Enhancing algal biomass, lipid and astaxanthin production by mix-cultivation of Haematococcus pluvialis with Simplicillium lanosoniveum DT06

To enhance algal lipid and astaxanthin synthesis, the astaxanthin-producing green alga Haematococcus pluvialis was mix-cultivated with the antibiotic-synthesizing fungus Simplicillium lanosoniveum DT06 under non-aseptic conditions (NM) in this study. Results showed that in contrast to aseptic pure culture (AH) and non-aseptic pure culture (NH) of H. pluvialis , the production of cell (biomass), lipids and astaxanthin increased 56% and 119%, 112.4% and 279%, 74% and 175%, reaching 2.45 g/l, 0.837 g/l and 88.84 mg/l respectively; the average growth rate and the average specific growth rate increased 60.8% and 133.1 %, 19% and 31.6%, reaching 194.2 mg l -1 d -1 and 0.25 d -1 respectively; and the average lipid synthesis rate and average specific lipid synthesis rate increased 112.5% and 278.66%, 36.15% and 97%, reaching 69.75 mg l -1 d -1 and 28.47 mg g -1 d -1 respectively; and also the content of C16-C18 fatty acids that are suitable for biofuels production increased to 83.19%. Therefore, NM provides an efficient and economical way for the production of biofuels.

Haematococcus pluvialis is a unicellular green alga, capable of synthesizing high-value astaxanthin along with lipids (13), and thus a suitable candidate for biofuel production (14)(15)(16). Whereas, H. pluvialis's low cell growth rate constrains its lipid productivity, which is attributed, at least partially, to its susceptibility to harmful bacteria and fungi that inhibit its growth severely (9,17). As a matter of fact, contamination of harmful microorganisms is a major impediment to any algae for substantial production of biofuels, because aseptic cultivation of algae outdoor on an industrial scale is unachievable. Therefore, suppressing or even eliminating harmful bacteria and fungi is essential to promote cell growth and lipid synthesis of H. pluvialis and other algae.
In the previous studies, we isolated a symbiotic fungus simplicillium lanosoniveum DT06 from the culture broth of cyanobacterium Chroococcus sp. (18). DT06 can synthesize a new antibiotic inhibiting gram-negative bacteria and some fungi (19) and promote Chlamydomonas reinhardtii's growth and lipid production in aseptic cultures (20). Therefore, in the present study, we mix-cultivated H. pluvialis with DT06 in non-aseptic cultures to mimic the actual outdoor culture regime of H.
pluvialis and find out if enhancements of cell, lipid and astaxanthin production can be achieved. 4

Microorganisms and media
H. pluvialis was obtained from the Institute of Hydrobiology of Chinese Academy of Science, and maintained at 4 o C in a liquid BBM medium (13). Seed culture was prepared by inoculating 5 ml activated H. pluvialis culture into 250 ml flasks containing 100 ml of BBM medium, and incubated in an orbital shaker with top cool white fluorescent lamps at 25 o C, 110 rpm, 60 μmol photons m -2 s -1 .
The fungus simplicillium lanosoniveum DT06 was isolated from the culture broth of cyanobacterium Chroococcus sp. (18), which was streaked on PDA (21) agar plate and incubated at 28 o C for 7 days to facilitate spore production. The fungal spores were collected from the agar plate with 20 ml sterile distilled water and counted using a hemacytometer under the light microscope.

Growth kinetics
The cell growth rate (g l -1 d -1 ) of H. pluvialis was calculated through Eq. (2). r g = X n -X 0 t n -t 0 The specific cell growth rate (d -1 ) of H. pluvialis was calculated through Eq. (3). μ= lnX n -lnX 0 t n -t 0 where X0 and Xn is the biomass (g l -1 ) at day t0 and tn respectively.

Lipid production
0.05 g of dried algal cells was blended with 5 ml of chloroform/methanol (2: 1) and the mixture was stirred at 2000 rpm on a magnetic stirrer for 20 min. The procedure was carried out 2 times. All the chloroform layers were collected, combined and evaporated to dryness at 60 o C. The total lipid (GL) was weighed on an analytical 6 balance.

Lipid synthesis kinetics
The total lipid content (mg g -1 ) of H. pluvialis was calculated through Eq. (4).
where GL is the total lipid (mg), V is the culture volume (l), X is biomass (g l -1 ).
The specific lipid synthesis rate of H. pluvialis was calculated through Eq. (6).
where X0 and Xn is the biomass (g l -1 ) at day t0 and tn, while G0 and Gn is the corresponding lipid content (mg g -1 ) at day t0 and tn, respectively.

Astaxanthin production
Astaxanthin was analyzed according to the method (13).

Nitrate nitrogen
Nitrate nitrogen was analyzed according to the method (24).

pH
pH was measured using a pH meter.

Biomass
The growth of H. pluvialis was promoted in NM but inhibited in NH in comparison with AH. As shown in Fig. 1, the biomass of AH increased slowly on the first 2 days (lag phase) and rapidly from then on till the 8th day and hereafter remained relative stable at 1.57 g/l (stationary phase). The biomass of NH changed in a manner similar to that of AH but was lower than those of AH and reached 1.12 g/l at the end of experiment, which was 29% lower than that of AH. By contrast, the biomass of NM elevated drastically from day2 till day10 and reached the highest value of 2.45 g/l on day12, which increased 56% and 119% compared to those of AH and NH, respectively. 8

Growth kinetic
As shown in Table 1

Lipid concentration and lipid content
As shown in Fig. 2, the highest lipid concentration (0.837 g/l) and lipid content (341.8 mg/g) were achieved in NM, which increased 112.4% and 36%, 279% and 73% in comparison with those of AH (0.394 g/l, 251.4 mg/g) and NH (0.221 g/l, 197.5 mg/g), respectively.

