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Comparisons of Growth and Biochemical Composition between Mixed Culture of Alga and Yeast and Monocultures
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 104, No. 5, 391–397. 2007 DOI: 10.1263/jbb.104.391
© 2007, The Society for Biotechnology, Japan
Comparisons of Growth and Biochemical Composition between Mixed Culture of Alga and Yeast and Monocultures ShiQing Cai,1 ChaoQun Hu,1* and ShaoBo Du1 Key Laboratory of Applied Marine Biology of Guangdong Province (LAMB), South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, People’s Republic of China1 Received 14 February 2007/Accepted 4 August 2007
The alga Isochrysis galbana 8701 and the yeast Ambrosiozyma cicatricosa were mix-cultivated in the same medium for 7 d to compare their growth performance and biochemical composition with those of the same organisms cultured under monoculture conditions. The specific growth rates of both species were significantly higher (p <0.05) in the mixed culture than in the monocultures during the corresponding experimental phases. At the end of experiment, the biomass concentration obtained in the mixed culture reached 1.32 ± 0.04 g/l of dry weight, which was significantly higher (p < 0.05) than those obtained in monocultures, and the alga I. galbana in the mixed culture dominated the cell numbers making up 96.64% of the cells. The biochemical profile of the mixed culture is similar to that of the I. galbana monoculture with some variations; The percentages of both the fatty acids 14:00 and 18:00 detected in the mixed culture were significantly higher (p < 0.05) than those detected in the I. galbana monoculture, while the content of the fatty acid 18:2(n-6) detected in the mixed culture was significantly lower (p <0.05) than that detected in the I. galbana monoculture. This study indicates the improved growth performance in mixed culture compared with monocultures and the similarities between the biochemical compositions of the mixed culture and the I. galbana monoculture. [Key words: Ambrosiozyma cicatricosa, biomass, Isochrysis galbana, mixed culture]
lopsis larvae, and Saccharomyces acidosaccharophill can be used as rotifer food. However, to date, yeast is still limited in terms of its use as a partial substitute for alga as food for marine organisms because of the deficiency of certain compositional components in yeast products, particularly the lack of highly unsaturated fatty acids (HUFAs), which are essential for the successful growth and development of many marine organisms (10). Many methods and techniques, such as the use of bioreactors, the heterotrophic culture of alga, and the mixed culture of microorganisms, have been developed to reduce the costs of aquatic food production, and/or for improving the nutritional value of aquatic food. Of these techniques, the mixed culture of microorganisms is a convenient solution for achieving the goals. When using a mixed culture, two or more preselected species of microorganism are synchronously cultivated within the same medium, where these microorganisms can mutually exploit complementary metabolic activities to survive, grow, and reproduce (11). Mixed cultures of heterotrophic microorganisms are often used for the treatment of waste materials discharged as a result of agricultural and industrial activities, as well as for the production of biomass and bioactive compounds (11). Although the fact that growth is prompted when algae and yeast are mixcultured in the same medium was confirmed in other fields
Marine microalgae have been widely used as an aquaculture food (1), because they can supply the energy and nutrients essential for the growth and development of marine organisms (2). Although marine microalgae have been appreciated as a food in aquacultures, alga production is subjected to the constraints of high operational costs (3, 4). Therefore, even a small improvement in alga culture techniques could result in substantial savings in the mass production of microalgae (5), and the major challenge facing algologists is the improvements of culture techniques to reduce production costs while maintaining alga production reliability (4). Yeast is an important unicellular organism existing extensively in seawater. Because it can be mass-produced at a relatively low cost and is high in nutritional value, yeast is gradually accepted by people and its employment as food in aquaculture is becoming common (6, 7). Furthermore, the potential numbers of species of yeast that can be applied in aquaculture are increasing, for example, six new strains of yeast were examined and evaluated as food for bivalves by Brown et al. (8). Agh and Sorgeloos (9) reported that the yeasts Saccharomyces cerevisiae, Rhodotorula, Zygosaccharomyces marina, Torulopsis candida var. marina, Toru* Corresponding author. e-mail: [email protected] phone/fax: +86-20-89023218 391
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(12, 13), little information is available on mixed cultures of algae and yeast being used as live food in aquacultures. The objective of this study is to investigate the growth performance and biochemical composition of a mixed culture of the alga I. galbana and the yeast A. cicatricosa and compare them with those under monoculture conditions, particularly to discuss the biochemical quality from the view point of aquaculture live food.
250°C at 10°C min–1, and kept at 250°C for 10 min. After that, samples of 1 µl were injected at a temperature of 275°C. Detection was performed with a flame ionization detector at a temperature of 280°C. The scan range was 29.0–450.0 amu with a scan rate of 1 s–1. The experimental results were compared statistically by oneway analysis of variance (ANOVA) (P ≤0.05) using software packages of SPSS 13.0.
RESULTS MATERIALS AND METHODS Strains and culture conditions The alga I. galbana 8701 used in this study was obtained from the Ocean University of China，and the yeast A. cicatricosa, which was one of strains confirmed to be useful as live food in aquacultures (14), was randomly selected for this study. The cells were in the size range of (length × width) 2–7 µm × 1–4 µm for the yeast A. cicatricosa, and 4–6 µm × 3–4 µm for the alga I. galbana. Two axenic microorganisms were separately cultivated to the exponential phase prior to use. Aged seawater autoclaved at 120°C for 20 min, enriched with f/2 medium (15) and supplemented with 2 g/l glucose, was used for a culture medium. Cultures were carried out in 5-l Erlenmeyer flasks containing 2000 ml of the culture medium. An equal concentration of 2.5 ×104 cells ml–1 was used for the start concentration of the two monocultures, while the inoculum of the mixed culture contained half of the number (1.25 ×104 cells ml–1) of each species. The culture time was 7 d with continuous illumination at an irradiance of 72 µmol m–2 s–1, provided by fluorescent lamps (75 W) in a temperature-controlled chamber (20 ±1°C). All treatments were carried out in triplicate. Analysis of growth rate The daily total number of cells in each treatment was determined with a haemocytometer after fixing with 5% formalin (5). In the case of the mixed culture, the cell density of the yeast was determined by plating differently diluted culture fluid on agar