J. Phycol. 34, 712–721 (1998) EFFECTS OF ENVIRONMENTAL CONDITIONS ON GROWTH AND LIPID ACCUMULATION IN NITZSCHIA COMMUNIS (BACILLARIOPHYCEAE)1 Thomas A. Dempster 2 and Milton R. Sommerfeld Department of Plant Biology, Arizona State University, Box 871601, Tempe, Arizona 85287-1601 ABSTRACT pogenic microalgae as the raw material for renewa- Microalgae from natural saline habitats in the south- ble alternative liquid fuel sources.western United States were isolated and screened for rapid Accumulation of large intracellular neutral lipidgrowth potential and ability to store intracellular neutral reserves in microalgae has been noted by several in-lipid, a precursor for renewable liquid fuel sources. After vestigators (Fogg and Collyer 1953, Milner 1953,preliminary screening of isolated strains, Nitzschia com- Opute 1974b). Triglyceride accumulation rangingmunis (Rabenhorst) was selected for optimization of from 40% to 80% of cell dry weight was reported ingrowth rate and lipid yield. Nitzschia communis was the diatom Nitzschia palea (Opute 1974a). Hydrocar-subjected to two media types (SERI I, II) with different bons in marine phytoplankton (Blumer et al. 1971)major ion compositions designed to mimic natural saline and fatty acid composition of various microalgaegroundwater aquifers in the arid Southwest. Growth rates have also been studied (Milner 1948, Otsuka andwere determined through 4 days of logarithmic growth, fol- Morimura 1966, Fisher and Schwarzenbach 1978,lowed by 2 days of physiological stress (nitrogen depriva- Ben-Amotz et al. 1985). These microalgal derivativestion) and subsequent measurement of neutral lipid accu- are suitable precursors for the production of variousmulation. Poststress intracellular neutral lipid increases fuels. Triglycerides, hydrocarbons, and fatty acidswere documented by utilizing the ﬂuorochrome Nile Red may be transformed into gasoline and diesel fuelwith ﬂuorometric analysis and epiﬂuorescent microscopy. substitutes via catalytic conversion and transesteriﬁ-Growth rate was slightly higher in SERI Type I medium, cation (McIntosh 1985b).whereas lipid yield was higher in SERI Type II medium. The southwestern United States has been consid-Rapid growth and appreciable lipid yields were observed ered the most appropriate region to concentrate mi-over a broad range of temperatures (20Њ–30Њ C) and spe- croalgal outdoor mass-culture efforts (Johnsonciﬁc conductances (10–70 mS·cmϪ1). The highest lipid 1987). This region offers favorable temperatures,yields were observed at elevated conductances (between 40 high levels of year-round incident solar radiation,mS·cmϪ1 and 70 mS·cmϪ1). Substitution of individual salt large expanses of unpopulated ﬂat land, and a largeconcentrations from SERI Type II into SERI Type I me- supply of water from natural saline groundwaterdium indicated that increased lipid yield in Type I medium aquifers that is unsuitable for human and livestockwas associated with a reduction in MgCl2 concentration. consumption, as well as other industrial and agri- cultural uses.Key index words: alternative liquid fuel; microalgae; To be exploited, microalgal candidates must pos-neutral lipid; Nitzschia communis; renewable liquid sess certain desirable characteristics. They must ex-fuel; storage lipid; triolein hibit rapid growth rates and appreciable lipid yields. These organisms must also be tolerant of the ex- The use of microalgae for a potential renewable treme environmental conditions found in the aridliquid fuel source was ﬁrst proposed by Meier Southwest and possess a life cycle that permits con-(1955). The concept arose from earlier work de- tinuous culturing (Neenan et al. 1986).signed to examine microalgal mass culturing in the The objective of this study was to evaluate the ef-production of high protein foods and waste water fects of media type, temperature, speciﬁc conduc-treatment (Burlew 1953). tance, and nutrient deﬁciency-induced stress on The energy crisis and the Arab oil embargo dur- growth and neutral lipid production in the microal-ing the early 1970s stimulated many countries to in- ga Nitzschia communis.vestigate renewable alternative energy sources (Mc- MATERIALS AND METHODSIntosh 1985a). Oil shortages and reports that manyconventional hydrocarbon fuel sources (i.e. fossil Culture conditions. Nitzschia communis (Strain 3013, ASU Culture Collection) was collected in 1985 from the Pecos Basin (Lat.fuel reserves) might be depleted early in the 21st 33Њ20Ј12Љ, Long. 104Њ19Ј54Љ) in Bottomless Lakes State Park, Cha-century prompted the Department of Energy vez County, New Mexico (Tyler 1989).(DOE) and the Solar Energy Research Institute Two basic media (SERI Types I and II) were used that mim-(SERI) to initiate the Aquatic Species Program icked the natural desert groundwater conditions in the south- western United States (Barclay et al. 1988). The composition for(ASP) in 1979 (Neenan et al. 1986). The ASP was a range of conductances (10–70 mS·cmϪ1) of SERI media isdesigned to determine the feasibility of utilizing li- shown in Table 1. All media were enriched with trace metals, vitamins, iron-EDTA, urea, sodium meta-silicate, and monobasic potassium phosphate (Tyler 1989). 1 Received 12 August 1996. Accepted 30 March 1998. Stock cultures were maintained in both culture media (55 2 Author for reprint requests; e-mail firstname.lastname@example.org. mS·cmϪ1) at 25Њ C. Flasks were kept in a Percival incubator (Mod- 712
