0 = 0.037 ± 0.006 g N/ gx. By considering that transition from qmax to q0 is determined under N-deplete conditions by the sole accumulation of compounds (mainly lipids and carbohydrates) inside the cells [37], we can determine a fattening factor (η) as follows:

=

qmax q0

(6)

This factor measures the biomass increment that can be achieved by a microalgal strain under N-deplete conditions when cell duplication is stopped. We expect that this factor is characteristic of every microalgal strain. For the T. obliquus strain here investigated, η is equal to 1.87. Based on literature data, it is hard to find η values to make a

4.2. Determination of the biomass to glucose yield factor In order to determine the biomass to glucose yield factor Yx/glu, batch heterotrophic cultivation experiments were performed under non-axenic N-deplete conditions. Microalgae inocula were harvested from photoautotrophic cultures maintained under N-replete condition (NO3− > 50 mg/L), ensuring that the nitrogen quota of the inoculum was qmax. Different tests were performed by varying the initial biomass concentration x0. However, no statistically significant difference was found between Yx/glu values (Eq. (2)) attained with different x0. The mean Yx/glu value was 0.40 g/g (SE = 0.04, SD = 0.12). This Yx/glu value is comparable with those obtained for other microalgae species under axenic conditions [14,34], which are between 0.4 and 0.8 g/g, and larger than that previously reported for the same species [35], which was between 0.15 – 0.23 g/g. In these preliminary heterotrophic tests, it was assumed that there was no relevant bacteria contamination during cultivation. The reliability of such assumption will be discussed in Section 4.3. During the batch heterotrophic cultivation under N-deplete conditions, independently of x0, the ratio x(t)/x0 increased reaching a constant value xf/x0 = 1.87 (the 95% confidence intervals ranged from 1.80 to 1.94) at t = 2 d (Fig. 1A, B and A1 – Supplementary data), where xf denotes the biomass concentration under the stationary phase. Notice that xf was not determined by glucose exhaustion. This was verified by noting that the culture was still replete in glucose after xf was attained (glu(t) measured at the end of the batch was only between 7 and 25% less than glu0). We did not investigate the evolution of the microalgae biochemical composition during this test, but it can be expected that it varied owing to the accumulation of carbohydrates and lipids [33,36]. Since heterotrophic cultivation experiments were performed under N-deplete conditions while maintaining replete conditions for every other nutrient, the nitrogen quota attained by microalgae following the achievement of the stationary phase can be assumed to be q0. In order to proceed with the computation of q0, we now assume that the nitrogen contained in the biomass inoculum was not lost by microalgae during the batch (no relevant extracellular losses). Biomass concentration data displayed in Fig. 1A can then be used to estimate the evolution of the internal nitrogen quota as follows:

q (t ) =

x 0 qmax x (t )

Fig. 1. A) Evolution of the ratio between the biomass concentration measured at different times of the batch (x(t)) and the initial biomass concentration (x0). Reported values correspond to the average values obtained performing the test with different x0 values ranging between 0.3 and 1 g/L. For every data ± SD are reported (n = 12). B) Relation among initial (x0) and final (xf) biomass concentration attained at the beginning and at the end (after 2 days) of heterotrophic batch cultivation in N-deplete. C) Relation among initial biomass concentration (x0) and average biomass productivity (Px) attained at the end (after 2 days) of heterotrophic batch cultivation in N-deplete.

(4)

where q(t) is the internal nitrogen quota at time t. Substituting x(t) = xf into Eq. (4) and taking into account that xf/x0 = 1.87 then gives the following estimation for the internal nitrogen quota q0 attained under the stationary phase: 131

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comparison, because most of the nitrogen quota values are typically reported as gN/gC (N to C ratio) rather than as gN/gx (N to dry weight ratio). For Pseudochlorococcum sp. a η = 3.4 was found [38]. The average biomass productivity (Px), calculated over the entire cultivation period tf = 2 d, required to attain the stationary phase, can be derived as follows:

Px =

xf

x0 tf

=

x 0 (1.87 2

1)

= 0.43x 0

This can be coupled to the nitrogen mass balance: xN,f qmax= xG,f q0 + ΔN to get the following expression for the assimilated nitrogen ΔN:

N=

(7)

x G, f (qmax (1

qmax (1

q0 ) fC ))

(10)

4 After N-deplete conditions were attained, the cycle was repeated restarting from instruction n° 2 above.

where, again, we have taken into account that xf/x0 = 1.87. The derived dependence of PX on x0 is confirmed by the experimental PX values reported in Fig. 1C, where linear regression gave PX = ax0 with a = 0.45 ± 0.02. In accordance with the illustrated analysis, the largest productivity PX = 6.1 g/Ld was attained with the largest considered initial biomass concentration x0 = 13.5 g/L, which gave a final biomass concentration xf ˜ 26 g/L. Such productivity is comparable with the productivities attained under conventional axenic heterotrophic cultivation of microalgae [14].

