Enhancing Docosahexaenoic Acid Production of Isochrysis galbana from Starch-Rich Food Processing Byproducts

Abstract

Leftover dough, a byproduct in steamed bread processing, is rich in starch, which is a carbohydrate source for microorganisms. Carbon and nitrogen are the two most abundant nutrients in the medium of Isochrysis galbana. In this study, the leftover dough hydrolysates were used as carbon resources for the cultivation of Isochrysis galbana for docosahexaenoic acid production under different NaNO₃ concentrations. The results showed that hydrolysates and NaNO₃ concentration affected cell growth and docosahexaenoic acid (DHA) accumulation significantly. The maximum biomass concentration of 4.18 g L⁻¹ and maximum DHA yield of 341.3 mg L⁻¹ were obtained with 50.0 mL L⁻¹ hydrolysates. The DHA yield of Isochrysis galbana with 300.0 mg L⁻¹ NaNO₃ was 8.9-fold higher than that of the control. The results showed that the DHA production of Isochrysis galbana from starch-rich food processing byproducts was enhanced.

1. Introduction

Steamed bread is very popular among Chinese people. Leftover dough with high starch content is a byproduct of the processing of steamed bread. The starch content of the leftover dough can reach 48% [1]. Direct disposal of leftover dough not only causes a waste of resources but also results in environmental pollution. The starch is a carbohydrate source for microorganisms. The main component in the leftover dough, starch, can be converted to glucose by certain methods [1], which could be used as carbon sources of Isochrysis galbana (I. galbana). Because reagent cost is a great part of the cost of microalgae production [2], the use of the leftover dough could lower the production cost of docosahexaenoic acid (DHA).

DHA contributes greatly to human health and plays an important role in children’s brain development [3]. Microalgal DHA is one of the most important sources of DHA [4]. There are some promising microalgal species for commercial applications of DHA production. DHA production of 65.5 mg L⁻¹ by I. galbana is obtained under mixotrophic conditions [1]. The highest DHA productivity of Cryptothecodinium cohnii can reach 1.47 mg L⁻¹ h⁻¹ using a temperature-shift strategy [5]. DHA accounts for 17% of the dry cell weight of Throustochytium sp. [6]. Cryptothecodinium cohnii and Throustochytium sp. can be cultured only indoors [5,6]. However, I. galbana can be cultured both indoors and outdoors [7]. Furthermore, I. galbana has been widely used as a bait microalga because of its small size, high DHA content, and cell wall deficiency [8]. I. galbana is a promising microalgal species for commercial applications of DHA production.

Enhanced DHA accumulation increases by utilizing I. galbana as a renewable feedstock to produce DHA. DHA accumulation of I. galbana is influenced by light, nutrients, temperature, and agitation [1,7,8,9,10]. The culture medium provides the necessary nutrients for microalgal growth and DHA biosynthesis. Carbon and nitrogen are the two most abundant nutrients in the medium of I. galbana. DHA accumulation of I. galbana is prompted under nitrogen limitation [9]. Carbon and nitrogen resources have a significant effect on DHA accumulation of I. galbana [1]. Searching low-cost culture media is essential to reduce the DHA production cost of I. galbana. The leftover dough can be used as low-cost culture media. However, there are few studies about DHA production of I. galbana from starch-rich food processing byproducts [1].

This study aimed to evaluate the DHA production of I. galbana from starch-rich food processing byproducts. In addition, the effects of leftover dough hydrolysates and NaNO₃ concentrations on microalgal growth and DHA accumulation were investigated.

2. Materials and Methods

2.1. Materials

Reagents were purchased from Merck Chemicals (Shanghai, China) Co., Ltd. The activities of α-amylase, α-1, 4-glucan glucohydrolase, and cellulase were 50,000, 100,000, and 80,000 U g⁻¹, respectively. The leftover dough was from Baodaren Company (Nanchang, China). The sample was stored at 4 °C before testing.