LIPID ACCUMULATION IN N. COMMUNIS 713TABLE 1. SERI Types I and II media composition for conductances ranging from 10 mS·cmϪ1 to 70 mS·cmϪ1. Salts Type I/II Type I/II Type I/II Type I/II Type I/II (g·LϪ1) (10) (25) (40) (55) (70)CaCl2·2H2O 0.00/0.037 3.93/0.037 5.62/0.037 7.61/0.037 8.43/0.037MgCl2·6H2O 4.11/1.95 11.84/3.03 22.79/3.92 35.31/4.36 42.23/4.23Na2SO4 0.00/2.67 2.93/5.87 3.31/15.72 3.71/23.31 3.62/28.36KCl 0.19/0.47 0.41/0.97 0.66/2.03 0.96/3.04 1.19/3.67NaHCO3 0.18/1.21 0.17/2.32 0.17/2.86 0.17/3.23 0.17/3.25NaCl 2.12/0.23 3.85/0.88 9.13/1.23 13.02/1.49 16.04/1.53CaSO4 1.69/1.51 0.00/8.08 0.00/12.96 0.00/20.59 0.00/26.08el I-35LLVL; Percival, Boone, Iowa) in a 12L:12D photoperiod 6 nm, bandwidth 40 Ϯ 8 nm). Standard lipid curves were pre-with cool white ﬂuorescent tubes at a photon ﬂux density of ϳ200 pared using dilutions of Triolein Standard (Sigma Diagnostics, St.mol·mϪ2·sϪ1. Louis, Missouri) and the Nile Red staining technique (Greenspan Experimental procedure. Initial optical densities (absorbances) of et al. 1985, Cooksey et al. 1987).ca. 0.002 were attained by transferring aliquots of logarithmically Variables and SERI media alterations. The effects of media typegrowing cultures into sterile 50-mL ﬂasks at the onset of each (SERI I and II), temperature, and speciﬁc conductance onexperiment. A 3-mL aliquot from each experimental culture was growth and lipid yield were collectively screened on a thermogra-immediately subjected to analysis in a Beckman Model DU-64 dient plate with a matrical design that allowed simultaneous ob-spectrophotometer (Beckman Instruments, Inc., Fullerton, Cali- servation of up to 36 treatments and the generation of contourfornia) at 750 nm to ensure uniform cell densities. plots using Sigma Plot (version 3.0). A range of temperatures Typical experiments had a duration of 6 days. Growth rates from 10Њ to 35Њ C in 5Њ C increments and a range of speciﬁcwere monitored during the ﬁrst 4 days (day 0–day 4). Prestress conductances from 10 mS·cmϪ1 to 70 mS·cmϪ1 in 15 mS·cmϪ1intracellular neutral lipid content was measured at the end of day increments were examined.4, and the cultures were pelleted in 15-mL centrifuge tubes using Substitutions of individual salt concentrations from Type II intoan MSE Super-Minor centrifuge (VWR Scientiﬁc, San Francisco, Type I medium were performed to determine whether these sub-California) (1500 ϫ g). Supernatant was poured off, and the cells stitutions contributed to an increase in growth and lipid produc-were resuspended in nutrient-depleted media. The process was tion (Table 2). For each substitution, temperature was main-repeated to minimize carryover of existing nutrient-replete me- tained at 25Њ C and pH was found to only vary from 7.2 to 7.5,dia. During the last 2 days of each experiment, cultures were with the exception of sodium carbonate substitution, which in-subjected to nitrogen deprivation. Poststress lipid accumulation creased the medium pH to 8.17. The diatom was also subjectedwas measured again on day 6. to a gradation of MgCl2 concentrations, from 4.36 g·LϪ1 to 35.31 Growth characterization. Optical densities were obtained at 24-h g·LϪ1 (control) in Type I medium.intervals from day 0 (time of inoculation) to day 4 for cultures in Statistical analyses. One-way ANOVA was used to determinelogarithmic growth and on day 6 (for cultures grown under whether signiﬁcant differences existed between growth rates andstressed conditions) to determine whether a relationship existed lipid yields from exposure to different environmental parameters.between growth and lipid accumulation. Growth rates were cal- Tukey multiple comparison tests were used to determine whichculated from optical densities and expressed in terms of dou- treatments were signiﬁcantly different when ANOVA revealed sig-blings per day (Sorokin 1973). niﬁcant differences. Lipid yield characterization. Epiﬂuorescent microscopy and theﬂuorochrome Nile Red were used to provide photomicrographic RESULTSevidence of intracellular neutral lipids (Cooksey et al. 1987). Prestress lipid yields were determined on day 4 after the loga- Growth. The highest cell density observed had anrithmic growth period. Poststress lipid accumulation was mea- optical density of 0.076, which corresponded to ca.sured on day 6 following 2 days of nitrogen deprivation. For bothpre- and poststress determination of lipid yield, a 4-mL aliquot 5 ϫ 105 cells·mLϪ1. A linear relationship existed be-from each culture was subjected to ﬂuorometric analysis. The tween cell numbers and culture optical density.cells were stained with 50 L of Nile Red (10 mg Nile Red·100 SERI media type. Growth of N. communis, on aver-mLϪ1 acetone). Analysis was completed 15 min after staining us- age, was slightly higher in Type I medium than ining a Turner Model 110 ﬂuorometer (Turner, Mountain View, Type II (Figs. 1, 2). Higher growth rates were alsoCalifornia) equipped with a blue lamp and high sensitivity door,a Ditric Optics, Inc. (Hudson, Massachusetts), three-cavity narrow observed at lower temperatures (10Њ–20Њ C) in SERIband excitation ﬁlter (center 480 Ϯ 2 nm, bandwidth 7.1 Ϯ 1.5 Type I medium (Fig. 1), whereas slightly betternm), and a wide band interference emission ﬁlter (center 550 Ϯ growth occurred at higher temperatures (25Њ–35Њ C)TABLE 2. Composition of SERI Type I medium at 55 mS·cmϪ1 speciﬁc conductance and altered Type I media with substitutions of major ion concen-trations from SERI Type II medium. Major ions (g·LϪ1) Medium CaCl2 MgCl2 Na2SO4 KCl NaHCO3 NaCl Na2CO3 1 Control (I/55) 7.61 35.31 3.71 0.96 0.17 13.02 0.00 2 ϪCaCl2 0.037 35.31 3.71 0.96 0.17 13.02 0.00 3 ϪMgCl2 7.61 4.36 3.71 0.96 0.17 13.02 0.00 4 ϩNa2SO4 7.61 35.31 23.31 0.96 0.17 13.02 0.00 5 ϩKCl 7.61 35.31 3.71 3.04 0.17 13.02 0.00 6 ϩNaHCO3 7.61 35.31 3.71 0.96 3.23 13.02 0.00 7 ϩNaCl 7.61 35.31 3.71 0.96 0.17 20.59 0.00 8 ϩNa2CO3 7.61 35.31 3.71 0.96 0.17 13.02 1.49
714 THOMAS A. DEMPSTER AND MILTON R. SOMMERFELD FIG. 1. Growth rates (doublings·day Ϫ1) ob-served on the thermogradient plate for N. commu-nis cultured in SERI Type I medium over a rangeof temperatures (10Њ–35Њ C) and speciﬁc conduc-tances (10–70 mS·cmϪ1). FIG. 2. Growth rates (doublings·dayϪ1) ob-served on the thermogradient plate for N. com-munis cultured in SERI Type II medium over arange of temperatures (10Њ–35Њ C) and speciﬁcconductances (10–70 mS·cmϪ1).