dishes (12). The specific growth rate (K) of each culture was calculated from the expression K = (ln Nt − ln N0)/t where Nt is the cell count at time t, N0 is the initial cell count at time zero, and t is time (d). Analysis of biomass concentration A known volume (3–10 ml) of individual culture fluid was filtered through predried Whatman GF/C filter paper (0.45 µm), then dried to a constant weight to determine daily biomass concentration according to method reported by Ip and Chen (16), and the first sampling was carried out 2 d after inoculation. At the end of the experiment, the cultures were harvested by centrifugation at 3000 rpm for 10 min, and the cell pellets were rinsed twice with distilled water, recentrifuged, and then freeze-dried for biochemical analysis. Determination of biochemical composition Proteins were extracted following the procedure of Rafiqul et al. (17) and quantified using the dye-binding method described by Braford (18). Lipids were extracted using the method of Bligh and Dye (19) and quantified following the method of Pande et al. (20). The carbohydrates were determined using the phenol-sulfuric acid method with glucose as a standard (21). The ash content was calculated after combusting samples at 450°C for 16 h. The amino acids were analyzed using an automatic amino acid analyzer (model 835-50; Hitachi, Tokyo). Prior to analysis, the samples were subjected to hydrolysis according to the method described by Sujak et al. (22). Fatty acid analysis was carried out following the method of Swaaf et al. (23); GC/MS with a column of 30 m × 0.25 mm × 0.25 µm was performed, the column temperature was raised from 150°C to
Growth The dynamics of the specific growth rates of the three cultures are shown in Fig. 1. The maximal specific growth rates of the three cultures (1.03 ±0.020 for mixed culture, 0.9 ± 0.019 for I. galbana monoculture and 0.55 ± 0.020 for A. cicatricosa monoculture) all appeared on day 2, and then decreased to a relatively low level (0.80 ± 0.030 for mixed culture, 0.73 ± 0.020 for I. galbana monoculture and 0.10 ±0.020 for A. cicatricosa monoculture) on day 3. Thereafter, the specific growth rates gradually decreased to a minimal level (0.64 ± 0.030 for mixed culture, 0.63 ± 0.010 for I. galbana monoculture and 0.02 ± 0.015 for A. cicatricosa monoculture) at the end of the experiment. Throughout the cultivation process, the A. cicatricosa monoculture had the lowest value for specific growth rate of the three cultures. The specific growth rates of the mixed culture were significantly higher (p < 0.05) than those of the I. galbana monoculture during the corresponding experimental phases except for that on day 7. On day 7, there was no significant difference in specific growth rate between the mixed culture (0.64 ± 0.030) and the I. galbana monoculture (0.63 ± 0.010). This may have resulted from the depletion of the nutrients in the culture medium used, leading to the decrease in the fast growth of the two species in the mixed culture. Between days 2 and 3, the specific growth rate of the A. cicatricosa monoculture decreased by 82%, which was a much more rapid change than those of two alga-containing cultures (22% for mixed culture and 25% for I. galbana monoculture). Within the system of the mixed culture (Fig. 2), from day
FIG. 1. Dynamics of specific growth rates of each culture. Data are expressed as mean ± standard deviation of three replicates. Filled squares, Mixed culture; empty lozenges, I. galbana monoculture; filled triangles, A. cicatricosa monoculture.
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FIG. 2. Dynamics of specific growth rates of two species in mixed culture. Data are expressed as mean ± standard deviation of three replicates. Lozenges, I. galbana; squares, A. cicatricosa.
FIG. 3. Dynamics of proportions of two species in mixed culture. Data are expressed as mean ± standard deviation of three replicates. Lozenges, I. galbana; squares, A. cicatricosa.
2 to day 7, the specific growth rates of the A. cicatricosa were 1.17 ± 0.026, 0.63 ± 0.020, 0.47 ± 0.032, 0.37 ± 0.050, 0.30 ± 0.029, and 0.26 ± 0.032, respectively, which were significantly higher than those obtained in the A. cicatricosa monoculture during the corresponding experimental phases (p < 0.05). As for the alga I. galbana, from day 3 to the end of the experiment, the specific growth rates were 0.92 ±0.020, 0.88 ± 0.032, 0.84 ± 0.049, 0.81 ± 0.028, and 0.74 ± 0.035, respectively, which were also significantly higher (p < 0.05) than those obtained in the I. galbana monoculture during the corresponding experimental phases. The specific growth rate of I. galbana obtained in the mixed culture on day 2
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FIG. 4. Variations in biomass concentration for each culture. Data are expressed as mean ± standard deviation of three replicates. Empty squares, Mixed culture; filled squares, I. galbana monoculture; filled triangles, A. cicatricosa monoculture.
(0.84 ± 0.025) was on the contrary, lowering (p <0.05) than that (0.98 ± 0.019) of I. galbana in monoculture on day 2. The changes in cell number proportion in the mixed culture are illustrated in Fig. 3. The accumulative trends of the cell numbers of the two species in the mixed culture were opposite. On day 2, the bulk of the cell number (65.88%) in the mixed culture was accounted for by the yeast A. cicatricosa, and then the proportion of yeast cell number decreased rapidly, accompanying the increase in the number of alga cells. At the end of the experiment, the proportion of yeast cells decreased to 3.36%, on the other hand, the proportion of alga cells amount increased to 96.64%. Biomass The variations in the biomass concentrations of the three cultures are shown in Fig. 4. The maximum biomass concentration (0.31 ± 0.050 g/l) of the A. cicatricosa monoculture was found on day 2, and, at the end of experiment, the value fell to 0.17 ± 0.090 g/l. Inversely, the biomass concentration of the I. galbana monoculture increased from 0.10 ± 0.089 g/l (on day 2) to 1.17 ± 0.024 g/l (on day 7). In the case of the mixed culture, a similar increase in biomass concentration was observed but the increase was relatively rapid, increasing from 0.11 ± 0.090 g/l (on day 2) to 1.32 ± 0.041 g/l (on day 7). From day 5 to the end of the experiment, the biomass concentrations (1.08 ± 0.021 g/l, 1.22 ± 0.020 g/l, and 1.32 ± 0.041 g/l, respectively) were significantly higher (p < 0.05) in the mixed culture than in the I. galbana monoculture (0.73 ± 0.040 g/l, 1.09 ± 0.060 g/l, and 1.17 ± 0.024 g/l, respectively) during the corresponding experimental phases. Biochemical composition There were significant differences in gross composition between the A. cicatricosa monoculture and the two alga-containing cultures (Table 1),
TABLE 1. Gross compositions of each culture (% of dry biomass) Culture method Protein Lipid Carbohydrate Ash 11.21 ± 3.01b 38.36 ± 0.94b 2.64 ± 0.37a Mixed culture 41.42 ± 2.06a I. galbana monoculture 38.51 ± 1.36a 15.11 ± 1.71b 41.82 ± 3.12b 2.18 ± 0.22a b a a A. cicatricosa monoculture 51.32 ± 2.10 6.35 ± 0.88 28.48 ± 2.81 6.37 ± 0.44b All the values represent mean ± S.D. and values that show the same superscript letter in the same column are not significantly different (P>0.05).