2.2. Preparation of Leftover Dough Hydrolysates

The sample was pretreated by enzymatic hydrolysis as follows. The leftover dough of 500.00 g for each sample was dissolved in 1 L distilled water to prepare wheat flour suspension in a 2.5-L flask. Suspension pH was adjusted to about 6.2 by 1.0 M Na₂CO₃. The α-amylase dosage was 2.0 g L⁻¹. The mixture was agitated (100 rpm, 3 h) on a magnetic stirrer at 95 °C. The enzyme was deactivated at 100 °C for 10 min. Hereafter, the solution was cooled to 55 °C. The pH was regulated to 4.5. Then 5.0 g α-1, 4-glucan glucohydrolase was added. The mixture was agitated on the stirrer at 55 °C at 100 rpm for 48 h. The enzyme was deactivated at 85 °C for 10 min. The hydrolysates were separated by centrifugation (7200× g, 12 min). After phase separation, the upper liquid layer was transferred to a new 2.5-L flask and stored at 4 °C prior to use.

2.3. Microalgal Strain and Cultivation Conditions

Microalgal strain I. galbana was described in [1]. It was preserved in f/2 medium [11]. The microalga was cultured in a 4.0 L photobioreactor at about 26 °C. The photobioreactor was made of glass and had a diameter of 10 cm. The illumination and photoperiod were 160 μmol photons m⁻² s⁻¹ and 14 h light/10 h dark, respectively. The medium was composed of (mg L⁻¹): KH₂PO₄·2H₂O 22.6, sea salt 28,000.0, different amounts of NaNO₃, and leftover dough hydrolysates. The pH was regulated to 7.0. The sterilization conditions were 121 °C and 30 min. The leftover dough hydrolysates were sterilized by sterile filtration. The inoculation ratio of I. galbana was 1:5 (v/v). The initial biomass was 0.25 g L⁻¹. The air aeration rate was 1 vvm. The cultures were cultivated for 7 days.

To ensure the adaptability of the microalgal cultures exposed to the leftover dough hydrolysates, the microalgal species was first cultivated in sterilized liquid f/2 medium and then transferred to sterile agar plates prepared with hydrolysates for 8 generations. After that, the microalga was transferred to a sterilized liquid medium with the hydrolysates. This process was repeated 6 times to ascertain the adaptability of the microalga to the hydrolysates. Both the agar plates and the liquid medium contained 25.0 mL L⁻¹ hydrolysates.

2.4. Experimental Design

For the experiment of leftover dough hydrolysates, different amounts of hydrolysates (25.0, 50.0, 100.0 and 200.0 mL L⁻¹) were tested. NaNO₃ of 300.0 mg L⁻¹ was used for all the treatments. The culture medium without hydrolysates (0.0 mL L⁻¹) was used as control.

For the experiment of NaNO₃ concentrations, different NaNO₃ concentrations (150.0, 300.0, 600.0 and 1200.0 mg L⁻¹) were tested. The amount of hydrolysates used for all the treatments was 50.0 mL L⁻¹. The culture medium without NaNO₃ (0.0 mg L⁻¹) was used as the control.

2.5. Analytical Methods

The sample was prepared and its elemental composition was measured by our previous method [12]. The limit of detection (LOD) values of Cu, Fe, B, Zn, Mn, Mo, Si, Co, Ca, Mg, K, Pb, P, Na, As, and Se were 0.05, 0.12, 0.12, 0.05, 0.05, 0.05, 0.13, 0.03, 0.13, 0.13, 0.13, 0.03, 0.12, 0.13, 0.03, and 0.03 mg L⁻¹, respectively. The limit of quantitation (LOQ) values of Cu, Fe, B, Zn, Mn, Mo, Si, Co, Ca, Mg, K, Pb, P, Na, As, and Se were 0.22, 0.37, 0.37, 0.22, 0.22, 0.22, 0.44, 0.13, 0.44, 0.44, 0.44, 0.13, 0.37, 0.44, 0.13, and 0.13 mg L⁻¹, respectively. The total carbohydrate in the leftover dough was measured according to a previous study [13]. The proteins in the samples were measured by the Bradford assay [14]. The glucose was measured according to a reference [15]. The LOD and LOQ values of the glucose were 7.3 and 32.4 mg L⁻¹, respectively. The pH of the hydrolysates was measured by a pH meter. The NO₃⁻ concentration of the culture medium was analyzed referred to a previous method [16].