LIPID ACCUMULATION IN N. COMMUNIS 715 FIG. 3. Epiﬂuorescent micrographs of N. communis taken (a) prior to nitrogen deprivation (prestress), and (b) after 2 days of nitrogendeprivation (poststress). Scale bars ϭ 10 m. Note the multiple small neutral lipid bodies comprising only a small portion of the totalcell volume in (a) and the two or three large lipid deposits comprising more than two-thirds of the total cell volume in (b).in Type II medium (Fig. 2). However, four of the higher in Type II (113 mg·LϪ1) than in Type I me-ﬁve highest growth rates were observed in Type II dium (37 mg·LϪ1)(Figs. 4, 5).medium (Fig. 2). Temperature. Optimal temperature for prestress Temperature. The highest growth rates on the ther- yield was between 25Њ and 30Њ C in both Type I andmogradient plate for Type I and II media both oc- Type II media. The highest poststress lipid yieldscurred at 30Њ C (Figs. 1, 2). In addition, N. communis occurred at 25Њ C, although substantial yields wereexhibited rapid growth over a broad range of tem- also observed at 20Њ and 30Њ C (Figs. 4, 5).peratures (20Њ–30Њ C) in both media types. The tem- Speciﬁc conductance. Optimal prestress yield oc-perature extremes (10Њ and 35Њ C) resulted in re- curred at a speciﬁc conductance of 25 mS·cmϪ1 induced growth in Type I medium (Fig. 1), whereas Type I and 40 mS·cmϪ1 in Type II medium. Post-almost no growth was observed at 10Њ or 15Њ C in stress lipid yield was greatest at ca. 55 mS·cmϪ1 inType II medium (Fig. 2). Type I and between 40 and 55 mS·cmϪ1 speciﬁc con- Speciﬁc conductance. The highest growth rates on ductance in Type II medium (Figs. 4, 5).the thermogradient plate were 2.14 doublings per Substitution of salt concentrations. Individual saltday observed at 55 mS·cmϪ1 in Type I medium and concentrations were substituted from Type II into2.25 doublings per day at 40 mS·cmϪ1 in Type II. Type I medium (Table 2) to determine whether spe-However, growth was not inﬂuenced as much by the ciﬁc salt concentrations affected lipid yield.range of speciﬁc conductances as it was by the range Growth was slightly higher than, but not signiﬁ-of temperatures investigated. cantly different from, the control in all substitutions Lipid yield. Epiﬂuorescent microscopy, utilized that increased salt concentrations (Fig. 6). Cultureswith the ﬂuorochrome Nile Red, revealed that neu- subjected to reduced major salt concentrations (i.e.tral lipids were a major form of carbon storage in CaCl 2 and MgCl 2 ) exhibited signiﬁcantly lowerN. communis. Prior to nitrogen deprivation (pre- growth rates than control cultures.stress), rapidly growing cells exhibited multiple (10– The average prestress lipid yield increased as15) small neutral lipid bodies comprising a small NaHCO3 concentration increased from 0.17 to 3.23portion (ca. 10%–20%) of total cell volume (Fig. g·LϪ1 and was signiﬁcantly higher than the prestress3a). Storage lipid bodies increased in size and coa- control with yields of 45.83 and 12.76 mg·LϪ1, re-lesced after 2 days of nitrogen deprivation (post- spectively (Fig. 7a). Other substitutions did not re-stress), creating two to three large neutral lipid de- sult in prestress yields that were statistically differentposits that made up 60%–75% of total cell volume from the control.(Fig. 3b). A signiﬁcantly higher poststress yield (142.36 SERI media type. Pre- and poststress lipid yields in compared to 50.38 mg·LϪ1) was observed whenN. communis were consistently higher in SERI Type MgCl2 concentration was reduced from the controlII medium than in Type I. Average prestress yields concentration of 35.31 to 4.36 g·LϪ1 (i.e. 0.37–were almost four times higher in Type II (38 0.046M) (Fig. 7b).mg·LϪ1) than in Type I medium (10 mg·LϪ1). Av- When MgCl2 concentration in Type I medium aterage poststress yields were more than three times 55 mS·cmϪ1 was systematically reduced from 35.31
716 THOMAS A. DEMPSTER AND MILTON R. SOMMERFELD FIG. 4. Poststress lipid yields (mg·LϪ1) observedon the thermogradient plate for N. communis cul-tured in SERI Type I medium over a range of tem-peratures (10Њ–35Њ C) and speciﬁc conductances(10–70 mS·cmϪ1). FIG. 5. Poststress lipid yields (mg·LϪ1) observedon the thermogradient plate for N. communis cul-tured in SERI Type II medium over a range of tem-peratures (10Њ–35Њ C) and speciﬁc conductances(10–70 mS·cmϪ1).