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TABLE 2. Amino acid compositions (% of total amino acids) of each culture I. galbana monoculture
Mixed culture
A. cicatricosa monoculture
Nonessential 8.50 ± 0.20a 6.58 ± 0.17b Ala 8.90 ± 0.39a Asp 10.74 ± 0.28a 10.78 ± 0.10a 10.19 ± 0.25b Glu 12.69 ± 0.08b 14.12 ± 0.50a 14.58 ± 0.14a Gly 6.43 ± 0.19b,c 6.15 ± 0.08c 6.77 ± 0.43a,b Ser 3.24 ± 0.21b 3.42 ± 0.26b 4.10 ± 0.29a Arg 6.51 ± 0.39b 6.15 ± 0.04b 7.52 ± 0.33a Pro 4.51 ± 0.32b,c 4.14 ± 0.06c 4.76 ± 0.14a,b Essential 0.37 ± 0.08a 0.50 ± 0.09a Cys 0.38 ± 0.07a His 1.92 ± 0.31a 1.95 ± 0.05a 2.10 ± 0.42a a a Ile 4.92 ± 0.32 5.05 ± 0.09 5.31 ± 0.22a Leu 9.15 ± 0.28a 9.06 ± 0.79a 7.76 ± 0.65b Lys 5.16 ± 0.15b 5.11 ± 0.92b 9.06 ± 1.18a Met 2.88 ± 0.74a 2.78 ± 0.44a 1.84 ± 0.93a Phe 5.08 ± 0.34b,c 5.29 ± 0.32a,b 4.53 ± 0.40c Tyr 3.57 ± 0.46a 3.45 ± 0.33a 2.38 ± 0.37b Thr 4.95 ± 0.31a 4.89 ± 0.60a 4.72 ± 0.11a Trp 1.95 ± 0.76a 1.82 ± 0.75a 1.20 ± 0.36a a a Val 7.09 ± 0.64 6.87 ± 0.64 6.04 ± 0.93a All the values represent mean ± S.D. and values that show the same superscript letter in the same line are not significantly different (P>0.05).
while no significant difference was found between the two alga-containing cultures. The two alga-containing cultures had similar compositions for both essential and nonessential amino acids except that the concentration of the amino acid glutamate (14.12 ± 0.50%) was significantly higher in mixed culture than in the I. galbana monoculture (12.69 ± 0.08%), as shown in Table 2. Methionine is an important sulphur-containing amino acid, and the proportions of methionine were 2.88 ± 0.74% for the I. galbana monoculture and 2.78 ± 0.44% for mixed culture. As for the fatty acids (Table 3), the dominant fatty acid present in all three cultures was 18:1(n-9). No HUFAn3 were detected in the A. cicatricosa monoculture, which was significantly different from the two alga-containing cultures, in which HUFAn3 were found in similar profile. Although at a low level, the two highly important fatty acids 20:5(n-3) and 22:6(n-3), were both detected in the two alga-containing cultures. In addition, the mixed culture was unique in its high contents of the fatty acids 14:00 (18.85 ± 0.84%) and 18:00 (9.03 ± 0.49%) compared with the contents in the I. galbana monoculture. However, the I. galbana monoculture contained a higher content of the fatty acid 18:2(n-6) (22.76 ±4.07%) than the mixed culture (8.00 ±0.76%). Taken together, the mixed culture was richer in saturated fatty acids (44.28 ±1.79%) than the two monocultures, whereas the contents of both monoenoic and polyenoic fatty acids were higher in the two monocultures than in the mixed culture. DISCUSSION Growth There are several interactions between species exhibited in mixed microorganism cultures, such as competition, predation, parasitism, and amensalism (11). Only those species exhibiting neutralism, commensalism, mutual-
TABLE 3. Fatty acid compositions (% of total fatty acids) of each culture I. galbana Mixed A. cicatricosa monoculture culture monoculture 12:00 – – 1.95 ± 0.67 18.85 ± 0.84b 1.95 ± 0.24c 14:00 3.40 ± 0.09a 15:00 – 0.43 ± 0.10 – 15.13 ± 0.19c 15.59 ± 0.57a,c 16:00 16.07 ± 0.26a,b 16:1(n-7) 0.98 ± 0.34a 1.05 ± 0.18a 8.77 ± 0.81b 17:00 0.41 ± 0.09b 0.04 ± 0.02a – 17:1(n-9) – 0.30 ± 0.12 – 9.03 ± 0.49b 9.46 ± 0.45b 18:00 5.79 ± 0.66a 18:1(n-9) 40.95 ± 2.49b 38.20 ± 1.46b 42.70 ± 3.06b 18:2(n-6) 22.76 ± 4.07b 8.00 ± 0.76c 15.37 ± 1.03a 18:3(n-3) 0.62 ± 0.20a 1.25 ± 0.34a 3.64 ± 0.62b a b 18:4(n-3) 1.04 ± 0.23 2.86 ± 0.90 – 0.46 ± 0.22a 0.35 ± 0.07a 20:00 1.21 ± 0.36b 20:1(n-9) 0.80 ± 0.29b – 0.22 ± 0.09a 20:3(n-3) 1.46 ± 0.52b 0.44 ± 0.13a – 0.53 ± 0.19b – 20:4(n-3) 0.27 ± 0.01a 0.38 ± 0.17b – 20:5(n-3) 0.11 ± 0.05a 0.34 ± 0.07a – 22:00 1.88 ± 0.74b 0.16 ± 0.11b – 22:3(n-3) 0.18 ± 0.12b 0.17 ± 0.06b – 22:4(n-6) 0.39 ± 0.19b 1.38 ± 0.33b – 22:6(n-3) 0.84 ± 0.47b 24:00 0.84 ± 0.16 – – 44.28 ± 1.79b 29.30 ± 2.00a Saturated 29.60 ± 2.36a Monoenoic 42.73 ± 3.12a 39.55 ± 1.76a 51.69 ± 3.96b Polyenoic 27.67 ± 5.95b 15.17 ± 2.99a 19.01 ± 1.65a HUFAn3 2.86 ± 1.26b 2.89 ± 0.93b – All the values represent mean ± S.D. and values that show the same superscript letter in the same line are not significantly different (P >0.05). Fatty acid
ism, or requiring different environmental parameters can be used for mixed cultures (24, 25). In this study, the two species used can be defined as exhibiting mutualism since their growths were promoted in the mixed culture compared with in the monocultures. According to Dong and Zhao (13), the promotion effect on growth in mixed cultures can be attributed to sufficient in situ oxygen and carbon dioxide transitions, since the alga acted as an oxygen generator for the yeast, while the yeast produces carbon dioxide for the alga. As a result, the stresses caused by carbon dioxide on the yeast and oxygen on the alga were alleviated or eliminated. Thus, the growth conditions were optimized for both species. Additionally, this sufficient in situ transition may maintain an O2/CO2 balance that enhances the photosynthesis of the alga. In the mixed culture system, the exception of low specific growth rate of the alga on day 2 may have been due to the initial rapid growth of the yeast A. cicatricosa, which might have resulted in an excessive accumulation of the metabolite carbon dioxide (13), sequentially suppressing the growth of the alga I. galbana, since the growth of the alga is known to be inhibited by a high concentration of carbon dioxide (26). Although the initial growth of the alga was slightly inhibited by the yeast in the mixed culture, the fast recovery on day 3 confirmed that the alga can rapidly overcome the inhibitory effect, and that the two species (alga and yeast) can adapt well to the coexisting circumstance and build up the beneficial balance of mutualism. Biomass The biomass concentration, directly reflect-