The maximum quantum yield of photosystem Π (F_v/F_m) was an indicator of photosynthetic efficiency [17]. Each 80.0 mL suspension was centrifugated at 9 600 rpm for 6 min. The microalga was washed with water three times. The microalga was then dried at 80 °C for 12 h. The biomass concentration (X, g L⁻¹), the biomass productivity (P, g L⁻¹ d⁻¹), and the specific growth rate (μ, d⁻¹) were calculated using the method of Zheng et al. [8].

Each 50.0-mL suspension was centrifugated at 9 600 rpm for 6 min. The microalga was washed with water three times. The microalgal pellets were then resuspended in 25.0 mL water in a glass test tube. Biomass was hydrolyzed by cellulase on the stirrer at 55 °C for 10 h prior to extracting the lipids. The pH was changed to 4.6. The cellulase concentration was 6.0 g L⁻¹. The lipids were extracted by the hexane-methanol extraction method [18]. The lipid content was defined as:

$$\mathrm{Lipid} \mathrm{content}=\frac{\mathrm{Weight} \mathrm{of} \mathrm{extracted} \mathrm{lipids}}{\mathrm{Weight} \mathrm{of} \mathrm{biomass}}\times 100\%$$

(1)

Fatty acids were analyzed by gas chromatography-mass spectrometry (GC-MS) (Thermo Finnigan, San Jose, CA, USA). Methylation of fatty acids was performed according to the method of Metchalfe and Schmitz [19]. The fatty acids were dissolved in hexane. Each sample of 1.0 μL was for GC-MS analysis. The GC-MS method was in accordance with a reference [20]. The DHA content was defined as:

$$\mathrm{DHA} \mathrm{content}=\frac{\mathrm{Weight} \mathrm{of} \mathrm{extracted} \mathrm{DHA}}{\mathrm{Weight} \mathrm{of} \mathrm{extracted} \mathrm{lipids}}\times 100\%$$

(2)

2.6. Data Analysis

The experiment was conducted in triplicate at random. The statistical analysis of variance (ANOVA) test and the Duncan’s multiple range test (when ANOVA indicated at least one significantly different result) were conducted by a statistical software SPSS 13.0 (SPSS Inc., Chicago, IL, USA) for data analysis according to our previous report [17].

3. Results and Discussion

3.1. Characteristics of Leftover Dough and Its Hydrolysates

The chemical composition of the leftover dough is presented in

Table 1. Chemical composition of leftover dough. Data are reported as means ± standard deviation (n = 3), the same as below.

Parameters	Composition (wt%)
Moisture	36 ± 1
Starch	51 ± 2
Protein	5 ± 0
Ash	8 ± 1

. Of all the components in the leftover dough, starch had the highest proportion, 51%. The results show that leftover dough is rich in starch. Moisture had a relatively high proportion, 36%. Starch cannot be degraded by microalgae [12,21]. It can be hydrolyzed into glucose, which is often used to grow microalgae. Therefore, leftover dough can be converted into available carbon resources to produce DHA from microalgae.

The starch was successfully hydrolyzed into glucose by enzymes. The characteristics of the hydrolyzates were analyzed (

Table 2. The characteristics of the enzymatic hydrolysates of leftover dough.

Parameters	Concentration	Parameters	Concentration
pH	6.3 ± 0.0	Si (mg L−1)	27.0 ± 1.0
Protein (g L−1)	45.6 ± 0.9	Co (mg L−1)	0.0 ± 0.0
Starch (g L−1)	0.0 ± 0.0	Ca (mg L−1)	92.0 ± 8.0
Glucose (g L−1)	248.0 ± 5.0	Mg (mg L−1)	176.0 ± 12.0
Cu (mg L−1)	33.0 ± 2.0	K (mg L−1)	450.0 ± 18.0
Fe (mg L−1)	52.0 ± 2.0	Pb (mg L−1)	0.0 ± 0.0
B (mg L−1)	11.0 ± 0.0	P (mg L−1)	397.0 ± 13.0
Zn (mg L−1)	24.0 ± 1.0	Na (mg L−1)	11.0 ± 0.0
Mn (mg L−1)	9.0 ± 0.0	As (mg L−1)	0.0 ± 0.0
Mo (mg L−1)	0.0 ± 0.0	Se (mg L−1)	8.0 ± 0.0

