00 200 180 (100) 150 120 邑 B 100 14BIeM 80 40 8101214161820 Time (days) Figure 1.Cultivation under control conditions in regular production medium(M-9 medium with 5 wt sucrose and without phytohormones):(biomass,(O)nitrate,(sucrose,and ( shikonin.Data represent means of duplicate experiments. period of 8 days following the addition of fresh C- study this issue,the reverse sequence of media additions rich medium.A maximum concentration of ~220 mg/L was tried(Figs.2C,D).Following cultivation under control was measured.Correspondingly,the concentration of conditions for about 13 days,N-rich medium was added to phenolics increased almost 3.5-fold over a 6-day period the culture on day 13(Fig.2C).While cell growth was following the addition of C-rich medium.A maximum of strongly stimulated (~43%increase),the yield of shikonin -550 mg/L was measured.Following dilution of the derivatives remained the same (~140 mg/L)during the culture with fresh C-rich medium,the cell concentration 8-9-day period following the addition of N-rich medium. dropped from 140 g/L to ~100/L,and subsequently a Soluble phenolics were not measured in this experiment. modest increase in cell fresh weight(~15%)over a period On day 23,C-rich medium was added to the culture.After of 8 days could be observed.The N-rich medium was added a lag of 24-48 h,fresh accumulation of shikonin was to the culture on day 24(Fig.2A)to further test the validity noticed.This increase continued for a period of 10 days.A of earlier observations regarding the inhibitory effect of maximum concentration of 200-220 mg/L was measured, high nitrate levels.Almost all the added nitrate and ~83% reflecting 35%increase over levels on day 23. of the added sucrose was consumed over a period of 9 days Given the evidence of the stimulatory effect of C-rich following the addition of N-rich medium(Fig.2B).There medium on shikonin productivity,a natural question arises. was a noticeable increase in cell fresh weight (~25%) Will successive additions of C-rich medium result in further over the fresh weight on day 24.Conspicuously,there product accumulation or is there a limit to the stimulatory was almost no further increase in the concentration of effect?Might there be some limitation imposed by the cell's soluble phenolics.A meager 3-4%increase was recorded. biosynthetic apparatus at some stage? Likewise,the shikonin concentration showed only a very To address these questions,C-rich medium was added to slight increase (~9%)over the level on day 24.These cultures after 12 days of cultivation under control condi- results further confirmed previous observations regarding tions(Fig.2E).The anticipated increase in shikonin yield the influence of excess nitrogen on shikonin production. was observed.Shikonin levels increased ~2.2-fold from They also reveal that shikonin production and general 150 to 325 mg/L over 9-10 days following addition of secondary metabolic activity are strongly stimulated by the C-rich medium.Cell growth increased by approximately addition of C-rich medium.Also,N-rich medium supported 25%in this experiment.However,further addition of growth of the culture while preventing further product C-rich medium on day 24 resulted in very little con- accumulation. sumption of the added sucrose (Fig.2F)and showed only Due to continuously changing conditions during batch 12%increase in shikonin yield.A 20%increase in cell culture,there is continuous variation in cellular physiology. fresh weight was observed.A medium preparation without It may be argued,therefore,that the diverse effects of C-rich sucrose (C-rich salts)was also added on day 24 to some and N-rich media on cell growth and shikonin production, flasks in a parallel experiment (data not shown).While when administered in alternating succession,are really cell concentration increased by about 11%,product levels effects caused by altered cellular physiology during this remained unchanged from levels on day 24.These results period rather than direct influences of the added media.To suggest the existence of some intrinsic limitation to the SRINIVASAN AND RYU:ALTERNATING CARBON AND NITROGEN FEEDING 79580 150 100 50 0 0 2 4 6 8 10 12 14 16 18 20 Time (days) Figure 1. Cultivation under control conditions in regular production medium (M-9 medium with 5 wt % sucrose and without phytohormones): (m) biomass, (0) nitrate, (0) sucrose, and (0) shikonin. Data represent means of duplicate experiments. period of 8 days following the addition of fresh C￾rich medium. A maximum concentration of -220 mg/L was measured. Correspondingly, the concentration of phenolics increased almost 3.5-fold over a 6-day period following the addition of C-rich medium. A maximum of -550 mg/L was measured. Following dilution of the culture with fresh C-rich medium, the cell concentration dropped from 140 g/L to -1OO/L, and subsequently a modest increase in cell fresh weight (-15%) over a period of 8 days could be observed. The N-rich medium was added to the culture on day 24 (Fig. 2A) to further test the validity of earlier observations regarding the inhibitory effect of high nitrate levels. Almost all the added nitrate and -83% of the added sucrose was consumed over a period of 9 days following the addition of N-rich medium (Fig. 2B). There was a noticeable increase in cell fresh weight (-25%) over the fresh weight on day 24. Conspicuously, there was almost no further increase in the concentration of soluble phenolics. A meager 3-4% increase was recorded. Likewise, the shikonin concentration showed only a very slight increase (-9%) over the level on day 24. These results further confirmed previous observations regarding the influence of excess nitrogen on shikonin production. They also reveal that shikonin production and general secondary metabolic activity are strongly stimulated by the addition of C-rich medium. Also, N-rich medium supported growth of the culture while preventing further product accumulation. Due to continuously changing conditions during batch culture, there is continuous variation in cellular physiology. It may be argued, therefore, that the diverse effects of C-rich and N-rich media on cell growth and shikonin production, when administered in alternating succession, are really effects caused by altered cellular physiology during this period rather than direct influences of the added media. To study this issue, the reverse sequence of media additions was tried (Figs. 2C, D). Following cultivation under control conditions for about 13 days, N-rich medium was added to the culture on day 13 (Fig. 2C). While cell growth was strongly stimulated (-43% increase), the yield of shikonin derivatives remained the same (-140 mg/L) during the 8-9-day period following the addition of N-rich medium. Soluble phenolics were not measured in this experiment. On day 23, C-rich medium was added to the culture. After a lag of 24-48 h, fresh accumulation of shikonin was noticed. This increase continued for a period of 10 days. A maximum concentration of 200-220 mg/L was measured, reflecting 35% increase over levels on day 23. Given the evidence of the stimulatory effect of C-rich medium on shikonin productivity, a natural question arises. Will successive additions of C-rich medium result in further product accumulation or is there a limit to the stimulatory effect? Might there be some limitation imposed by the cell's biosynthetic apparatus at some stage? To address these questions, C-rich medium was added to cultures after 12 days of cultivation under control condi￾tions (Fig. 2E). The anticipated increase in shikonin yield was observed. Shikonin levels increased -2.2-fold from 150 to 325 mg/L over 9-10 days following addition of C-rich medium. Cell growth increased by approximately 25% in this experiment. However, further addition of C-rich medium on day 24 resulted in very little con￾sumption of the added sucrose (Fig. 2F) and showed only 12% increase in shikonin yield. A 20% increase in cell fresh weight was observed. A medium preparation without sucrose (C-rich salts) was also added on day 24 to some flasks in a parallel experiment (data not shown). While cell concentration increased by about 11%, product levels remained unchanged from levels on day 24. These results suggest the existence of some intrinsic limitation to the SRlNlVASAN AND RYU: ALTERNATING CARBON AND NITROGEN FEEDING 795