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ing the accumulation rate of biomass, can act as a criterion for evaluating the productivity of a culture method. In this study, two alga-containing cultures displayed the analogous increasing trends in biomass concentration, on the contrary, a slow decreasing trend was found in the A. cicatricosa monoculture. The higher biomass concentration obtained in the mixed culture suggests that the mixed culture had a higher productivity than the two monocultures. However, the contributions to the higher productivity in the mixed culture were obviously different between two species, since the alga I. galbana dominated the mixed culture in terms of number of cells (Fig. 3). Therefore, it is reasonable to postulate that the alga I. galbana benefited more from the mutualism under the present culture conditions. Thus, the improvement in the biomass in the mixed culture mainly resulted from the promoted growth of the alga I. galbana. Biochemical composition The biochemical composition of mixed culture closely approximates that of the I. galbana monoculture (Tables 1–3). In the mixed culture, since the biomass was mainly composed of alga cells (Fig. 3), it was deduced that the biochemical characteristics of the dry biomass of the mixed culture was essentially the same as those of the alga I. galbana. This may account for the similarities in the biochemical composition between the mixed culture and the I. galbana monoculture. Regarding the gross composition of microorganisms, it is widely accepted that differences in the species, culture conditions and/or analytical methods make it difficult to compare the results presented by different authors and thus to compare our results with those in literature. However, according to the data available, it was reported that microalgae used in an aquaculture typically contain 30% to 40% protein, 10% to 20% lipid and 5% to 15% carbohydrate (2, 27, 28), and our results (except for carbohydrate) for mixed culture closely matched the ranges reported. Some researchers have suggested that these gross compositions may not always correlate directly with nutritional value (29, 30), for example, two alga, namely, Phaeodactylum tricornutum and Nannochloris atomus, which are rich in protein and carbohydrate, respectively, were considered to have low nutritional value (30). However, Brown et al. (27) implied that when other specific essential nutrients (e.g., polyunsaturated fatty acids and vitamins) are in adequate proportions, the differences may become important. With respect to the carbohydrate, the value attained in this study is relatively higher than the range reported. However, in application, algal diets with high levels of carbohydrate were reported to produce the best growth for juvenile oysters of Ostrea edulis (31). The nutritional value of protein depends highly on the amino acid composition and is also intimately related to both specific feeding organisms and the developmental phases of feeding animals (32). Interestingly, there is evidence indicating that protein quality depends on the presence of sulphur-containing amino acids, mainly methionine, which is necessary for the synthesis of cysteine, as well as phenylalanine, needed for the synthesis of tyrosine (33, 34). It was also found that no significant changes in nutritional value were observed when a suitable protein source that resembled the amino acid profile of the feeding animal larvae was available (35). In the mixed culture, both the sulphur-con-
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taining amino acids methionine and cysteine were detected, and the profile of essential amino acids was very similar to those of oyster spat and larvae reported by Brown et al. (8), suggesting that a rich source of essential amino acids in the mixed culture of the alga I. galbana and the yeast A. cicatricosa could be provided for bivalve cultures (8). The amino acid composition of the mixed culture also closely matched those of microalgae used widely in aquacultures (2). The yeast has an inferior profile of fatty acids since it lacks HUFAs, which makes it unsuitable as a complete diet for larval cultures (8). In this study, no HUFAs were found in the A. cicatricosa monoculture, while the two alga-containing cultures contained these fatty acids. Additionally, a higher concentration of the fatty acid 14:00 (18.85%) was detected in the mixed culture than in the two monocultures. Thompson et al. (36) found that diets high in fatty acids (14:00 + 16:00) could make obtaining energy more efficient, which is helpful for rapidly growing larvae. As for HUFAs, the two alga-containing cultures all showed small quantities, which may have been due to the low nutrition level in the f/2 medium used. For example, similar results on HUFAs were reported by Fernández-Reiriz et al. (37) and Sánchez et al. (38) who cultivated the alga I. galbana under similar culture conditions. In addition, it is widely reported that the fatty acid composition of a given species of alga can vary with the growing conditions (29, 39) and also change according to the growth phases of the alga (37). Therefore, an optimization of the culture conditions for the mixed culture of alga and yeast is expected to result in an improvement in composition. Thus, further investigation of this mixed culture technique is desirable. In summary, this mixed culture strategy led to significant improvements in growth and biomass concentration, which means that more biomass will be achieved in a given time when compared with the biomass achieved in monocultures. Meanwhile, the biochemical profiles of the dry biomass of the mixed culture closely resembled those of alga, implicating that the biomass produced in the mixed culture has an acceptable nutritional value. Although the contents of some secondary ingredients in mixed culture were relatively lower than those of the alga monoculture, they are compensated in terms of the higher biomass concentration. In addition, there is evidence that mixed diets have a better balance of nutrients and are applied in aquacultures regularly (40–43). Furthermore, enhanced weight gain, feed efficiency and disease resistance have been observed in marine organisms when following the dietary administration of yeast food supplement (44, 45). Thus, this study could represent a basic step in the development of a new technology for replacing traditional monocultures. ACKNOWLEDGMENTS The authors are grateful to Professor ShiChun Sun of the Ocean University of China for providing the alga used in this study.