). The hydrolyzates were of partial acidity with a pH of 6.3. The glucose in the hydrolyzates was 248.0 g L⁻¹. The starch in the hydrolyzates was 0.0 g L⁻¹. The results show that starch in leftover dough can be converted into glucose by enzymes. The protein concentration was 45.6 g L⁻¹. The most abundant element was K (450.0 mg L⁻¹). Two relatively abundant elements were P and Mg. Mo, Co, Pb, and As were 0.0 mg L⁻¹. The glucose and the elements played an important role in the algal growth. The hydrolyzates can be used as renewable nutrients to produce DHA from microalgae.

3.2. Effects of Different Amounts of Hydrolysates and NaNO₃ Concentrations on Cell Growth of I. galbana

3.2.1. Effects of Different Amounts of Hydrolysates

I. galbana was grown in different amounts of leftover dough hydrolysates with 300.0 mg L⁻¹ NaNO₃ (

Table 3. Effect of different amounts of leftover dough hydrolysates and NaNO₃ concentrations on cell growth of I. galbana.

Parameters	Amount of Leftover Dough Hydrolysates (mL L−1)					NaNO3 Concentration (mg L−1)
	0.0	25.0	50.0	100.0	200.0	0.0	150.0	300.0	600.0	1200.0
Xmax (g L−1)	0.39 ± 0.00	2.37 ± 0.02	4.18 ± 0.02	2.26 ± 0.06	0.58 ± 0.00	0.86 ± 0.03	2.48 ± 0.00	4.29 ± 0.04	2.56 ± 0.07	0.51 ± 0.02
Pmax (g L−1 d−1)	0.05 ± 0.00	0.53 ± 0.05	0.96 ± 0.06	0.45 ± 0.02	0.09 ± 0.00	0.13 ± 0.01	0.55 ± 0.07	0.99 ± 0.06	0.49 ± 0.03	0.09 ± 0.00
μmax (d−1)	0.04 ± 0.00	0.84 ± 0.06	1.45 ± 0.08	0.74 ± 0.04	0.11 ± 0.01	0.15 ± 0.00	0.89 ± 0.09	1.42 ± 0.05	0.77 ± 0.02	0.10 ± 0.01

X_max—maximum biomass concentration; P_max—maximum biomass productivity; μ_max—maximum specific growth rate.

and Figure 1). The biomass accumulation was time-dependent. The biomass concentration of the cultures with 0.0 and 200 mL L⁻¹ hydrolysates decreased slightly at the late growth stage. The maximum biomass concentration (X_max, g L⁻¹), biomass productivity (P_max, g L⁻¹ d⁻¹), and specific growth rate (μ_max, d⁻¹) of the optimal amounts of the hydrolysates (50.0 mL L⁻¹) were 4.18 g L⁻¹, 0.96 g L⁻¹ d⁻¹, and 1.45 d⁻¹, respectively. The biomass concentration of Schizochytrium limacinum was 18.5 g L⁻¹ under heterotrophic growth conditions [22]. The biomass concentration of Cryptothecodinium cohnii was 2.42 g L⁻¹ under autotrophic growth conditions [5]. The maximum biomass concentration of Schizochytrium sp. was 65 g L⁻¹ under heterotrophic growth conditions [23]. The difference in biomass concentration was mainly caused by different species under autotrophic/mixotrophic/heterotrophic growth conditions. X_max, P_max, and μ_max were enhanced with increasing hydrolysates from 0.0 to 50.0 mL L⁻¹. X_max, P_max, and μ_max decreased with increasing hydrolysates from 50.0 to 200.0 mL L⁻¹. This showed that cell growth was highly inhibited with 0.0 mL L⁻¹ hydrolysates compared to 50.0 mL L⁻¹ hydrolysates. This is because the culture with 0.0 mL L⁻¹ hydrolysates (0.0 g L⁻¹ glucose) had no organic carbon glucose to support cell growth and only used inorganic carbon CO₂ in the air to grow. The results showed that cell growth was inhibited with 25.0 mL L⁻¹ hydrolysates compared with 50.0 mL L⁻¹ hydrolysates. The possible reason is that the culture with 25.0 mL L⁻¹ hydrolysates had a low concentration of glucose. There was no sufficient organic carbon resource for I. galbana. Compared to 50.0 mL L⁻¹ hydrolysates, X_max, P_max, and μ_max of the culture (200.0 mL L⁻¹ hydrolysates) decreased markedly. The results showed that a high amount of hydrolysates (200.0 mL L⁻¹) decrease cell growth and that an excessive amount of hydrolysates was not conducive to the cell growth of I. galbana. The results were consistent with a previous study [24]. The possible reason for this is that the excessive amount of hydrolysates, causing substrate inhibition, decreased glucose metabolism and ATP production. ATP was necessary to cell growth and its decrease resulted in biomass reduction [25].

