Heterotrophic Culture Conditions Research Articles

Optimization of Heterotrophic Culture Conditions for the Microalgae Euglena gracilis to Produce Proteins.

Euglena gracilis is one of the few permitted edible microalgae. Considering consumer acceptance, E. gracilis grown heterotrophically with yellow appearances have wider food industrial applications such as producing meat analogs than green cells. However, there is much room to improve the protein content of heterotrophic culture cells. In this study, the effects of nitrogen sources, temperature, initial pH, and C/N ratios on the protein production of E. gracilis were evaluated under heterotrophic cultivation. These results indicated that ammonium sulfate was the optimal nitrogen source for protein production. The protein content of E. gracilis cultured by ammonium sulfate increased by 113% and 44.7% compared with that cultured by yeast extract and monosodium glutamate, respectively. The manipulation of the low C/N ratio further improved E. gracilis protein content to 66.10% (w/w), which was 1.6-fold of that in the C/N = 25 group. Additionally, amino acid analysis revealed that the nitrogen-to-protein conversion factor (NTP) could be affected by nitrogen sources. A superior essential amino acid index (EAAI) of 1.62 and a balanced amino acid profile further confirmed the high nutritional value of E. gracilis protein fed by ammonium sulfate. This study highlighted the vast potency of heterotrophic cultured E. gracilis as an alternative dietary protein source.

Optimization of Heterotrophic Culture Conditions for the Algae Graesiella emersonii WBG-1 to Produce Proteins.

The aim of this study was to improve the protein content and yield of heterotrophic microalgal cultivation and establish a simple, economical, and efficient method for microalgal protein production using the novel green alga, Graesiella emersonii WBG-1, which has not been previously reported for heterotrophic cultivation. Through batch heterotrophic cultivation of this alga, we observed that glucose was the optimal carbon source, while it could not use sucrose as a carbon source. Biomass production and protein content were significantly reduced when sodium acetate was used as the carbon source. Compared with nitrate, protein content increased by 93% when urea was used as the nitrogen source. Cultivation temperature had a significant impact on biomass production and protein content. The optimal conditions were glucose as the carbon source at an initial concentration of 10 g/L, urea as the nitrogen source at an initial concentration of 1.62 g/L, and a culture temperature of 35 °C. On the second day of batch cultivation, the highest protein content (66.14%) was achieved, which was significantly higher than that reported in heterotrophic cultures of Chlorella and much higher than that reported for specially established technologies aimed at increasing the protein content, such as two-stage heterotrophic, heterotrophy-dilution-photoinduction, and mixotrophic processes. These results demonstrate the great potential of the heterotrophic cultivation of G. emersonii WBG-1 for protein production.

Effects of shear stress and shear protectants on heterotrophic culture of Haematococcus pluvialis

A strategy named “Sequential Heterotrophy-Dilution-Photoinduction” is used for commercial production of astaxanthin in Haematococcus pluvialis. However, shear stress during heterotrophic culture has become the technical bottleneck for the wide application and amplification of this technology in microalgae culture, especially in the heterotrophic culture of H. pluvialis. To clarify the effects of shear stress, the influence of shear stress generated by different aeration rates on the heterotrophic growth of H. pluvialis was first investigated. The results showed that shear stress could cause the transformation of motile cells to non-motile cells, resulting in decrease in growth rate. The critical shear stress was produced by a superficial gas velocity of >3.21 × 10−3 m·s−1. The further analysis based on computational fluid dynamics indicated that shear stress-induced damage to cells was mainly caused by bubble rupture. To reduce the negative effects of shear stress, 0.05 % of Pluronic F-68 was selected for shear protection. Finally, it was successfully applied to the heterotrophic culture of H. pluvialis in a 500 L fermenter, increasing the cell yield by 30.39 %. This study could provide a reference and basis for the control of heterotrophic culture conditions and scale-up of H. pluvialis, and a feasible strategy for reducing the shear stress-induced damage during the heterotrophic process.

Simultaneous probing of dual intracellular metabolites (ATP and paramylon) in live microalgae using graphene oxide/aptamer nanocomplex.