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20. Pande, S. V., Parvin, R., and Venkitasubramanian, T. A.: Microdetermination of lipids and serum total fatty acids. Anal. Biochem., 6, 415–425 (1963). 21. Kochert, G.: Carbohydrate determination by phenol-sulfuric acid method, p. 95–97. In Hellebust, J. A. and Craige, J. S. (ed.), Handbook of physiological and biochemical methods. Cambridge University Press, London (1978). 22. Sujak, A., Kotlarz, A., and Strobel, W.: Compositional and nutritional evaluation of several lupin seeds. Food Chem., 98, 711–719 (2006). 23. Swaaf, M. E. D., Rijk, T. C. D., Eggink, G., and Sijtsma, L.: Optimisation of docosahexaenoic acid production in batch cultivations by Crypthecodinium cohnii. J. Biotechnol., 70, 185–192 (1999). 24. Hahn-Hagerdal, B.: Co-immobilization involving cells, organelles, and enzymes, p. 79–94. In Mattiasson, B. (ed.), Immobilised cells and organelles II. CRC Press, Boca Raton, FL (1983). 25. Kurosawa, H. and Tanaka, H.: Advances in immobilized cell culture: Development of co-immobilized mixed culture system of aerobic and anaerobic micro-organisms. Process Biochem., 25, 189–196 (1990). 26. Lee, Y. K. and Tay, H. S.: High CO2 partial pressure depresses productivity and bioenergetic growth yield of Chorella pyrenoidosa culture. J. Appl. Phycol., 3, 95–101 (1991). 27. Brown, M. R., Jeffrey, S. W., Volkman, J. K., and Dunstan, G. A.: Nutritional properties of microalgae for mariculture. Aquaculture, 151, 315–331 (1997). 28. Renaud, S. M., Thinh, L. V., and Parry, D. L.: The gross composition and fatty acid composition of 18 species of tropical Australian microalgae for possible use in mariculture. Aquaculture, 170, 147–159 (1999). 29. Webb, K. L. and Chu, F. E.: Phytoplankton as a food source for bivalve larvae, p. 272–291. In Pruder, G. D., Langdon, C. J., and Conklin, D. E. (ed.), Proceedings of the Second International Conference on Aquaculture Nutrition: Biochemical and Physiological Approaches to Shellfish Nutrition. Louisiana State University, Baton Rouge, LA (1982). 30. Brown, M. R., Jeffrey, S. W., and Garland, C. D.: Nutritional aspects of microalgae used in mariculture: a literature review, p. 1–44. CSIRO Marine Laboratories, Hobart (1989). 31. Enright, C. T., Newkirk, G. F., Craigie, J. S., and Castell, J. D.: Evaluation of phytoplankton as diets for juvenile Ostrea edulis. J. Exp. Mar. Biol. Ecol., 96, 1–13 (1986). 32. Sheen, S. S. and Huang, H. T.: The effects of different protein sources on the survival of grass shrimp, Penaeus monodon (Fabricius, 1978) larvae from the zoea to postlarva (Decapoda, Natania). Crustaceana, 71, 909–924 (1998). 33. James, K. A. and Hove, E. L.: The ineffectiveness of supplementary cysteine in legume-based rat diets. J. Nutr., 110, 1736–1744 (1980). 34. Molvig, L., Tabe, L. M., Eggum, B. O., Moore, A. E., Craig, S., Spencer, D., and Higgins, T. J. V.: Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (Lupinus angustifolius L.) expressing a sunflower seed albumin gene. Proc. Natl. Acad. Sci., 94, 8393–8398 (1997). 35. Roustaian, P., Kamarudin, M. S., Omar, H. B., Saad, C. R., and Ahmad, M. H.: Amino acid composition of developing larval freshwater prawn Macrobrchium rosenbergii. J. World Aquac. Soc., 31, 130–136 (2000). 36. Thompson, P. A., Guo, M., and Harrison, P. J.: The influence of irradiance on the biochemical compostion of three phytoplankton species and their nutritional value for larvae of the Pacific oyster (Crassostrea gigas). Mar. Biol., 117, 259– 268 (1993). 37. Fernández-Reiriz, M. J., Perez-Camacho, A., Ferreiro, M. J., Blanco, J., Planas, M., Campos, M. J., and Labarta,
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© 2007, The Society for Biotechnology, Japan
Comparisons of Growth and Biochemical Composition between Mixed Culture of Alga and Yeast and Monocultures ShiQing Cai,1 ChaoQun Hu,1* and ShaoBo Du1 Key Laboratory of Applied Marine Biology of Guangdong Province (LAMB), South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, People’s Republic of China1 Received 14 February 2007/Accepted 4 August 2007
The alga Isochrysis galbana 8701 and the yeast Ambrosiozyma cicatricosa were mix-cultivated in the same medium for 7 d to compare their growth performance and biochemical composition with those of the same organisms cultured under monoculture conditions. The specific growth rates of both species were significantly higher (p <0.05) in the mixed culture than in the monocultures during the corresponding experimental phases. At the end of experiment, the biomass concentration obtained in the mixed culture reached 1.32 ± 0.04 g/l of dry weight, which was significantly higher (p < 0.05) than those obtained in monocultures, and the alga I. galbana in the mixed culture dominated the cell numbers making up 96.64% of the cells. The biochemical profile of the mixed culture is similar to that of the I. galbana monoculture with some variations; The percentages of both the fatty acids 14:00 and 18:00 detected in the mixed culture were significantly higher (p < 0.05) than those detected in the I. galbana monoculture, while the content of the fatty acid 18:2(n-6) detected in the mixed culture was significantly lower (p <0.05) than that detected in the I. galbana monoculture. This study indicates the improved growth performance in mixed culture compared with monocultures and the similarities between the biochemical compositions of the mixed culture and the I. galbana monoculture. [Key words: Ambrosiozyma cicatricosa, biomass, Isochrysis galbana, mixed culture]
lopsis larvae, and Saccharomyces acidosaccharophill can be used as rotifer food. However, to date, yeast is still limited in terms of its use as a partial substitute for alga as food for marine organisms because of the deficiency of certain compositional components in yeast products, particularly the lack of highly unsaturated fatty acids (HUFAs), which are essential for the successful growth and development of many marine organisms (10). Many methods and techniques, such as the use of bioreactors, the heterotrophic culture of alga, and the mixed culture of microorganisms, have been developed to reduce the costs of aquatic food production, and/or for improving the nutritional value of aquatic food. Of these techniques, the mixed culture of microorganisms is a convenient solution for achieving the goals. When using a mixed culture, two or more preselected species of microorganism are synchronously cultivated within the same medium, where these microorganisms can mutually exploit complementary metabolic activities to survive, grow, and reproduce (11). Mixed cultures of heterotrophic microorganisms are often used for the treatment of waste materials discharged as a result of agricultural and industrial activities, as well as for the production of biomass and bioactive compounds (11). Although the fact that growth is prompted when algae and yeast are mixcultured in the same medium was confirmed in other fields
Marine microalgae have been widely used as an aquaculture food (1), because they can supply the energy and nutrients essential for the growth and development of marine organisms (2). Although marine microalgae have been appreciated as a food in aquacultures, alga production is subjected to the constraints of high operational costs (3, 4). Therefore, even a small improvement in alga culture techniques could result in substantial savings in the mass production of microalgae (5), and the major challenge facing algologists is the improvements of culture techniques to reduce production costs while maintaining alga production reliability (4). Yeast is an important unicellular organism existing extensively in seawater. Because it can be mass-produced at a relatively low cost and is high in nutritional value, yeast is gradually accepted by people and its employment as food in aquaculture is becoming common (6, 7). Furthermore, the potential numbers of species of yeast that can be applied in aquaculture are increasing, for example, six new strains of yeast were examined and evaluated as food for bivalves by Brown et al. (8). Agh and Sorgeloos (9) reported that the yeasts Saccharomyces cerevisiae, Rhodotorula, Zygosaccharomyces marina, Torulopsis candida var. marina, Toru* Corresponding author. e-mail: [email protected] phone/fax: +86-20-89023218 391
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(12, 13), little information is available on mixed cultures of algae and yeast being used as live food in aquacultures. The objective of this study is to investigate the growth performance and biochemical composition of a mixed culture of the alga I. galbana and the yeast A. cicatricosa and compare them with those under monoculture conditions, particularly to discuss the biochemical quality from the view point of aquaculture live food.
250°C at 10°C min–1, and kept at 250°C for 10 min. After that, samples of 1 µl were injected at a temperature of 275°C. Detection was performed with a flame ionization detector at a temperature of 280°C. The scan range was 29.0–450.0 amu with a scan rate of 1 s–1. The experimental results were compared statistically by oneway analysis of variance (ANOVA) (P ≤0.05) using software packages of SPSS 13.0.