The above detailed UHS strategy was initially implemented with fc = 0, namely, it was initially assumed that no biomass was consumed to assimilate NO3−. This corresponds to assuming that the microalgae biomass concentration was not reduced during C-deplete/N-replete phase but that it only increased owing to nitrogen uptake. However, as evidenced by preliminary tests (Fig. A2 – Supplementary data), there was a relevant biomass decrease during C-deplete/N-replete phase. Consequently, it was not possible to recover with fc = 0 N-deplete conditions. With the NO3− amount determined by Eq. (10) and fc = 0, microalgae indeed attained the internal nitrogen quota qmax when NO3− in the medium was not exhausted yet (Fig. A2 - Supplementary data). Biomass and nitrogen concentration data reported in Fig. A2 (Supplementary data) were then used to get a more reliable approximation for fc. To this purpose, Eq. (8) was solved in fc with xN,f and xG,f equal to the biomass concentration values attained at the end of the Ndeplete and of the successive C-deplete phase, respectively, and ΔN equal to the variation in the nitrogen concentration attained over the Cdeplete phase. This gave fc = 17 gx/gN (SD = 6). This value indicates the amount of biomass that should be degraded for every gram of N assimilated during N-replete/C-deplete phase. The reported value was adopted to run the successive experiments. The evolutions of biomass, microalgae cells, NO3− and glucose concentrations obtained by implementing the UHS strategy with the above estimated fc are compared in Fig. 3 with those recorded during the application of the CS strategy. As it is apparent from Fig. 3E, with the proposed UHS strategy, two different cultivation phases periodically repeated: a phase with NO3- in the medium (N-replete) without glucose (C-deplete) and a phase with glucose in the media (C-replete) without NO3− (N-replete). These results confirm that the amounts of glucose and NO3− supplied to the culture by the UHS strategy were sufficient to sustain microalgae biomass and cell growth, and could be entirely consumed during the C-replete and N-replete phases, respectively (Fig. 3A and C). This allowed alternately recovering N-deplete and C-deplete conditions. The carbohydrate content in the biomass was monitored during the different cultivation phases. The carbohydrates biomass fraction

4.3. Heterotrophic fed-batch cultivation Following the determination of Yx/glu, Yx / NO3 , qmax and q0, cultivation experiments were performed to evaluate the application of the UHS strategy under heterotrophic non-axenic conditions. Two main issues were investigated: the ability of microalgae to grow under the cyclic alternation of the N-deplete/C-replete and the N-replete/C-deplete phases induced by the UHS strategy and the bacteria contamination control by UHS strategy. The results attained with the UHS strategy were compared with those attained by the simultaneous supply of carbon and nitrogen, i.e., the CS strategy. The CS strategy is intended to represent a negative control, where increased bacteria contamination and inhibited microalgae growth are expected as compared to the UHS strategy. In order to enforce the alternation of N-deplete/C-replete and Nreplete/C-deplete cultivation phases, the UHS strategy was implemented as follows: 1 Inoculum preparation: the microalgae inoculum was harvested from a photoautotrophic culture previously maintained under Nreplete conditions. This ensured that internal nitrogen quota of the inoculum was qmax. The inoculum was suspended in a medium without nitrogen at an initial concentration x0. 2 C-replete/N-deplete phase: the culture was supplied with a glucose amount equal to V(xG,f – x0)/Yx/glu, where xG,f is the maximum biomass concentration that can be attained under N-deplete/C-replete conditions and V is the culture volume. Based on the analysis presented in the previous section, we assumed VxG,f = 1.87x0V. At the end of this phase a biomass having q = q0 is expected (Fig. 2). 3 C-deplete/N-replete phase: after the supplied glucose was consumed by microalgae, the nitrogen amount required to bring the microalgae nitrogen quota from q0 to qmax was supplied (Fig. 2). In order to compute this nitrogen amount, it is important to notice that the variation in the microalgae biomass concentration is determined over the C-deplete/N-replete phase by the nitrogen uptake and by the consumption of biomass. The consumption of biomass allows deriving the energy to sustain nitrogen uptake. It can then be assumed that the amount of consumed biomass ΔxR is proportional to the assimilated nitrogen ΔN, i.e., ΔxR = fcΔN. Therefore, the overall variation in the biomass concentration Δx = xN,f - xG,f attained over the C-deplete/N-replete phase, xN,f denoting the final biomass concentration, can be expressed as follows: xN,f – xG,f = ΔN(1 - fc)