The effect of different amounts of hydrolysates on glucose and NO₃⁻ uptake is shown in Figure 2. I. galbana could accumulate biomass with glucose from the hydrolysates. Both the residual glucose and the NO₃⁻ concentrations of all the cultures except for 0.0 mL L⁻¹ hydrolysates decreased with increasing culture time. Different amounts of hydrolysates affected the residual glucose and the NO₃⁻ concentrations significantly (p < 0.05). The results suggest that amounts of hydrolysates have a great effect on glucose and NO₃⁻ uptake. After a 7-day cultivation, the culture with 200.0 mL L⁻¹ hydrolysates had the highest residual glucose concentration of 46.8 g L⁻¹ and a residual NO₃⁻ concentration of 187.0 mg L⁻¹. This showed that glucose and NO₃⁻ uptake was greatly inhibited. The residual glucose concentrations of the cultures with 25.0 and 50.0 mL L⁻¹ hydrolysates were 0.0 g L⁻¹. This showed that glucose in the hydrolysates was completely consumed by I. galbana. A previous study showed that there were detrimental effects of high glucose concentration on the nutrient uptake system of microalgae [26]. The residual NO₃⁻ concentration of the culture with 50.0 mL L⁻¹ hydrolysates was 0.0 mg L⁻¹. The results showed that overly low or overly high amounts of hydrolysates inhibited glucose and NO₃⁻ uptake.

Different amounts of hydrolysates affected the F_v/F_m of the cultures significantly (p < 0.05) (Figure 3). The F_v/F_m of I. galbana with 25.0, 50.0, and 100.0 mL L⁻¹ hydrolysates reached peak values on Day 5. Similar results were obtained in [17,21]. The F_v/F_m of I. galbana with 25.0, 50.0, and 100.0 mL L⁻¹ hydrolysates was relatively low at the initial growth stage and high at the late growth stage. It grew under heterotrophic or mixotrophic conditions at the initial growth stage and grew under autotrophic or mixotrophic conditions at the late growth stage. The F_v/F_m (200.0 mL L⁻¹ hydrolysates) decreased with increasing culture time. The microalga cultured with 50.0 mL L⁻¹ hydrolysates obtained the highest value of maximum F_v/F_m of 0.798. This indicated that an overly low or overly high amount of hydrolysates inhibited the photosynthesis of I. galbana.

3.2.2. Effects of NaNO₃ Concentration

I. galbana was grown under different NaNO₃ concentrations with 50.0 mL L⁻¹ hydrolysates (Table 3 & Figure 1). The biomass accumulated with culture time. The biomass concentration of the cultures with 0.0 and 1200 mg L⁻¹ NaNO₃ reached peak values on Day 4. The NaNO₃ concentrations affected cell growth significantly (p < 0.05). The X_max, P_max, and μ_max all increased at first. Then they decreased with a further increase in the NaNO₃ concentrations. The culture with 300.0 mg L⁻¹ NaNO₃ had the highest X_max of 4.29 g L⁻¹, a P_max of 0.99 g L⁻¹ d⁻¹, and a μ_max of 1.42 d⁻¹. The results show that a low or high concentration of NaNO₃ decreased the X_max, P_max, and μ_max and that a low or high NaNO₃ concentration inhibited microalgal growth. The results are consistent with a previous study [27]. A lower biomass concentration of I. galbana (1.25 g L⁻¹) is obtained under autotrophic growth conditions [28]. The X_max, P_max, and μ_max of the culture with 0.0 mg L⁻¹ NaNO₃ were 0.86 g L⁻¹, 0.13 g L⁻¹ d⁻¹, and 0.15 d⁻¹. The results show that the microalga could grow without the addition of NaNO₃. This might be because I. galbana used nitrogen resources in the hydrolysates to grow. The results show that an excessive concentration of NaNO₃ greatly inhibited microalgal growth because of substrate inhibition of the NaNO₃.