RESULTS MATERIALS AND METHODS Strains and culture conditions The alga I. galbana 8701 used in this study was obtained from the Ocean University of China，and the yeast A. cicatricosa, which was one of strains confirmed to be useful as live food in aquacultures (14), was randomly selected for this study. The cells were in the size range of (length × width) 2–7 µm × 1–4 µm for the yeast A. cicatricosa, and 4–6 µm × 3–4 µm for the alga I. galbana. Two axenic microorganisms were separately cultivated to the exponential phase prior to use. Aged seawater autoclaved at 120°C for 20 min, enriched with f/2 medium (15) and supplemented with 2 g/l glucose, was used for a culture medium. Cultures were carried out in 5-l Erlenmeyer flasks containing 2000 ml of the culture medium. An equal concentration of 2.5 ×104 cells ml–1 was used for the start concentration of the two monocultures, while the inoculum of the mixed culture contained half of the number (1.25 ×104 cells ml–1) of each species. The culture time was 7 d with continuous illumination at an irradiance of 72 µmol m–2 s–1, provided by fluorescent lamps (75 W) in a temperature-controlled chamber (20 ±1°C). All treatments were carried out in triplicate. Analysis of growth rate The daily total number of cells in each treatment was determined with a haemocytometer after fixing with 5% formalin (5). In the case of the mixed culture, the cell density of the yeast was determined by plating differently diluted culture fluid on agar dishes (12). The specific growth rate (K) of each culture was calculated from the expression K = (ln Nt − ln N0)/t where Nt is the cell count at time t, N0 is the initial cell count at time zero, and t is time (d). Analysis of biomass concentration A known volume (3–10 ml) of individual culture fluid was filtered through predried Whatman GF/C filter paper (0.45 µm), then dried to a constant weight to determine daily biomass concentration according to method reported by Ip and Chen (16), and the first sampling was carried out 2 d after inoculation. At the end of the experiment, the cultures were harvested by centrifugation at 3000 rpm for 10 min, and the cell pellets were rinsed twice with distilled water, recentrifuged, and then freeze-dried for biochemical analysis. Determination of biochemical composition Proteins were extracted following the procedure of Rafiqul et al. (17) and quantified using the dye-binding method described by Braford (18). Lipids were extracted using the method of Bligh and Dye (19) and quantified following the method of Pande et al. (20). The carbohydrates were determined using the phenol-sulfuric acid method with glucose as a standard (21). The ash content was calculated after combusting samples at 450°C for 16 h. The amino acids were analyzed using an automatic amino acid analyzer (model 835-50; Hitachi, Tokyo). Prior to analysis, the samples were subjected to hydrolysis according to the method described by Sujak et al. (22). Fatty acid analysis was carried out following the method of Swaaf et al. (23); GC/MS with a column of 30 m × 0.25 mm × 0.25 µm was performed, the column temperature was raised from 150°C to
Growth The dynamics of the specific growth rates of the three cultures are shown in Fig. 1. The maximal specific growth rates of the three cultures (1.03 ±0.020 for mixed culture, 0.9 ± 0.019 for I. galbana monoculture and 0.55 ± 0.020 for A. cicatricosa monoculture) all appeared on day 2, and then decreased to a relatively low level (0.80 ± 0.030 for mixed culture, 0.73 ± 0.020 for I. galbana monoculture and 0.10 ±0.020 for A. cicatricosa monoculture) on day 3. Thereafter, the specific growth rates gradually decreased to a minimal level (0.64 ± 0.030 for mixed culture, 0.63 ± 0.010 for I. galbana monoculture and 0.02 ± 0.015 for A. cicatricosa monoculture) at the end of the experiment. Throughout the cultivation process, the A. cicatricosa monoculture had the lowest value for specific growth rate of the three cultures. The specific growth rates of the mixed culture were significantly higher (p < 0.05) than those of the I. galbana monoculture during the corresponding experimental phases except for that on day 7. On day 7, there was no significant difference in specific growth rate between the mixed culture (0.64 ± 0.030) and the I. galbana monoculture (0.63 ± 0.010). This may have resulted from the depletion of the nutrients in the culture medium used, leading to the decrease in the fast growth of the two species in the mixed culture. Between days 2 and 3, the specific growth rate of the A. cicatricosa monoculture decreased by 82%, which was a much more rapid change than those of two alga-containing cultures (22% for mixed culture and 25% for I. galbana monoculture). Within the system of the mixed culture (Fig. 2), from day
FIG. 1. Dynamics of specific growth rates of each culture. Data are expressed as mean ± standard deviation of three replicates. Filled squares, Mixed culture; empty lozenges, I. galbana monoculture; filled triangles, A. cicatricosa monoculture.
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FIG. 2. Dynamics of specific growth rates of two species in mixed culture. Data are expressed as mean ± standard deviation of three replicates. Lozenges, I. galbana; squares, A. cicatricosa.
FIG. 3. Dynamics of proportions of two species in mixed culture. Data are expressed as mean ± standard deviation of three replicates. Lozenges, I. galbana; squares, A. cicatricosa.
2 to day 7, the specific growth rates of the A. cicatricosa were 1.17 ± 0.026, 0.63 ± 0.020, 0.47 ± 0.032, 0.37 ± 0.050, 0.30 ± 0.029, and 0.26 ± 0.032, respectively, which were significantly higher than those obtained in the A. cicatricosa monoculture during the corresponding experimental phases (p < 0.05). As for the alga I. galbana, from day 3 to the end of the experiment, the specific growth rates were 0.92 ±0.020, 0.88 ± 0.032, 0.84 ± 0.049, 0.81 ± 0.028, and 0.74 ± 0.035, respectively, which were also significantly higher (p < 0.05) than those obtained in the I. galbana monoculture during the corresponding experimental phases. The specific growth rate of I. galbana obtained in the mixed culture on day 2
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FIG. 4. Variations in biomass concentration for each culture. Data are expressed as mean ± standard deviation of three replicates. Empty squares, Mixed culture; filled squares, I. galbana monoculture; filled triangles, A. cicatricosa monoculture.