(9)

(8)

Fig. 2. Qualitative description of biomass concentration evolution induced by the uncoupled heterotrophic strategy (UHS). 132

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Fig. 3. In A and C, the biomass concentration and cell concentration are reported respectively for microalgae cultivated with the uncoupled NO3− (greys area) and glucose (white area) addition in the uncoupled heterotrophic strategy (UHS) as detailed in (E). In B and D, the biomass concentration and cell concentration are reported respectively for microalgae cultivated with the coupled NO3− and glucose addition in the coupled strategy (CS) (F). ± SD are reported (n = 2).

result and evidence, in addition, that microalgae can restrict NO3− assimilation to the C-deplete phase alone. Considering that NO3− uptake requires energy [41], it can be argued that respiration of internal stored biomass by microalgal cells can give enough energy to support NO3− assimilation. The determined fc factor (17 gx/gN) depends on the energy demand associated to nitrogen uptake: the lower the fc, the lower the energy demand for the specific nitrogen source. NH4+ should be tested in place of NO3− because it requires less energy to be assimilated [42] and could therefore reduce biomass consumption during C-deplete/N-replete phase as compared to the application of NO3−. It is worth noting that, with the UHS strategy there was a temporal separation between biomass and cell production, which occurred separately during N-deplete and C-deplete phase, respectively, i.e., they were uncoupled. In fact, as reported in Fig. 4, during the N-deplete phase, there was a relevant biomass production, while the cell

increased during the C-replete/N-deplete phase by the consumption of glucose, and it decreased during the C-deplete/N-replete phase being consumed to derive the energy needed to uptake NO3− and sustain the metabolism. Nonetheless, it was found that the average carbohydrates fractions attained during C-deplete/N-replete and C-replete/N-deplete phases barely deviated from 22 ± 3% and 31 ± 2%, respectively. Consequently, no statistically significant variation was attained in the biomass carbohydrate fraction during the experiment. It can then be derived that the carbohydrates accumulated during the C-replete phases were almost completely consumed during the successive C-deplete phase. It should be remarked that is already known that T. obliquus can use starch accumulated inside cells as the only energy source [39,40]. In fact, this is the common way microalgae survive during the night over day/night cycles (starch is stored during the light phase and consumed during the night phase). Data reported in Fig. 3 confirm this

Fig. 4. Biomass production (A) and cell production (B) calculated for every different phase (N-deplete/C-replete and N-replete/C-deplete) of the uncoupled heterotrophic strategy (UHS). xf and x0, cellf and cell0 indicate initial and final biomass concentration and initial and final cell concentration for every phase of the cultivation. ± SD are reported (n = 2).

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concentration remained substantially constant (no significant variation was detected). During the C-deplete phase, there was instead a relevant cell production, while biomass concentration slightly decreased. This latter decrease was about – 25%, calculated as (xN,f – xG,f)/xG,f. These data confirm that, during the heterotrophic N-deplete phase, microalgae stopped to duplicate, while the biomass concentration increased due to accumulation of starch (and/or lipids), thus increasing average cell size (Fig. 5) as previously found under N-deplete photoautotrophic conditions [37]. This observed behaviour originates by cell cycle arrest induced by nitrogen deficiency [43]. On the other hand, during the Cdeplete/N-replete phase, microalgae used internal starch and lipids as the only source of energy to duplicate and to sustain the uptake of NO3−, which led to a reduction of dry weight and average cell size (Fig. 5). This result is in agreement with what found for heterotrophic metabolism of microalgae during night under day/night cycles (circadian rhythm). It has been proved that some species (including T. obliquus) regulate their metabolism to have cell duplication during the night, by using the accumulated starch as energy source [39,40,44]. The temporal separation between biomass production and cell duplication should generate a variation in mean cell weight. This was determined during the application of the UHS strategy as follows:

mC =

x (t ) 103 cell (t )