The NaNO₃ concentrations affected glucose and NO₃⁻ uptake significantly (p < 0.05) (Figure 4). The optimal NaNO₃ concentration for glucose and NO₃⁻ uptake was 300.0 mg L⁻¹. After a 7-day cultivation, the culture with 300.0 mg L⁻¹ NaNO₃ had the lowest residual glucose and NO₃⁻ concentrations of 0.0 g L⁻¹/mg L⁻¹. This showed that the glucose and the NO₃⁻ uptake was enhanced. The results show that the glucose in the hydrolysates and the NO₃⁻ were completely consumed by I. galbana. The culture with 1200.0 mg L⁻¹ NaNO₃ had the highest residual glucose concentration, 10.1 g L⁻¹, as well as the highest residual NO₃⁻ concentration, 812.0 mg L⁻¹. The results show that the glucose and the NO₃⁻ uptake was hindered with a high NaNO₃ concentration. The culture with 0.0 mg L⁻¹ NaNO₃ had relatively a high residual glucose concentration, 8.8 g L⁻¹. This shows that the glucose uptake was highly restrained without the addition of NaNO₃.

The NaNO₃ concentration significantly (p < 0.05) affected the photosynthesis of I. galbana (Figure 5). The F_v/F_m of I. galbana with 0.0 mg L⁻¹ NaNO₃ reached peak values on Day 3. Similar results were obtained by Wang et al. [8]. The reason for this may be that a lack of nitrogen reduced chlorophyll biosynthesis, which lowered the photosynthesis [29]. Compared with 300.0 mg L⁻¹ NaNO₃, the F_v/F_m of the microalga with an overly high concentration of NaNO₃ (600.0 and 1 200.0 mg L⁻¹) gradually decreased. The results show that the photosynthesis of the microalga was inhibited. Excessive NaNO₃ would affect the fixation of CO₂, thereby inhibiting photosynthesis [30]. The F_v/F_m of I. galbana with 150.0 and 300.0 mg L⁻¹ NaNO₃ reached peak values on Day 5. The microalga cultivated with 300.0 mg L⁻¹ NaNO₃ owned the highest value of maximum F_v/F_m of 0.795. This shows that an overly low or overly high concentration of NaNO₃ decreases microalgal photosynthesis.

3.3. Effects of Different Amounts of Hydrolysates and NaNO₃ Concentrations on Lipid Accumulation and DHA Production of I. galbana

3.3.1. Effects of Different Amounts of Hydrolysates

The microalga converted the glucose into lipids and DHA with different amounts of hydrolysates with 300.0 mg L⁻¹ NaNO₃ (

Table 4. Effect of different amounts of leftover dough hydrolysates and NaNO₃ concentrations on lipid accumulation and DHA production of I. galbana.

Parameters	Amount of Leftover Dough Hydrolysates (mL L−1)					NaNO3 Concentration (mg L−1)
	0.0	25.0	50.0	100.0	200.0	0.0	150.0	300.0	600.0	1200.0
Lipid content (%)	14 ± 0	37 ± 0	35 ± 2	15 ± 1	16 ± 0	33 ± 1	35 ± 0	37 ± 2	12 ± 0	10 ± 0
Lipid productivity (mg L−1d−1)	7.8 ± 0.0	203.5 ± 1.0	343.4 ± 4.5	78.6 ± 1.1	11.0 ± 0.0	35.8 ± 1.2	202.0 ± 3.7	366.3 ± 6.4	71.5 ± 2.3	5.9 ± 0.0
Maximum lipid yield (mg L−1)	54.6 ± 1.4	1461.5 ± 8.7	2436.0 ± 10.0	565.5 ± 5.2	92.8 ± 3.0	283.8 ± 4.5	1449.0 ± 9.0	2601.1 ± 9.7	512.4 ± 6.9	51.0 ± 1.0
DHA content (%)	7 ± 0	14 ± 0	14 ± 1	8 ± 1	8 ± 0	12 ± 1	11 ± 0	13 ± 1	5 ± 1	4 ± 0
Maximum DHA yield (mg L−1)	3.8 ± 0.0	204.6 ± 1.9	341.3 ± 4.6	45.2 ± 1.0	7.4 ± 0.1	34.1 ± 0.2	159.4 ± 5.8	338.1 ± 2.6	25.6 ± 0.0	2.0 ± 0.0