LIPID ACCUMULATION IN N. COMMUNIS 717 FIG. 6. Comparison of growth rates in SERIType I medium (control) and SERI Type I mediumwith substitutions of individual salt concentrationsfrom SERI Type II medium (mean Ϯ 1 SD, n ϭ 3).Individual salt concentration increases and decreas-es are denoted by ‘‘ϩ’’ and ‘‘Ϫ’’, respectively. Thex-axis labels represent the following: 1 ϭ I/55, 2 ϭϪCaCl2, 3 ϭ ϪMgCl2, 4 ϭ ϩNa2SO4, 5 ϭ ϩKCl, 6ϭ ϩNaHCO3, 7 ϭ ϩNaCl, and 8 ϭ ϩNa2CO3.to 4.36 g·LϪ1, prestress yields ranged from 5.39 DISCUSSIONmg·LϪ1 (at 4.36 g·LϪ1) to 18.37 mg·LϪ1 (at 28 g·LϪ1), Growth. Nitzschia communis grew well in both SERIbut were not statistically different (Fig. 8a). Howev- media. Growth was, on average, only slightly higherer, reduction in MgCl2 concentration resulted in in- in Type I medium than in Type II. Sommerfeld andcreased poststress neutral lipid yield. A concentra- Ellingson (1987) and Sommerfelt et al. (1987) alsotion of 12 g·LϪ1 stimulated a signiﬁcantly higher observed no signiﬁcant differences between growthpoststress lipid yield (200.26 mg·LϪ1) than other in Type I and II media by 62 strains of microalgae.concentrations (Fig. 8b). In contrast, Amphora sp. and Ankistrodesmus sp. ex- FIG. 7. Comparison of lipid yields (a) before(prestress), and (b) after (poststress) nitrogen deple-tion in SERI Type I medium (control) and SERI TypeI media with substitutions of individual salt concen-trations from SERI Type II medium (mean Ϯ 1 SD,n ϭ 3). Individual salt concentration increases anddecreases are denoted by ‘‘ϩ’’ and ‘‘Ϫ’’, respectively.The x-axis labels represent the following: 1 ϭ I/55, 2ϭ ϪCaCl2, 3 ϭ ϪMgCl2, 4 ϭ ϩNa2SO4, 5 ϭ ϩKCl, 6ϭ ϩNaHCO3, 7 ϭ ϩNaCl, and 8 ϭ ϩNa2CO3.
718 THOMAS A. DEMPSTER AND MILTON R. SOMMERFELD FIG. 8. Effect of reduced magnesium chlorideconcentrations in SERI Type I medium on (a) pres-tress, and (b) poststress lipid yield (mean Ϯ 1 SD, nϭ 5).hibited signiﬁcantly higher growth rates in SERI growth temperature for most microalgae under con-Type II than Type I medium (Barclay et al. 1985). sideration in the ASP ranged between 20Њ and 35ЊBarclay et al. (1987) also reported optimal growth C (Barclay et al. 1985, Tadros 1985, Johansen et al.for two Amphora spp. and one Monoraphidium sp. in 1987).Type II medium and optimal growth for one Cyclo- Nitzschia communis satisﬁed another desirabletella sp. in Type I. Growth in SERI media (Type I or characteristic for potential mass-culture efforts byII) appeared to vary signiﬁcantly among genera, spe- being euryhaline with good growth over a conduc-cies, and strains of microalgae (Barclay et al. 1986). tance range from 10 to 70 mS·cmϪ1. Tolerance of Growth in the two SERI media also differed N. communis to a wide range of speciﬁc conduc-among algal classes and divisions. Type II medium tances would allow this strain to grow rapidly in mostsupported growth of all diatoms, but Type I often of the natural saline groundwater aquifers in theinhibited diatom growth (Tadros 1987). Ellingson Southwest. In a survey of 106 saline waters in Ari-et al. (1989) reported that cyanophyte and chryso- zona, California, New Mexico, Texas, and Utah, con-phyte growth was equivalent in both SERI media, ductance averaged 22.7 mS·cmϪ1 (Ellingson andbut chlorophyte growth was signiﬁcantly higher in Sommerfeld 1992).Type II. Optimal speciﬁc conductance for growth also var- Nitzschia communis grew rapidly over a range of ies substantially between microalgal genera, species,temperatures from 20Њ to 30Њ C, with the highest and strains, as well as within strains, with respect togrowth rates observed at 30Њ C. Rapid growth in el- type of medium. This suggests that total dissolvedevated temperatures (thermophily) and over a salts may not be as important as the relative pro-broad range of temperatures (eurythermy) are de- portions of speciﬁc ions. For example, optimalsirable characteristics for microalgae to possess to growth for Cyclotella cryptica was observed at 25have potential for mass culture (Neenan et al. mS·cmϪ1 in SERI Type I and at 40 mS·cmϪ1 in Type1986). II (Johansen et al. 1987). Barclay et al. (1986) listed Optimal temperature for growth, like media type numerous microalgal strains that required different(I, II), is genera, species, and even strain speciﬁc conductances of each SERI medium for optimal(Burlew 1953, Barclay et al. 1986). Guillard and Ry- growth. Conversely, the same conductance for op-ther (1962) isolated strains of Cyclotella nana from timal growth in both SERI media has also been re-near-shore and offshore locations and reported tem- ported (Barclay et al. 1985, Sommerfeld et al. 1987).perature optima of 25Њ and 15Њ C, respectively. Sim- For example, Amphora sp. and Ankistrodesmus sp.ilar to N. communis, Navicula saprophila exhibited op- grew best at 25 mS·cmϪ1 in both SERI media (Bar-timal growth at 30Њ C (Chelf et al. 1987). Optimal clay et al. 1985). In addition, some strains grew rap-