(0.84 ± 0.025) was on the contrary, lowering (p <0.05) than that (0.98 ± 0.019) of I. galbana in monoculture on day 2. The changes in cell number proportion in the mixed culture are illustrated in Fig. 3. The accumulative trends of the cell numbers of the two species in the mixed culture were opposite. On day 2, the bulk of the cell number (65.88%) in the mixed culture was accounted for by the yeast A. cicatricosa, and then the proportion of yeast cell number decreased rapidly, accompanying the increase in the number of alga cells. At the end of the experiment, the proportion of yeast cells decreased to 3.36%, on the other hand, the proportion of alga cells amount increased to 96.64%. Biomass The variations in the biomass concentrations of the three cultures are shown in Fig. 4. The maximum biomass concentration (0.31 ± 0.050 g/l) of the A. cicatricosa monoculture was found on day 2, and, at the end of experiment, the value fell to 0.17 ± 0.090 g/l. Inversely, the biomass concentration of the I. galbana monoculture increased from 0.10 ± 0.089 g/l (on day 2) to 1.17 ± 0.024 g/l (on day 7). In the case of the mixed culture, a similar increase in biomass concentration was observed but the increase was relatively rapid, increasing from 0.11 ± 0.090 g/l (on day 2) to 1.32 ± 0.041 g/l (on day 7). From day 5 to the end of the experiment, the biomass concentrations (1.08 ± 0.021 g/l, 1.22 ± 0.020 g/l, and 1.32 ± 0.041 g/l, respectively) were significantly higher (p < 0.05) in the mixed culture than in the I. galbana monoculture (0.73 ± 0.040 g/l, 1.09 ± 0.060 g/l, and 1.17 ± 0.024 g/l, respectively) during the corresponding experimental phases. Biochemical composition There were significant differences in gross composition between the A. cicatricosa monoculture and the two alga-containing cultures (Table 1),
TABLE 1. Gross compositions of each culture (% of dry biomass) Culture method Protein Lipid Carbohydrate Ash 11.21 ± 3.01b 38.36 ± 0.94b 2.64 ± 0.37a Mixed culture 41.42 ± 2.06a I. galbana monoculture 38.51 ± 1.36a 15.11 ± 1.71b 41.82 ± 3.12b 2.18 ± 0.22a b a a A. cicatricosa monoculture 51.32 ± 2.10 6.35 ± 0.88 28.48 ± 2.81 6.37 ± 0.44b All the values represent mean ± S.D. and values that show the same superscript letter in the same column are not significantly different (P>0.05).
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TABLE 2. Amino acid compositions (% of total amino acids) of each culture I. galbana monoculture
Mixed culture
A. cicatricosa monoculture
Nonessential 8.50 ± 0.20a 6.58 ± 0.17b Ala 8.90 ± 0.39a Asp 10.74 ± 0.28a 10.78 ± 0.10a 10.19 ± 0.25b Glu 12.69 ± 0.08b 14.12 ± 0.50a 14.58 ± 0.14a Gly 6.43 ± 0.19b,c 6.15 ± 0.08c 6.77 ± 0.43a,b Ser 3.24 ± 0.21b 3.42 ± 0.26b 4.10 ± 0.29a Arg 6.51 ± 0.39b 6.15 ± 0.04b 7.52 ± 0.33a Pro 4.51 ± 0.32b,c 4.14 ± 0.06c 4.76 ± 0.14a,b Essential 0.37 ± 0.08a 0.50 ± 0.09a Cys 0.38 ± 0.07a His 1.92 ± 0.31a 1.95 ± 0.05a 2.10 ± 0.42a a a Ile 4.92 ± 0.32 5.05 ± 0.09 5.31 ± 0.22a Leu 9.15 ± 0.28a 9.06 ± 0.79a 7.76 ± 0.65b Lys 5.16 ± 0.15b 5.11 ± 0.92b 9.06 ± 1.18a Met 2.88 ± 0.74a 2.78 ± 0.44a 1.84 ± 0.93a Phe 5.08 ± 0.34b,c 5.29 ± 0.32a,b 4.53 ± 0.40c Tyr 3.57 ± 0.46a 3.45 ± 0.33a 2.38 ± 0.37b Thr 4.95 ± 0.31a 4.89 ± 0.60a 4.72 ± 0.11a Trp 1.95 ± 0.76a 1.82 ± 0.75a 1.20 ± 0.36a a a Val 7.09 ± 0.64 6.87 ± 0.64 6.04 ± 0.93a All the values represent mean ± S.D. and values that show the same superscript letter in the same line are not significantly different (P>0.05).
while no significant difference was found between the two alga-containing cultures. The two alga-containing cultures had similar compositions for both essential and nonessential amino acids except that the concentration of the amino acid glutamate (14.12 ± 0.50%) was significantly higher in mixed culture than in the I. galbana monoculture (12.69 ± 0.08%), as shown in Table 2. Methionine is an important sulphur-containing amino acid, and the proportions of methionine were 2.88 ± 0.74% for the I. galbana monoculture and 2.78 ± 0.44% for mixed culture. As for the fatty acids (Table 3), the dominant fatty acid present in all three cultures was 18:1(n-9). No HUFAn3 were detected in the A. cicatricosa monoculture, which was significantly different from the two alga-containing cultures, in which HUFAn3 were found in similar profile. Although at a low level, the two highly important fatty acids 20:5(n-3) and 22:6(n-3), were both detected in the two alga-containing cultures. In addition, the mixed culture was unique in its high contents of the fatty acids 14:00 (18.85 ± 0.84%) and 18:00 (9.03 ± 0.49%) compared with the contents in the I. galbana monoculture. However, the I. galbana monoculture contained a higher content of the fatty acid 18:2(n-6) (22.76 ±4.07%) than the mixed culture (8.00 ±0.76%). Taken together, the mixed culture was richer in saturated fatty acids (44.28 ±1.79%) than the two monocultures, whereas the contents of both monoenoic and polyenoic fatty acids were higher in the two monocultures than in the mixed culture. DISCUSSION Growth There are several interactions between species exhibited in mixed microorganism cultures, such as competition, predation, parasitism, and amensalism (11). Only those species exhibiting neutralism, commensalism, mutual-
TABLE 3. Fatty acid compositions (% of total fatty acids) of each culture I. galbana Mixed A. cicatricosa monoculture culture monoculture 12:00 – – 1.95 ± 0.67 18.85 ± 0.84b 1.95 ± 0.24c 14:00 3.40 ± 0.09a 15:00 – 0.43 ± 0.10 – 15.13 ± 0.19c 15.59 ± 0.57a,c 16:00 16.07 ± 0.26a,b 16:1(n-7) 0.98 ± 0.34a 1.05 ± 0.18a 8.77 ± 0.81b 17:00 0.41 ± 0.09b 0.04 ± 0.02a – 17:1(n-9) – 0.30 ± 0.12 – 9.03 ± 0.49b 9.46 ± 0.45b 18:00 5.79 ± 0.66a 18:1(n-9) 40.95 ± 2.49b 38.20 ± 1.46b 42.70 ± 3.06b 18:2(n-6) 22.76 ± 4.07b 8.00 ± 0.76c 15.37 ± 1.03a 18:3(n-3) 0.62 ± 0.20a 1.25 ± 0.34a 3.64 ± 0.62b a b 18:4(n-3) 1.04 ± 0.23 2.86 ± 0.90 – 0.46 ± 0.22a 0.35 ± 0.07a 20:00 1.21 ± 0.36b 20:1(n-9) 0.80 ± 0.29b – 0.22 ± 0.09a 20:3(n-3) 1.46 ± 0.52b 0.44 ± 0.13a – 0.53 ± 0.19b – 20:4(n-3) 0.27 ± 0.01a 0.38 ± 0.17b – 20:5(n-3) 0.11 ± 0.05a 0.34 ± 0.07a – 22:00 1.88 ± 0.74b 0.16 ± 0.11b – 22:3(n-3) 0.18 ± 0.12b 0.17 ± 0.06b – 22:4(n-6) 0.39 ± 0.19b 1.38 ± 0.33b – 22:6(n-3) 0.84 ± 0.47b 24:00 0.84 ± 0.16 – – 44.28 ± 1.79b 29.30 ± 2.00a Saturated 29.60 ± 2.36a Monoenoic 42.73 ± 3.12a 39.55 ± 1.76a 51.69 ± 3.96b Polyenoic 27.67 ± 5.95b 15.17 ± 2.99a 19.01 ± 1.65a HUFAn3 2.86 ± 1.26b 2.89 ± 0.93b – All the values represent mean ± S.D. and values that show the same superscript letter in the same line are not significantly different (P >0.05). Fatty acid
ism, or requiring different environmental parameters can be used for mixed cultures (24, 25). In this study, the two species used can be defined as exhibiting mutualism since their growths were promoted in the mixed culture compared with in the monocultures. According to Dong and Zhao (13), the promotion effect on growth in mixed cultures can be attributed to sufficient in situ oxygen and carbon dioxide transitions, since the alga acted as an oxygen generator for the yeast, while the yeast produces carbon dioxide for the alga. As a result, the stresses caused by carbon dioxide on the yeast and oxygen on the alga were alleviated or eliminated. Thus, the growth conditions were optimized for both species. Additionally, this sufficient in situ transition may maintain an O2/CO2 balance that enhances the photosynthesis of the alga. In the mixed culture system, the exception of low specific growth rate of the alga on day 2 may have been due to the initial rapid growth of the yeast A. cicatricosa, which might have resulted in an excessive accumulation of the metabolite carbon dioxide (13), sequentially suppressing the growth of the alga I. galbana, since the growth of the alga is known to be inhibited by a high concentration of carbon dioxide (26). Although the initial growth of the alga was slightly inhibited by the yeast in the mixed culture, the fast recovery on day 3 confirmed that the alga can rapidly overcome the inhibitory effect, and that the two species (alga and yeast) can adapt well to the coexisting circumstance and build up the beneficial balance of mutualism. Biomass The biomass concentration, directly reflect-