). Different amounts of hydrolysates affected lipid content significantly (p < 0.05). The culture with 25.0 mL L⁻¹ hydrolysates had the highest lipid content, 37%. The lipid content of I. galbana (71.1%) is about two times that in this study [28]. A higher lipid content of I. galbana (44.93%) was obtained [10]. The culture with 50.0 mL L⁻¹ hydrolysates had a relatively high lipid content, 35%. This was caused by nitrogen limitation. I. galbana was under nitrogen limitation at the later 3 days (Figure 2B). The lipid content greatly increased under nitrogen limitation [31,32]. The optimal amount of hydrolysates was 50.0 mL L⁻¹ with the highest maximum lipid and DHA yield, 2436.0 and 341.3 mg L⁻¹, respectively. The culture with 0.0 mL L⁻¹ hydrolysates had the lowest maximum lipid and DHA yields, 54.6 and 3.8 mg L⁻¹, respectively. A lower or higher amount of hydrolysates reduced lipid and DHA accumulation significantly (p < 0.05). This shows that an amount of hydrolysates below 25.0 mL L⁻¹ or above 100.0 mL L⁻¹ could be harmful to lipid and DHA accumulation.

The composition of the fatty acids of I. galbana with different amounts of hydrolysates was analyzed (

Table 5. Effect of different amounts of leftover dough hydrolysates and NaNO₃ concentrations on fatty acid composition of I. galbana.

Parameters	Amount of Leftover Dough Hydrolysates (mL L−1)					NaNO3 Concentration (mg L−1)
	0.0	25.0	50.0	100.0	200.0	0.0	150.0	300.0	600.0	1200.0
C14:0	15 ± 0	10 ± 0 *	11 ± 0	12 ± 0	14 ± 0	13 ± 0	10 ± 0	10 ± 0	15 ± 0	17 ± 0
C16:0	13 ± 1	9 ± 0	7 ± 0	11 ± 0	13 ± 1	9 ± 1	7 ± 0	8 ± 0	13 ± 0	14 ± 1
C16:1	7 ± 0	3 ± 0	4 ± 0	5 ± 0	7 ± 0	6 ± 0	6 ± 0	4 ± 0	2 ± 0	6 ± 0
C18:0	6 ± 0	3 ± 0	3 ± 0	4 ± 0	4 ± 0	4 ± 0	2 ± 0	2 ± 0	5 ± 0	4 ± 0
C18:1	17 ± 0	12 ± 0	13 ± 0	15 ± 1	16 ± 0	15 ± 1	16 ± 0	14 ± 0	10 ± 1	15 ± 0
C18:2	3 ± 0	5 ± 0	4 ± 0	3 ± 0	3 ± 0	6 ± 0	5 ± 0	4 ± 0	2 ± 0	5 ± 0
C18:3	8 ± 0	12 ± 0	11 ± 0	10 ± 0	9 ± 0	11 ± 0	13 ± 0	12 ± 0	14 ± 0	6 ± 0
C18:4	8 ± 0	14 ± 0	15 ± 0	11 ± 0	8 ± 0	10 ± 0	15 ± 0	16 ± 0	14 ± 0	8 ± 0
C20:0	6 ± 0	7 ± 0	5 ± 0	8 ± 0	8 ± 0	5 ± 0	4 ± 0	6 ± 0	8 ± 0	10 ± 0
C22:5	3 ± 0	6 ± 0	7 ± 0	5 ± 0	3 ± 0	3 ± 0	6 ± 0	6 ± 0	5 ± 0	2 ± 0
C22:6	7 ± 0	14 ± 0	14 ± 0	8 ± 0	8 ± 0	12 ± 0	11 ± 0	13 ± 0	5 ± 0	4 ± 0
Others	7 ± 0	5 ± 0	6 ± 0	8 ± 1	7 ± 0	6 ± 0	5 ± 0	5 ± 0	7 ± 1	9 ± 0
Unsaturated fatty acids	54 ± 1	66 ± 0	68 ± 0	57 ± 0	54 ± 1	63 ± 1	72 ± 0	69 ± 0	52 ± 0	46 ± 1
Saturated fatty acids	38 ± 0	29 ± 1	26 ± 1	35 ± 1	38 ± 0	31 ± 1	23 ± 1	26 ± 1	41 ± 1	45 ± 0