LIPID ACCUMULATION IN N. COMMUNIS 719idly over a broad range of conductances in both me- two- to threefold increases in the lipid content ofdia (Barclay et al. 1986, Chelf et al. 1987, Tadros chlorophytes, whereas both increases and decreases1987). were observed in diatoms after nitrogen deprivation. Lipid yield. Intracellular neutral lipid production Barclay et al. (1985) reported that lipid contentin N. communis was signiﬁcantly higher in SERI Type more than doubled in Amphora sp., but increasedII than Type I medium. Ellingson et al. (1989) also only slightly in Ankistrodesmus sp. after nitrogen de-reported that mean lipid yield in Type II was nearly privation. Similarly, postnitrogen stress lipid yield in-twofold higher than in Type I for 102 strains ex- creased for approximately 10 days in Cyclotella sp.,amined. However, the optimal SERI media type for but ﬂuctuated in Ankistrodesmus sp., Chlorella sp., andlipid production also differed between genera, spe- Isochrysis sp. (Benemann and Tillett 1987).cies, and strains of microalgae (Barclay et al. 1986, Neenan et al. (1986) reported that rapidly grow-Johansen et al. 1987). ing microalgae consisted of 50%–70% membrane- Although average lipid yield in N. communis was bound polar lipids, but shifted to a predominantnot signiﬁcantly different at temperatures between neutral lipid composition (up to 60%) when de-20Њ and 30Њ C, slightly higher lipid yields were ob- prived of nitrogen. Suen et al. (1987) reported aserved at 30Њ C. Others have shown that lipid yield metabolic shift in Nannochloropsis sp. from polar lip-usually increased as temperature increased up to 35Њ id biosynthesis under nitrogen sufﬁciency to neutralC (Johansen et al. 1987, Tadros 1987). Opute lipid biosynthesis under nitrogen deﬁciency. How-(1974a) reported optimal fat synthesis in Nitzschia ever, both polar and neutral lipid yields increasedpalea at 35Њ C, but a drastic reduction in lipid pro- after nitrogen or silicon deﬁciency in Chaetoceros sp.duction at temperatures below 15Њ C and above 40Њ (Sriharan and Bagga 1987), Cyclotella sp., and Mon-C. Chelf et al. (1987) observed total lipid increases oraphidium minitum (Sriharan et al. 1987). Cookseyin Navicula saprophila as temperature increased from et al. (1989) proposed that nitrogen deﬁciency did17Њ to 32Њ C. Polar and neutral lipid contents were not directly induce triglyceride synthesis in microal-signiﬁcantly higher for Chaetoceros sp., Cyclotella sp., gae but caused cell division to cease, which reducedand Monoraphidium minitum when cultured at 30Њ C, intracellular energy demand on storage lipids. Hecompared to 25Њ C (Sriharan and Bagga 1987, Sri- suggested that triglycerides are synthesized at theharan et al. 1987). same rate throughout the cell cycle, and triglyceride Although N. communis exhibited appreciable lipid accumulation varies in response to energy demandyields over a wide range of speciﬁc conductances, at different stages of the cell cycle.optimal speciﬁc conductance for lipid production Inﬂuence of major salts. Substitution of salt concen-was between 40 and 55 mS·cmϪ1. Lipid yield was trations among the two media generally did not leadusually higher in salt-tolerant microalgae as conduc- to increased neutral lipid storage in N. communis.tance of SERI media increased (Barclay et al. 1986). However, neutral lipid production was noticeably in-Increased lipid yield was observed as increased salt ﬂuenced by speciﬁcally altering the MgCl2 concen-concentration caused physiological stress in Botry- tration in the culture medium. Roessler (1989) re-ococcus braunii and Isochrysis sp. (Ben-Amotz et al. ported that the activity of acetyl-CoA carboxylase1985) and Chlorella sp. (Tadros 1985). (ACC), an enzyme utilized early in fatty acid synthe- Nitrogen deprivation. Dramatic intracellular neutral sis, was dependent on the presence of divalent metallipid (triglyceride) increases occurred after nitrogen cations, especially magnesium (Mgϩϩ). Roessler ob-deprivation. Both nitrogen deﬁciency, a decrease in served reduced ACC activity when manganesethe amount of available nitrogen, and nitrogen de- (Mnϩϩ) was the only divalent metal present and noprivation, the absence of nitrogen in culture media, ACC activity when only cobalt (Coϩϩ) was present.have been closely related to increased lipid accu- This study has attempted to evaluate some of themulation in microalgae. For example, lipid produc- factors that may inﬂuence the growth and lipid yieldtion increased as nitrogen concentration decreased of a common diatom, Nitzschia communis, which mayin cultures of Chlorella vulgaris (Fogg and Collyer be a potential candidate for future efforts to develop1953), Nitzschia palea (Opute 1974a), Ankistrodesmus alternative renewable liquid fuel sources. In the lab-sp., Chlorella sp., Navicula saprophila (Chelf et al. oratory, N. communis exhibited rapid growth, appre-1987), Chaetoceros sp. (Sriharan and Bagga 1987), Cy- ciable lipid yield, tolerance of extreme conditionsclotella sp., and Monoraphidium minitum (Sriharan et similar to those found in the arid southwestern Unit-al. 1987) and Nannochloropsis sp. (Suen et al. 1987). ed States, and a life cycle that permitted continuousThe effect of nitrogen deﬁciency on lipid produc- culturing. Field operations will be necessary to de-tion varies among and within algal divisions. Two termine whether N. communis exhibits outdoorgreen algae exhibited increased lipid content in re- growth and lipid production characteristics compa-sponse to nitrogen deﬁciency, whereas four cyano- rable to those observed in the laboratory.bacteria did not show any signiﬁcant changes in lip-id composition when exposed to reduced nitrogen Barclay, B., Nagle, N. & Terry, K. 1987. Screening microalgaeconcentrations (Piorreck et al. 1984). Shifrin and for biomass production potential: protocol modiﬁcationChisholm (1981) examined 30 species and reported and evaluation. In Johnson, D. A. [Ed.] FY 1986 Aquatic