The development of an intracellular metabolite imaging platform for live microorganisms has been a challenge in the study of microbes. Herein, we performed metabolite imaging in live microalgal cells using a graphene oxide (GO)/aptamer complex. The properties of the GO were characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM), which were determined to have 140 ± 3nm in mean diameter. An ATP-specific aptamer was mixed with GO to form a GO/aptamer complex, and the feasibility of the complex was tested in vitro. The high correlation between the fluorescence intensity and concentration of ATP was observed in the range 0-10mM. Next, the feasibility of the complex was confirmed in vivo. Under both phototrophic and heterotrophic culture conditions, Euglena gracilis internalized the complex, and bright fluorescence was observed as the aptamer was bound to the target metabolite (ATP). The fluorescence intensity of cells was correlated to the ATP concentration in the cells. Imaging of dual intracellular metabolites (ATP and paramylon) was achieved by simply using two different aptamers (ATP-specific aptamer and paramylon-specific aptamer) together, showing the great potential of the complex as a dual-sensing/imaging platform. In addition, the GO/aptamer complex exhibited low cytotoxicity; the proliferation and viability of E. gracilis cells were not significantly affected by the complex. Our results suggested that this new imaging platform can be efficiently used for detecting dual intracellular metabolites in live microalgal cells.

Evaluation of internal reference genes in Auxenochlorella protothecoides under continuous heterotrophic culture conditions at normal, low and high temperatures

Real-time reverse quantitative transcription PCR (RT-qPCR) is a commonly used method for gene expression analysis. However, the accuracy of RT-qPCR analysis greatly depends on the stability of the reference genes selected for data normalization. Unfortunately, there have been no systematic evaluation studies on reference genes in the unicellular green alga Auxenochlorella protothecoides. Ten genes (ACT, COX6B, CYB5R4, EF1α, GAPDH, rbcL, RPL19, RPL32, RPS10 and TUA) were chosen as candidate reference genes. The expression stability of each reference gene was determined by applying geNorm, NormFinder, BestKeeper and comprehensive ranking based on geomean ranking values. In order to validate the suitability and reliability of the candidate reference genes selected for data normalization, two key functional genes related to lipid metabolism were also selected as target genes. The results revealed that RPL32 was the most stably expressed and suitable reference genes under all experimental conditions of the analyses; CYB5R4 was also a suitable reference gene under continuous heterotrophic culture condition, and RPL19 was top-ranked and stably expressed under both low and high temperature stress. These results provided a list of suitable reference genes for accurate normalization of heterotrophic A. protothecoides time-course samples under different temperature stress in qPCR experiments. This study will be of very great reference value to assess the stability of candidate reference genes in other heterotrophically grown algae.

<b>Screening of an adapted culture medium composed by different carbon sources for heterotrophic cultivation of <i>Chlorella vulgaris</i> using a microplate assay

The ability of microalgae to grow heterotrophically lies in the ability to oxidize organic compounds present in the external environment. The aim of this work was to evaluate the growth of Chlorella vulgaris under heterotrophic culture conditions using Basal Bold and NPK media supplemented with different sole carbon sources (glucose, fructose, sucrose, glycerol or acetate). The kinetic parameters obtained were maximum specific growth rate (µ max ), doubling time (DT), maximal absorbance, which was also converted to cell concentration values using a linear relation, and cell productivity (P X ). Among all the treatments analyzed, the highest maximum specific growth rate found was 0.030 hour -1 (0.72 day -1 ) in the treatment using Basal Bold medium supplemented with glucose. The highest cellular concentration and cell productivity were also found for this same treatment (4.03 x 10 6 cell mL -1 and 64.0 x 10 6 cell L -1 day -1 , respectively). It was concluded that that the Basal Bold medium was more efficient for Chlorella vulgaris growth, since it induced higher values of µ max and cellular concentration. Results obtained were very reproducible using microplate assay.