1.6 to 0.03. These latter data evidence that, by the application of the UHS strategy under non-axenic heterotrophic cultivation conditions, bacteria contamination was not merely maintained unchanged, but even reduced. As discussed in the introduction section, a key difference between bacteria and microalgae, which likely allows the UHS strategy to work, is that microalgae can grow when either the organic carbon or the nitrogen are not available in the medium by consuming the internal carbon and nitrogen quota, respectively. Instead, as reported by Sterner and Elser [47], most bacteria generally grow under higher homeostasis with respect to microalgae, corresponding to a lower variability in their biochemical composition in response to environmental variations (e.g. concentration of nutrients). That’s why bacteria grow is generally represented by Monod model. However, this is not true for all bacteria. Some bacteria species (e.g. phosphate accumulating bacteria) can accumulate relevant amount of substrates as well. Some other bacteria, as chemolithotrophs, could grow in the absence of organic substrates by using inorganic compounds (e.g. ammonium) as energy source. Consequently, it should be considered that also other factors could determine the obtained result, as: the strategy was tailored on the properties of T. obliquus, which means that also if some bacteria could grow following a similar mechanism, they did not grow because the strategy (inoculum condition, time intervals between nutrients feeding, etc.) was not calibrated for them. Else, inorganic salts used were species already oxidized (NO3−, PO43-, SO42-) which likely prevent chemolithotrophs growth. We notice that a similar result was found for microorganisms storing PHA, whose growth could be also favored by the uncoupled addition of N and C sources [48]. The measured bacteria concentration was used to estimate the fraction of the produced biomass that can be imputed to bacteria. Microalgae generally live in non-axenic cultures [16,49], but bacteria do not generally represent an issue because they are present in a limited amount under conventional photoautotrophic conditions. To have an idea of the bacterial contamination impact on the produced biomass during the tested heterotrophic processes, the mass of bacteria was estimated by considering an average bacterial cell weight equal to 1 pg/ cell [50] and by assuming that biomass dry weight determined with 0.7 μm filters only measured microalgal biomass. Based on these assumptions, it was found that bacteria/microalgae biomass dry weight ratio was 2.7% at the beginning of the cultivation, which is the condition found in the photoautotrophic pilot plant where the inoculum was harvested. During the heterotrophic cultivation, the ratio increased to 50% with the CS strategy, while it decreased to 0.05% with the UHS strategy. The pH variation during cultivation gave also useful indications about the bacteria contamination. With the UHS strategy, a slight alkalization occurred requiring HCl addition to maintain the pH at 7, while, with the CS strategy, the pH dropped to acidic values, requiring

(11) 6

where mc is the mean cell weight (pg/cell), and cell(t) (10 /mL) and x (t) (g/L) are the cell concentration and the biomass concentration at time t, respectively. Microalgae cell weight fluctuated under the implementation of the UHS strategy ranging between 25 and 100 pg/cell. These values are in agreement with those already found for the same species in previous studies [37,45]. As shown in Fig. 5, the oscillation was regular during the cultivation, with microalgae cell size increasing during the C-replete/N-deplete phase and decreasing during the C-deplete/N-replete phase. These data confirm that, under C-replete/N-deplete conditions, microalgae stopped duplication and the increased biomass concentration was the result of the cell size increase, while, during the C-deplete/N-replete phase, they duplicated and thus reduced their size. It is now important to notice that lower biomass and cell concentration were attained with the CS strategy (where glucose and NO3− were added simultaneously) as compared to the UHS strategy (Fig. 3). Unlike the UHS strategy, the CS strategy did not induce temporal separation between biomass growth and cell growth (Fig. 3B and D), due to the simultaneous presence of NO3- and glucose in the cultivation medium (Fig. 3F). The average cell weight was also constant during the cultivation at 65 ± 25 pg/cell. The main element explaining the reduced biomass production attained with the CS strategy, as compared to the UHS strategy, is bacteria contamination. In heterotrophic experiment, the inocula used (coming from photoautotrophic cultivation) were characterized by a bacteria/microalgae cells ratio with an average value of 1.6. This ratio is comparable with those found under photoautotrophic cultivation conditions for Tisochrysis lutea [46]. As shown in Fig. 6, when glucose and NO3− were simultaneously supplied to the cultivation medium by the CS strategy, bacteria growth was favored and eventually outclassed microalgae growth. Particularly, with the CS strategy, the bacteria concentration increased from 30⋅106 cell/mL to 4000⋅106 cell/mL at the end of the test, which corresponds to an increase in the bacteria/ microalgae cells ratio from 1.6 to 40. In future research the possibility to test different CS strategies in which organic and nitrogen substrates are added more frequently at lower concentration values should be considered too. The frequency of substrate additions could be modulated reducing the difference between bacteria and microalgae growth rate, by reducing the average nutrient concentration in the medium. In contrast, during UHS strategy cultivation, bacteria concentration was reduced from 30⋅106 cell/mL to 5⋅106 cell/mL at the end of the test, corresponding to a reduction in the bacteria/microalgae cells ratio from