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ing the accumulation rate of biomass, can act as a criterion for evaluating the productivity of a culture method. In this study, two alga-containing cultures displayed the analogous increasing trends in biomass concentration, on the contrary, a slow decreasing trend was found in the A. cicatricosa monoculture. The higher biomass concentration obtained in the mixed culture suggests that the mixed culture had a higher productivity than the two monocultures. However, the contributions to the higher productivity in the mixed culture were obviously different between two species, since the alga I. galbana dominated the mixed culture in terms of number of cells (Fig. 3). Therefore, it is reasonable to postulate that the alga I. galbana benefited more from the mutualism under the present culture conditions. Thus, the improvement in the biomass in the mixed culture mainly resulted from the promoted growth of the alga I. galbana. Biochemical composition The biochemical composition of mixed culture closely approximates that of the I. galbana monoculture (Tables 1–3). In the mixed culture, since the biomass was mainly composed of alga cells (Fig. 3), it was deduced that the biochemical characteristics of the dry biomass of the mixed culture was essentially the same as those of the alga I. galbana. This may account for the similarities in the biochemical composition between the mixed culture and the I. galbana monoculture. Regarding the gross composition of microorganisms, it is widely accepted that differences in the species, culture conditions and/or analytical methods make it difficult to compare the results presented by different authors and thus to compare our results with those in literature. However, according to the data available, it was reported that microalgae used in an aquaculture typically contain 30% to 40% protein, 10% to 20% lipid and 5% to 15% carbohydrate (2, 27, 28), and our results (except for carbohydrate) for mixed culture closely matched the ranges reported. Some researchers have suggested that these gross compositions may not always correlate directly with nutritional value (29, 30), for example, two alga, namely, Phaeodactylum tricornutum and Nannochloris atomus, which are rich in protein and carbohydrate, respectively, were considered to have low nutritional value (30). However, Brown et al. (27) implied that when other specific essential nutrients (e.g., polyunsaturated fatty acids and vitamins) are in adequate proportions, the differences may become important. With respect to the carbohydrate, the value attained in this study is relatively higher than the range reported. However, in application, algal diets with high levels of carbohydrate were reported to produce the best growth for juvenile oysters of Ostrea edulis (31). The nutritional value of protein depends highly on the amino acid composition and is also intimately related to both specific feeding organisms and the developmental phases of feeding animals (32). Interestingly, there is evidence indicating that protein quality depends on the presence of sulphur-containing amino acids, mainly methionine, which is necessary for the synthesis of cysteine, as well as phenylalanine, needed for the synthesis of tyrosine (33, 34). It was also found that no significant changes in nutritional value were observed when a suitable protein source that resembled the amino acid profile of the feeding animal larvae was available (35). In the mixed culture, both the sulphur-con-
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taining amino acids methionine and cysteine were detected, and the profile of essential amino acids was very similar to those of oyster spat and larvae reported by Brown et al. (8), suggesting that a rich source of essential amino acids in the mixed culture of the alga I. galbana and the yeast A. cicatricosa could be provided for bivalve cultures (8). The amino acid composition of the mixed culture also closely matched those of microalgae used widely in aquacultures (2). The yeast has an inferior profile of fatty acids since it lacks HUFAs, which makes it unsuitable as a complete diet for larval cultures (8). In this study, no HUFAs were found in the A. cicatricosa monoculture, while the two alga-containing cultures contained these fatty acids. Additionally, a higher concentration of the fatty acid 14:00 (18.85%) was detected in the mixed culture than in the two monocultures. Thompson et al. (36) found that diets high in fatty acids (14:00 + 16:00) could make obtaining energy more efficient, which is helpful for rapidly growing larvae. As for HUFAs, the two alga-containing cultures all showed small quantities, which may have been due to the low nutrition level in the f/2 medium used. For example, similar results on HUFAs were reported by Fernández-Reiriz et al. (37) and Sánchez et al. (38) who cultivated the alga I. galbana under similar culture conditions. In addition, it is widely reported that the fatty acid composition of a given species of alga can vary with the growing conditions (29, 39) and also change according to the growth phases of the alga (37). Therefore, an optimization of the culture conditions for the mixed culture of alga and yeast is expected to result in an improvement in composition. Thus, further investigation of this mixed culture technique is desirable. In summary, this mixed culture strategy led to significant improvements in growth and biomass concentration, which means that more biomass will be achieved in a given time when compared with the biomass achieved in monocultures. Meanwhile, the biochemical profiles of the dry biomass of the mixed culture closely resembled those of alga, implicating that the biomass produced in the mixed culture has an acceptable nutritional value. Although the contents of some secondary ingredients in mixed culture were relatively lower than those of the alga monoculture, they are compensated in terms of the higher biomass concentration. In addition, there is evidence that mixed diets have a better balance of nutrients and are applied in aquacultures regularly (40–43). Furthermore, enhanced weight gain, feed efficiency and disease resistance have been observed in marine organisms when following the dietary administration of yeast food supplement (44, 45). Thus, this study could represent a basic step in the development of a new technology for replacing traditional monocultures. ACKNOWLEDGMENTS The authors are grateful to Professor ShiChun Sun of the Ocean University of China for providing the alga used in this study.
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