720 THOMAS A. DEMPSTER AND MILTON R. SOMMERFELD Species Program Annual Report, Publ. SERI/SP-231–3071. So- Report, Publ. SERI/SP-231–3071. Solar Energy Research lar Energy Research Institute, Golden, Colorado, pp. 23– Institute, Golden, Colorado, 342 pp. 40. Maddux, W. S. & Jones, R. F. 1964. Some interactions of tem-Barclay, B., Nagle, N., Terry, K. & Roessler, P. 1985. Collecting perature, light intensity, and nutrient concentration dur- and screening microalgae from shallow, inland saline hab- ing the continuous culture of Nitzschia closterium and Te- itats. In McIntosh, R. P. [Ed.] Aquatic Species Program Re- traselmis sp. Limnol. Oceanogr. 9:79–86. view: Proceedings from the March 1983 Principal Investigators McIntosh, R. P. 1985a. Aquatic Species Program Review: Proceed- Meeting, Publ. SERI/CP-231–2700. Solar Energy Research ings of the March 1985 Principal Investigators Meeting, Publ. Institute, Golden, Colorado, pp. 52–68. SERI/CP-231–2700. 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Phytochemistry 23:207–16. Species Program Annual Review Meeting. Solar Energy Re- Roessler, P. G. 1989. Puriﬁcation and characterization of ace- search Institute, Golden, Colorado, pp. 139–58. tyl-CoA carboxylase from the diatom Cyclotella cryptica. InCooksey, K. E., Guckert, J. B., Williams, S. A. & Callis, P. R. Aquatic Species Program Annual Review Meeting. Solar Energy 1987. Fluorometric determination of the neutral lipid Research Institute, Golden, Colorado, pp. 125–38. content of microalgal cells using nile red. J. Microbiol. Shifrin, N. S. & Chisholm, S. W. 1981. Phytoplankton lipids: Methods 6:333–45. inter-speciﬁc differences and effects of nitrate, silicate andEllingson, S. B. & Sommerfeld, M. R. 1992. Ionic composition light-dark cycles. J. Phycol. 17:374–84. of high conductance waters of the arid Southwest. J. Ariz.- Sommerfeld, M. R. & Ellingson, S. B. 1987. Collection of high Nev. Acad. 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LIPID ACCUMULATION IN N. COMMUNIS 721 Total lipid production of the green alga Nannochloropsis sp. QII 1987. Screening and characterizing oleaginous microal- under different nitrogen regimes. J. Phycol. 29:289–96. gal species from the southeastern United States. In Johnson,Tadros, M. G. 1985. Screening and characterizing oleaginous mi- D. A. [Ed.] FY 1986 Aquatic Species Program Annual Report, croalgal species from the southeastern United States. In Mc- Publ. SERI/SP-231–3071. Solar Energy Research Institute, Intosh, R. P. [Ed.] Aquatic Species Program Review: Proceedings Golden, Colorado, pp. 67–89. from the March 1983 Principal Investigators Meeting, Publ. SERI/ Tyler, P. L. 1989. Microalgae of Inland Saline Waters: Distribution, CP-231–2700. Solar Energy Research Institute, Golden, Col- Diversity and Lipid Accumulation. M.Sc. thesis, Arizona State orado, pp. 28–42. University, Tempe, 90 pp. BOOK REVIEWJ. Phycol. 34, 721–723 (1998) MOLECULAR PHYLOGENY OF THE ALGAE gins of Algae and Their Plastids. This superb book dis-Bhattacharya, Debashish, ed. 1997. Origins of Algae cusses in great depth what research using the newand Their Plastids. Springer, Wien. 287 pp. 270 DM molecular methods has revealed about the evolution(approximately US$180.00), ISBN: 3-211-83036-7. of algae and their plastids in the decade since So- gin’s ﬁrst surprising phylogenetic trees were pub- lished. The construction of phylogenetic trees based on This is a weighty book. It contains 14 chaptersthe sequence of the ribosomal RNA of the small sub- written by 34 authors and coauthors. Bhattacharyaunit (SSU) of eukaryotic ribosomes has revolution- has chosen young researchers, all molecular biolo-ized the systematics of the algae. For me, this revo- gists actively working with the algae they discuss, tolution began with Mitch Sogin’s classic 1987 and write the chapters. The chapters are all very detailed1989 papers (Proc. Natl. Acad. Sci. USA 84: and contain extensive bibliographies. The bibliog-5823–5827; Science 243:75–77), which showed raphies are outstanding, giving complete titles inthat Giardia lamblia was the earliest diverging eu- large, easy-to-read print, a feature those of us in thekaryotic lineage, an honor it still holds, although its older generation, who can barely read the refer-lack of mitochondria is no longer considered prim- ences in Science and Nature, will especially appreci-itive since it has recently been shown to contain the ate. The references are also up to date, a difﬁcultgene for chaperonin 60, a mitochondrial protein. feat to manage in a multiauthored book. The bookSlightly higher up the tree, trypanosomes and their was published in late 1997, and the bibliographiesclose relative, Euglena gracilis, branched off together, contain numerous 1996 references and even someconﬁrming Euglena’s distinctiveness from other al- 1997 ones. Although molecular evolution is a rap-gae and lending credence to my hypothesis that Eu- idly moving ﬁeld, in no case am I aware of a majorglena obtained its chloroplasts secondarily from sym-biotic green algae. Dictyostelium branched off next, 1997 or 1998 discovery that diminishes a chapter’sbut after that, the tree had a bushy top, with this importance. Bhattacharya has produced the rightcrown radiation producing virtually all the other eu- book at the right time. I congratulate the publisherskaryotes. In this crown group, the chrysophyte alga on the very attractive format of the book and theOchromonas was on the same branch as the oomycete promptness with which it was published.Achlya, a relationship phycologists had long suspect- I have only two small complaints. The chaptersed because of the ﬁne structure of their heterokont are not numbered. Authors do refer to other chap-ﬂagella. Dinoﬂagellates, to my surprise, were ﬁrst ters by number, but usually by the wrong number.cousins of the ciliates. I wouldn’t have been sur- Also, the book is only available in hardcover, evenprised if I had looked carefully at the ﬁne structure though it was