*—% total fatty acids.

). There were 11 kinds of fatty acids, and their relative content was different. The same eleven varieties of fatty acids in I. galbana were identified in a previous study [8]. There are only six varieties of fatty acids without DHA in I. galbana [10]. The amount of hydrolysates affected DHA accumulation significantly (p < 0.05). I. galbana with 25.0 and 50.0 mL L⁻¹ hydrolysates had the highest DHA content, 14%. The results suggest that a lower or higher amount of hydrolysates is not conductive to DHA accumulation.

3.3.2. Effects of NaNO₃ Concentrations

Nitrogen resources are important for the lipid accumulation of microalgae [33,34]. The effect of NaNO₃ concentration on lipid production was investigated when the amount of hydrolysates was 50.0 mL L⁻¹ (Table 4). The NaNO₃ concentration affected lipid and DHA accumulation significantly (p < 0.05). The culture with 300.0 mg L⁻¹ NaNO₃ obtained the highest lipid and DHA content, 37% and 13%, respectively. The optimal NaNO₃ concentration was 300.0 mg L⁻¹ with maximum lipid and DHA yields of 366.3 and 338.1 mg L⁻¹, respectively. The culture with 0.0 mg L⁻¹ NaNO₃ obtained the maximum DHA yield of 34.1 mg L⁻¹. The results show that the maximum DHA yield of I. galbana with 300.0 mg L⁻¹ NaNO₃ concentration was 8.9-fold higher than that of the control. The culture with 1200.0 mg L⁻¹ NaNO₃ had the lowest lipid and DHA yield. The lipid and DHA yield enhanced with increasing NaNO₃ concentration from 0.0 to 300.0 mg L⁻¹ (Table 4). Triacylglycerol synthesis in the microalgae was prompted under nitrogen limitation by enhancing carbon flux toward glycerol-3-phosphate and acyl-CoA [35,36]. However, an overly high concentration of NaNO₃ decreased lipid and DHA production.

There were also 11 kinds of fatty acids, the relative content of which was different (Table 5). The contents of unsaturated and saturated fatty acids and DHA contents were significantly affected (p < 0.05) by the NaNO₃ concentrations. There were also 11 varieties of fatty acids including C20:2, C20:3, C20:4, and C20:5 in the I. galbana without C14:0 and C16:0 [8]. The difference in fatty acids composition may be caused by different culture days. The culture with 300.0 mg L⁻¹ NaNO₃ had the highest DHA content of 13%. Compared with 300.0 mg L⁻¹ NaNO₃, the DHA content decreased with high NaNO₃ concentrations (600.0 and 1200.0 mg L⁻¹). Compared to 300.0 mg L⁻¹ NaNO₃, the DHA content also decreased without the addition of NaNO₃ (0.0 mg L⁻¹).

4. Conclusions

The microalgae-based byproduct recycling pathway is a promising DHA production strategy. DHA biosynthesis is prompted by the hydrolysates from starch-rich food processing byproducts. The glucose in the hydrolysates could be used as an organic carbon resource to produce DHA by I. galbana under different NaNO₃ concentrations. As shown in our study, the optimal amount of hydrolysates for microalgal growth and the DHA production of I. galbana was 50.0 mL L⁻¹. The DHA production of I. galbana is enhanced 8.9-fold with 300.0 mg L⁻¹ NaNO₃.