Utilisation of Desmodesmus subspicatus LC172266 for simultaneous remediation of cassava wastewater and accumulation of lipids for biodiesel production

ABSTRACTIn view of the current demand for bioenergy and environmental sustainability, utilisation of Desmodesmus subspicatus LC172266 for simultaneous remediation of cassava wastewater and accumulation of lipids for biodiesel production was investigated. The microalga was grown under photoautotrophic, heterotrophic and mixotrophic culture conditions using Bold's Basal Medium (BBM) and cassava wastewater as substrates. Results showed that biomass accumulation under unoptimised autotrophic, heterotrophic and mixotrophic growths conditions were 0.217, 0.63 and 1.042 g/L, respectively. The lipid contents were 15.86, 21.40 and 24.70%, respectively. Fatty acid profiles revealed that although the most abundant compounds under autotrophic conditions were those recommended for biodiesel production, oleic acid eluted under mixotrophic (19.61%) and heterotrophic (10.25%) conditions exhibited more acceptable biodiesel quality. The physical properties of the fatty acid methyl esters (FAMEs) also meet the required biodiesel standards. The percentage reduction in total dissolved solids (TDS, 84.04), electrical conductivity (EC, 82.31), biochemical oxygen demand (BOD, 85.85) and chemical oxygen demand (COD, 89.04) under heterotrophic cultivation were more favourable than their respective mixotrophic values (80.56, 74.63, 62.22, and 51.39%). Although the lipid accumulation of the microalga under mixotrophic conditions was greater, bioremediation potential of cassava wastewater was more favourable under heterotrophy.

Characterization of the sulfur-formation (suf) genes in Synechocystis sp. PCC 6803 under photoautotrophic and heterotrophic growth conditions.

The sulfur-formation ( suf ) genes play important roles in both photosynthesis and respiration of cyanobacteria, but the organism prioritizes Fe-S clusters for respiration at the expense of photosynthesis. Iron-sulfur (Fe-S) clusters are important to all living organisms, but their assembly mechanism is poorly understood in photosynthetic organisms. Unlike non-photosynthetic bacteria that rely on the iron-sulfur cluster system, Synechocystis sp. PCC 6803 uses the Sulfur-Formation (SUF) system as its major Fe-S cluster assembly pathway. The co-expression of suf genes and the direct interactions among SUF subunits indicate that Fe-S assembly is a complex process in which no suf genes can be knocked out completely. In this study, we developed a condition-controlled SUF Knockdown mutant by inserting the petE promoter, which is regulated by Cu2+ concentration, in front of the suf operon. Limited amount of the SUF system resulted in decreased chlorophyll contents and photosystem activities, and a lower PSI/PSII ratio. Unexpectedly, increased cyclic electron transport and a decreased dark respiration rate were only observed under photoautotrophic growth conditions. No visible effects on the phenotype of SUF Knockdown mutant were observed under heterotrophic culture conditions. The phylogenetic distribution of the SUF system indicates that it has a co-evolutionary relationship with photosynthetic energy storing pathways.

Heterotrophic growth and nutrient productivities of Tetraselmis chuii using glucose as a carbon source under different C/N ratios

The genus Tetraselmis includes various species that are widely used as food microalgae in aquaculture. However, the mass cultivation of Tetraselmis spp. has been restricted to traditional photoautotrophic culture. Here, we investigated the heterotrophic culture condition for Tetraselmis chuii and assessed growth rate, morphology, and biomass productivity of T. chuii under heterotrophic culture conditions. Protein, polysaccharide, chlorophyll, total lipids, and fatty acid compositions under different initial C/N ratios were also analyzed. Tetraselmis chuii was found to be able to grow using glucose as the sole carbon source under strict heterotrophic conditions. Moreover, T. chuii showed a better heterotrophic growth status in the medium with 10 g L−1 glucose compared to the autotrophic condition. Comparatively high optical density and biomass were obtained at C/N ratios of 16 and 12. The dry weights of T. chuii under C/N ratios of 16 and 12 were 1.37 ± 0.04 and 1.26 ± 0.06 mg mL−1, respectively, significantly higher than those of the autotrophic group. In addition, T. chuii synthesized more polysaccharide under heterotrophic conditions, especially at a C/N ratio of 24. Tetraselmis chuii cultured under C/N ratios of 4 and 8 produced the highest percentages of C16:0 (43.36 %) and C18:1 (47.49 %) fatty acids, and more fatty acids, including C14:0, C18:0, C20:2, and C20:4, were detected in the groups with C/N ratios of 4 and 24. These findings may provide the basis for the mass culture of heterotrophic T. chuii by highlighting sources of plentiful nutrient substances, especially polysaccharides, for aquaculture.