Fig. 5. Mean cell weight (mc) measured during time for T. obliquus cultivated during the fed-batch uncoupled heterotrophic strategy (UHS). The white squares indicate N-deplete/C-replete phases, while grey squares indicate Cdeplete/N-replete phases. ± SD are reported (n = 2). 134

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Fig. 6. Microalgal cell concentration and bacteria cell concentration measured at different times for fed-batch cultivation with the uncoupled heterotrophic strategy (UHS) (A) and the coupled strategy (CS) (B) strategy. ± SD are reported (n = 2).

[36], exploiting the ability of microalgae to store organic carbon obtaining a xf/x0 that was about 2 times, confirming the reported values.

Table 2 Process parameters calculated for the two fed-batch strategies compared. The data that differs significantly between coupled strategy (CS) and uncoupled heterotrophic strategy (UHS) (α = 0.05) are evidenced. ± SD are reported (n = 2).

Px (g/Ld) Px , max (g/Ld) xmax (g/L) Yx / glu (g/g) Yx / NO3 (g/g)

cellmax (106/mL) Pcell (106/mLd) Pcell, max (106/mLd) Ycell/ glu (106/g) Ycell/ NO3 (106/g)

CS

UHS

0.2 ± 0.1 2±2 5±2 0.06 ± 0.04

0.51 ± 0.03 4.0 ± 0.1 11.1 ± 0.3 0.40 ± 0.02

90 ± 40 2±1 26 ± 17 0.8 ± 0.4

180 ± 60 8±1 42 ± 2 6.2 ± 0.4

0.8 ± 0.5

10 ± 4

5. Conclusions The proposed UHS strategy can enforce the periodic alternation of N-deplete/C-replete and N-replete/C-deplete phases. Such strategy sustained the heterotrophic non-axenic cultivation of microalgae by reducing bacteria contamination. This allowed obtaining productivities comparable to those attained by a conventional (axenic) heterotrophic process. The proposed strategy uncoupled cell duplication, taking place under N-replete (C-deplete) phase, and biomass growth, which was attained under N-deplete (C-replete) phase. The ability of the described strategy to control bacteria contamination can pave the way to the large scale application of nonaxenic heterotrophic cultivations ruling out the need of culture and reactor sterilization.

7.9 ± 0.5

120 ± 20

NaOH addition to maintain it at 7. The acidification of the medium observed with the CS strategy might be a consequence of the organic acids production by bacterial metabolism, suggesting that pH monitoring could be exploited to check bacteria contamination. In Table 2, the overall performances of the two supply strategies are compared. In agreement with data on bacteria contamination, it was found that UHS strategy led to statistically significantly larger yield factors Yx / glu , Yx / NO3 , Ycell / glu , Ycell / NO3 and maximum biomass concentration xmax. Larger values were also found with the UHS strategy for cells and biomass productivity, and for cell concentrations, but the differences were, in this case, not statistically significant. The value Yx / glu = 0.40 ± 0.02 confirms the previous preliminary estimation obtained based on the results of batch experiments (0.40 ± 0.04, see Section 4.2). The value found for the NO3− to biomass yield factor, Yx / NO3 = 7.9 ± 0.5, was in contrast about two-fold larger than the value found based on photoautotrophic cultivation tests under N-replete conditions (3.3 ± 0.4) (see Section 4.1). This difference can be explained by noting that during the application of the UHS strategy, Ndeplete cultivation phases were also included. In N-deplete phase biomass concentration increased by about two times without nitrogen uptake, which led to double the Yx / NO3 with respect to that one found during the preliminary photoautotrophic cultivation tests (in which only N-replete condition was considered). These results provide valuable information for the design of heterotrophic non-axenic cultivation. If the application of wastewaters is considered, the implementation of the UHS strategy should be tailored to the wastewater composition. Particularly, the effect of C/N ratio should be taken into account [51]. Moreover, since the UHS strategy can uncouple biomass production and cell duplication, it could be implemented to cultivate microalgae with wastewater containing compounds inhibiting cell duplication, including, for example, phenols [52,53]. Accordingly, it has been proved that olive mill wastewater (C/N ˜ 40) supply was more efficient in microalgae biomass production when it was added in N-deplete phase

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