originally published as a supplementof ciliates and seen the close similarity between their to Plant Systematics and Evolution, and there it wasalveolar sacs and the amphiesmal vesicles of dino- produced in softcover. Springer-Verlag should makeﬂagellates, but in those premolecular days, most the book available to everyone in softcover and at aphycologists paid scant attention to protozoa and price more phycologists could afford.fungi. Today’s molecular phylogenetic trees, how- This book has two main themes. In a number ofever, clearly show that the ‘‘Algae’’ are a group of the chapters, the authors’ main concern is what mo-unrelated protists and oomycete fungi, which have lecular sequences reveal about the evolution of theacquired their chloroplasts either directly or indi- algal class discussed and how subclasses, orders, andrectly. at times families are related to each other. The con- Thus, I was very pleased to be asked to review clusions are usually based on SSU rRNA trees, butDebashish Bhattacharya’s important new book, Ori- some chapters include protein-based trees where
722 BOOK REVIEWavailable. In the other chapters, the main concern role in earlier classiﬁcations of the green algae thatis the evolution of chloroplasts. Here, the crucial I suspect many morphologically oriented phycolo-questions are: did a cyanobacterium become a chlo- gists will be unhappy with Friedl’s revisions. I feelroplast only once or more than once (the consensus SSU rRNA data must be supported by other phylo-is that this primary symbiosis occurred only once), genetic trees based on protein sequences or mito-and how many times did chloroplasts arise second- chondrial genes before we discount ultrastructuralarily from a eukaryotic endosymbiont? In the two characteristics. Huss and Krantz’s short and emi-groups where the symbiont’s nucleus persists, the nently readable chapter on the charophytes showscryptomonads and chlorarachniophytes, the authors that contrary to popular opinion, the Charales areask why the nucleomorph has persisted and what a distinct and ancient lineage within the group.genes it contains. However, which of the other orders of the Charo- In the ﬁrst chapter, Bhattacharya describes con- phyta is the ancestor to land plants is still an opencisely and clearly for readers such as myself the question.three common methods of determining phyloge- The remaining chapters deal in large part withnetic trees from sequence data, namely distance, the evolution of chloroplasts in the algae. Start bymaximum parsimony, and maximum likelihood. I reading Delwiche and Palmer’s superb chapter,suspect this chapter will also be useful to those start- ‘‘The origin of plastids and their spread via second-ing to use these methods, for it gives the speciﬁc ary symbiosis.’’ This comprehensive chapter gives aname of the computer programs one can buy to detailed and balanced discussion of the data for andconstruct trees by each technique. against a single primary endosymbiosis forming the Chapters that discuss primarily the evolutionary chloroplasts of the red and green algae as well asrelationships within a single class of algae are Tur- the cyanelles of the glaucocystophytes. They con-ner’s on the cyanobacteria, Saunders and Kraft’s on clude that plastid sequence data support a mono-the red algae, Saunders and coauthors’ on the di- phyletic origin, that mitochondrial data supportnoﬂagellates, Druehl and colleagues’ on the brown monophyly of the red and green algae (no mito-algae, Friedl’s on the green algae, and Huss and chondrial data are available for the glaucocystophy-Kranz’s on the Charophyta. Although these chapters tes), and that the nuclear data are inconclusive.will mainly be of interest to specialists in each group, They and Turner emphasize the need for muchthey also contain enough general information to more data on the cyanobacteria, and both Turnermake them worthwhile reading for all phycologists and Delwiche and Palmer emphasize that althoughinterested in evolution. plastids appear as a monophyletic branch on the cy- Turner’s chapter shows clearly that each of the anobacterial tree, this does not exclude the possi-three known prochlorophytes, Prochloron didemni, bility that the same or two or more closely relatedProchlorothrix hollandica, and Prochlorococcus marinus, cyanobacteria could have separately given rise to pri-belongs to a different subgroup of the cyanobacter- mary chloroplasts, what Palmer calls ‘‘cryptic poly-ia. Whether chlorophyll b has evolved independently phyly.’’ Delwiche and Palmer continue with a con-a number of times, as ﬁrst believed, is made less cise description of all the algal groups which havecertain by the recent discovery that P. marinus has secondary chloroplasts, that is, chloroplasts thatboth phycoerythrin and chlorophyll b, an observa- evolved from endosymbiotic eukaryotic algae. Thesetion that raises the possibility of multiple pigment even include the Apicomplexa, for Iain Wilson andlosses. Donald Williamson have discovered that the malar- I was disappointed that the chapter on red algal ial parasite Plasmodium has a secondary chloroplast.evolution did not include a summary of current Wilson (Mol. Gen. Genet. 