Mixotrophic cultivation of green microalgae Scenedesmus obliquus on cheese whey permeate for biodiesel production

Microalgae mass cultivation for biodiesel production might very well become the next marketable biofuel. The main challenge to overcome however is the development of high efficiency strategies for the large-scale production of oleaginous microalgae at low costs. In the present work, the use of cheese whey permeate (WP) in mixotrophic microalgae cultures is proposed. Pure lactose, the main constituent of WP (>80% w/w of the total dissolved solids), can support Scenedesmus growth under heterotrophic culture conditions (absence of light). Substituting 40% (v/v) of the culture medium with WP significantly stimulates Scenedesmus obliquus growth under mixotrophic (μmax=1.083±0.030day−1) and heterotrophic (μmax=0.702±0.025day−1) conditions, compared to photoautotrophic control cultures (μmax=0.267±0.083day−1). As growth occurs in the presence of lactose, a significant reduction of its concentration is observed, while the galactose and glucose concentrations actually increase in the culture medium. Culture medium analyses showed complete exhaustion of extracellular nitrogen (nitrate and ammonium), while intracellular lipid analyses showed neutral lipid (NL) accumulation, particularly under conditions of high pH (>9.5). Photoautotrophic control cultures accumulated more lipids (per dry weight) than WP-supplemented cultures, an aspect which is discussed in the context of lipid enrichment strategies. A fast and simple method for NL cellular content estimation is also described.

Heterotrophic growth of Neochloris oleoabundans using glucose as a carbon source

BackgroundIn comparison with phototrophic growth, heterotrophic conditions can significantly increase growth rates, final cell number and cell mass in microalgae cultures. Neochloris oleoabundans is a microalga of biotechnological interest that accumulates lipids under phototrophic and nitrogen-limited conditions. Heterotrophic flask culture experiments were conducted to identify carbon sources that can be metabolized by N. oleoabundans, and bioreactor batch and fed-batch (nitrate pulse additions) cultures supplemented with glucose were performed to study the cellular composition of the microalgae under balanced and high C/N ratios (glucose/nitrate).ResultsN. oleoabundans was able to grow using glucose and cellobiose as sole carbon sources under strict heterotrophic conditions. Under a balanced C/N ratio of 17 and using bioreactor batch cultures containing 3 g/L glucose, a maximal cell mass of 1.72 g/L was found, with protein being the major cell component (44% w/w). A maximal cell mass of 9.2 g/L was obtained using batch cultures at a C/N ratio of 278. Under these conditions, lipid accumulation was promoted (up to 52% w/w) through N-limitation, resulting in high lipid productivity (528.5 mg/L/day). Fed-batch cultures were performed at a C/N ratio of 278 and with nitrate pulse additions. This condition allowed a maximal cell mass of 14.2 g/L to be achieved and switched the metabolism to carbohydrate synthesis (up to 54% of dry weight), mainly in the form of starch. It was found that transmembrane transport under these conditions was dependent on a proton-motive force, indicating that glucose is transported by a symporter.ConclusionsN. oleoabundans was able to grow under strict heterotrophic culture conditions with glucose or cellobiose as the only carbon source. The glucose used is transported by a symporter system. Batch cultures with a balanced C/N ratio accumulate proteins as the major cellular component; a high C/N ratio significantly increased the dry cell mass and resulted in a high lipid content, and a high cell density was achieved using fed-batch cultures promoting carbohydrate accumulation. These results suggest heterotrophic batch cultures of N. oleoabundans as an alternative for the production of proteins or lipids with simple culture strategies and minimal-mineral media supplemented with glucose.

Isolation of a novel strain of Monoraphidium sp. and characterization of its potential application as biodiesel feedstock

A novel green microalgae strain from Lake Fuxian has been isolated and identified as a potential feedstock for biodiesel production. The novel strain was named Monoraphidium sp. FXY-10 based on its morphological and genomic characterization. The lipid productivities, fatty acid profiles, and microalgae recovery efficiency (ηa) of FXY-10 were investigated and compared under autotrophic and heterotrophic conditions. FXY-10 under autotrophic conditions exhibited a higher cellular lipid content (56.8%) than those under heterotrophic conditions (37.56%). However, FXY-10 growing under heterotrophic conditions exhibited more than 20-fold increase in lipid productivity compared with that under autotrophic conditions (148.74mgL−1d−1 versus 6.88mgL−1d−1). Moreover, higher saturated and monounsaturated fatty acids (77.5%) of FXY-10 was obtained under heterotrophic culture conditions, suggesting its potential as a biodiesel feedstock. Gravity sedimentation was proposed as the harvesting biomass method based on the 97.9% microalgae recovery efficiency of heterotrophic cells after settling for 24h.