Lipid synthesis kinetics
The results of kinetic analyses were listed in Table 2. The average lipid synthesis rate (69.75 mg l -1 d -1 ) and average specific lipid synthesis rate (28.47 mg g -1 d -1 ) of NM were the highest among the three cultures, which were 112.5% and 36.15% higher than those of AH (32.83 mg l -1 d -1 , 20.91 mg g -1 d -1 ) and 278.66% and 97% higher than those of NH (18.42 mg l -1 d -1 , 14.45 mg g -1 d -1 ), respectively.

Astaxanthin production / Astaxanthin content
The astaxanthin production (concentration) and astaxanthin content in three cultures ( Fig. 3) were consistent with the lipid production and content (Table 2), which were highest in NM (88.84 mg/l, 36.26 mg/g) followed AH (51.04 mg/l, 32.51 mg/g) and NH (32.31 mg/l, 28.85 mg/g), and thus the astaxanthin production and astaxanthin 9 content of NM were 74%, 11.53% and 175%, 25.68% higher than those of AH and NH, respectively.

Fatty-acid composition
As results shown in Table 3, the fatty acids profiles of the total lipids of H. pluvialis in all cultures were in the range of C14-C22 and mainly C16-C18 that are suitable for biodiesel production (25). The content of C16-C18 fatty acids of NM (83.19%) was higher than those of AH (82.6%) and NH (80.71%), and more importantly oleic acid

Nitrate nitrogen
Variation of nitrate nitrogen concentration of three cultures (Fig. 4) followed a similar pattern. The nitrate nitrogen concentration of AH and NH declined drastically within the first 2 days, from 250 mg/l to 73.43 mg/l and 106.43 mg/l, and then slowly to 11.34 mg/l and 18.53 mg/l respectively at the end of the experiment. By contrast, the nitrate nitrogen concentration of NM decreased more drastically than those of AH and NH to 40.56 mg/l on the 2th day, and became undetectable on the 8th day.

pH
As shown in Fig. 5, the pH of AH and NH elevated quickly and continuously with the onset of experiment and reached 9.67 and 9.98 respectively at the end of the experiment. By contrast, the pH of NM increased slowly to 8.52 after 8 days, and then remained relatively constant in the range of 8.48 to 8.57.

Discussion
The results of this study demonstrated that the production of cell (biomass) (Fig. 1), lipid (Fig. 2) and astaxanthin (Fig. 3) of H. pluvialis elevated in NM in comparison with those of AH and NH.
(1) The elevation of H. pluvialis biomass in NM was caused definitely by the enhancement of cell growth as evidenced by the elevation of the average cell growth rate and specific growth rate (Table 1), which, in turn, might be attributed to the following factors: (a) DT06 suppressed or even eliminated the harmful microorganisms (bacteria and fungi) by synthesizing antibiotic. Since NM and NH were both non-aseptic cultures, the decrease of biomass in NH compared to that of AH (aseptic culture) indicted the existence and inhibition of harmful microorganisms against H. pluvialis; while, on the other hand, the fact that the biomass of NM was higher than that of AH implied the suppression or even elimination of the harmful microorganisms in NM.  11 those in other alga-microorganism co-cultures (13,27,28,29,30).
(c) The metabolism of DT06 stabilized the pH of NM. The quickly elevated pHs in AH and NH (Fig. 5) inhibited cell growth, which were resulted primarily from the uptake of physiologically alkaline salts e.g., NaNO3 (Fig. 4) and secretion of NH4 + (31) by H. pluvialis; while in NM, the release of CO2 and uptake of NH4 + by DT06 lowered and relatively stabilized the culture pH (Fig. 5), which was in favor of H.
pluvialis growth. Additionally, it is noteworthy that after the uptake of NH4 + , DT06 probably secreted organic nitrogen for the growth of H. pluvialis, which could explain the reason for the growth of H. pluvialis after day8 ( Fig. 1) when nitrate nitrogen was depleted (Fig. 4).
(2) The elevation of lipid production in NM was ascribed to (a) the elevated biomass of H. pluvialis (Fig. 1) that set the cell base for enhanced synthesis of lipid, and (b) the enhancement of lipid synthesis as evidenced by the elevation of lipid content (Fig.   2), average lipid synthesis rate and specific lipid synthesis rate of H. pluvialis ( Table   2). The reasons for this seemed to come from two aspects: firstly, the quickly depletion of nitrate nitrogen (Fig. 4), which resulted in nitrogen starvation that promoted lipid synthesis by directing the carbon flux toward the specific pathway (32), and secondly CO2 generated by DT06 increased CO2 concentration, which not only facilitated lipid synthesis (33)(34)(35) but also influenced the composition of fatty acid (36)(37)(38), and thus might be the cause for the enrichment of C16-C18 fatty acids, particularly oleic acid in NM (Table 3) as well.
(3) The reasons for the elevated astaxanthin production in NM (Fig. 3) were similar to 12 or even the same as those for the enhanced lipid yield (Fig. 2). Since astaxanthin is lipid-soluble dispersing in lipid droplets in H. pluvialis cells (15,16,39), it is not surprising that lipid and astaxanthin syntheses are closely associated, and conditions favor lipid synthesis also enhance astaxanthin production (40)(41)(42).

Conclusion
Mix-cultivation of the antibiotic-synthesizing fungus simplicillium lanosoniveum DT06 with the astaxanthin-producing green alga Haematococcus pluvialis in non-aseptic condition promotes algal growth as well as lipid and astaxanthin syntheses, and thus provides an efficient way for co-production of biofuels and high-value products.