243:249–252) hasknowledge on the Bangiophycidae, but instead the presented evidence that the plastid of Plasmodiumchapter referred the reader to Mark Ragan’s 1994 evolved from a symbiotic red alga, but Delwiche, inpaper on the topic (Proc. Natl. Acad. Sci. USA a recent paper (Science 275:1485–1489) on91:7276–7280). The big advantage of a com- another apicomplexan parasite, suggests the plastidprehensive book like the present one is that it was originally a symbiotic green alga.should collect all current data in one convenient Several very informative chapters extend the sec-place. ondary endosymbiosis story. Fraunholz and col- Friedl’s chapter on the evolution of the green al- leagues’ chapter on the cryptomonads show justgae based on SSU rRNA sequences presents once how far our knowledge of cryptomonad nucleo-again a new classiﬁcation of the green algae, includ- morphs has advanced since Marcelle Gillott’s pio-ing a new class, the Trebouxiophyceae. Friedl’s SSU neering study (J. Phycol. 16:558–568) on nu-rRNA data indicate that counterclockwise basal body cleomorph ultrastructure. The sequence of nucleo-orientation, the phycoplast type of mitosis, and morph SSU rRNA shows that the symbiont that gaveprobably also MLS rootlets are symplesiomorphic rise to the chloroplast was closely related to the redcharacters, that is, similar primitive features that are algae. The authors have shown that the nucleo-presently shared by independent lineages. Phyco- morph genome consists of three small chromo-plasts and MLS rootlets have played such a major somes with a total length of 600 kb. A collaborative
BOOK REVIEW 723project to sequence this genome is underway, and a Loeffelhardt and colleagues’ on the cyanelle ge-number of genes have already been identiﬁed. nome of Cyanophora paradoxa are both essential Goeff McFadden, in an excellent chapter written reading for anyone concerned with whether chlo-with his usual ﬂair, discusses the Chlorarachnioph- roplasts, including the cyanelles, are monophyletic.yta, ﬁlose amoebae whose chloroplasts have ﬁnally Although the evidence presented in these two chap-been shown by the sequence of the SSU rRNA of ters strongly suggests that only a single symbiotictheir nucleomorphs to have evolved from green al- event gave rise to the cyanelles of the glaucocysto-gae. Surprisingly, the nucleomorphs of Chlorarach- phytes and the plastids of red and green algae, wenion reptans also have three small chromosomes. still need more data. The enigmatic amoeba Pauli-McFadden and his team are rapidly characterizing nella chromatophora, which contains two sausage-these chromosomes. At the time he wrote the chap- shaped cyanelles, is not related to either the glau-ter, he had shown that each chromosome is capped cocystophytes or to the red and green algae. If itsby telomeres and each has a ribosomal RNA cistron cyanelles are not simply symbiotic cyanobacteria butinterior to each telomere. He had also found eight true plastids (of all the cyanelles, those of Paulinellaprotein genes, including heat shock protein 70, a most closely resemble free-living cyanobacteria), itsubunit of RNA polymerase, an RNA helicase, and seems very unlikely that these cyanelles arose froma chloroplast protease. Although McFadden was the the same symbiotic event that gave rise to the otherﬁrst person to show by in situ hybridization that the cyanelles and the chloroplasts of red and green al-plastid DNA of Toxoplasma, a close relative of Plas- gae. But more importantly, I would like to see datamodium, is located in a small spherical body, sur- on the plastid DNA of what Saunders calls typicalrounded, I believe, by three or four membranes, his dinoﬂagellate chloroplasts, plastids surrounded byshort chapter on the plastids of the Apicomplexa is three membranes that contain the unique solublenot up to his usual standards. I recommend that peridinin–chlorophyll a-protein in addition to theanyone interested in this fascinating story read Wil- peridinin–chlorophyll a/c light-harvesting protein,son and Williamson’s excellent 1997 review (Micro- and a nuclear-coded form II RUBISCO, consistingbiol. Mol. Biol. Rev. 61:1–16). of two large subunits only. Although the presence The chloroplast chapter I found most interesting of three surrounding membranes suggests that theseand most original in the sense of being a new syn- chloroplasts have evolved from a secondary symbi-thesis is that of Linda Medlin and her colleagues on osis, perhaps they did not. Possibly, they evolved di-the phylogeny of the golden algae. This chapter rectly from a symbiotic prokaryote. If so, these plas-draws together an immense amount of information tids have so many unique characteristics that theyon the heterokont groups and the haptophytes: his- easily could have arisen from a different symbiosistory of their nomenclature, morphology, pigments, than the one that gave rise to other primary chlo-phylogenetic trees based on the sequences of SSU roplasts. Isolating and sequencing the plastid DNArRNA, actin, and the light-harvesting proteins as well of one of these typical dinoﬂagellate chloroplastsas plastid phylogenetic trees based on three differ- should be a high priority for this generation of mo-ent chloroplast genes, to answer a set of clearly de- lecular phycologists.ﬁned questions on the evolution of both the host This review is dedicated to the memory of Marcelle A. Gillott, ancells and their chloroplasts. In brief, Medlin shows enthusiastic phycologist, a superb electron microscopist, and athat the haptophytes and the pigmented hetero- loyal friend, who died tragically in a freak accident on Novemberkonts are separate nonrelated groups. Each inde- 23, 1995.pendently acquired its plastid from a eukaryotic sym- SARAH P. GIBBSbiont at or shortly before the Permian–Triassic Department of Biologyboundary. Each group endocytosed a different alga. McGill University The two remaining chapters of the book, Bhatta- 1205 Dr. Penﬁeld Avenuecharya and Schmidt’s on the Glaucocystophyta, and Montreal, Quebec H3A 1B1, Canada