Growth characteristics of the cyanobacterium Nostoc flagelliforme in photoautotrophic, mixotrophic and heterotrophic cultivation

Nostoc flagelliforme is a terrestrial cyanobacterium with high economic value. Dissociated cells separated from a natural colony of N. flagelliforme were cultivated for 7 days under either phototrophic, mixotrophic or heterotrophic culture conditions. The highest biomass, 1.67 g L−1 cell concentration, was obtained under mixotrophic culture, representing 4.98 and 2.28 times the biomass obtained in phototrophic and heterotrophic cultures, respectively. The biomass in mixotrophic culture was not the sum as that in photoautotrophic and heterotrophic cultures. During the first 4 days of culture, the cell concentration in mixotrophic culture was lower than the sum of those in photoautotrophic and heterotrophic cultures. However, from the 5th day, the cell concentration in mixotrophic culture surpassed the sum of those obtained from the other two trophic modes. Although the inhibitor of photosynthetic electron transport DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] efficiently inhibited autotrophic growth of N. flagelliforme cells, under mixotrophic culture they could grow by using glucose. The addition of glucose changed the response of N.flagelliforme cells to light. The maximal photosynthetic rate, dark respiration rate and light compensation point in mixotrophic culture were higher than those in photoautotrophic cultures. These results suggest that photoautotrophic (photosynthesis) and heterotrophic (oxidative metabolism of glucose) growth interact in mixotrophic growth of N. flagelliforme cells.

Growth-associated biosynthesis of astaxanthin in heterotrophic Chlorella zofingiensis (Chlorophyta)

The green microalga Chlorella zofingiensis can produce the ketocarotenoid astaxanthin under heterotrophic culture conditions. Here we report the growth-associated biosynthesis of astaxanthin in this biotechnologically important alga. With glucose as sole carbon and energy source, C. zofinginesis grew fast in the dark with rapid exhaustion of nitrogen and carbon sources from media, leading to a high specific growth rate (0.034 h(-1)). Cultures started at a cell concentration of about 3.4 x 10(9) cells l(-1) reached, after 6 days, standing biomass values of 1.6 x 10(11) cells or 8.5 g dry weight l(-1). Surprisingly, the biosynthesis of astaxanthin was found to start at early exponential phase, independent of cessation of cell division. A general trend was observed that the culture conditions benefiting cell growth also benefited astaxanthin accumulation, indicating that astaxanthin was a growth-associated product in this alga. The maximum cell dry biomass and astaxanthin yield were 11.75 g l(-1) and 11.14 mg l(-1) (about 1 mg g(-1)), simultaneously obtained in the fed-batch culture with a combined glucose-nitrate mixture addition, which were the highest ever reported in dark-heterotrophic algal cultures. The possible reasons why dark-heterotrophic C. zofingiensis could produce astaxanthin during the course of cell growth were discussed.

Optimization of heterotrophic culture conditions for n-alkane utilization and phylogenetic position based on the 18S rDNA sequence of a thermotolerant Prototheca zopfii strain

This study reports on the optimization of the culture conditions for a thermotolerant eukaryotic algal strain, Prototheca zopfii RND16, which can effectively degrade and assimilate n-alkanes at elevated temperatures. RND16 was able to grow on 1% (v/v) n-alkanes (C 14–C 17) at temperatures up to 38°C. This ability differs from a previous finding that P. zopfii did not grow on hydrocarbons under temperature conditions above 25°C. Increasing the temperature from 25°C to 30–35°C resulted in an increase in the rate of n-alkanes consumption during growth of RND16 in quiescent culture. In shaking culture at 35°C, RND16 removed a 1% n-alkanes mixture almost completely at the basal medium salinity within 8 d. However, an appreciable decrease in the extent of hydrocarbon utilization was observed with increasing salinity and substrate concentration in the medium. The slow consumption of the n-alkanes in the early stage of degradation at 25°C was improved by supplementing 0.1% glucose. A comparative study on the nuclear small subunit rDNA (18S rDNA) sequences of three strains within the genus Prototheca revealed that both P. zopfii ATCC 30253, which utilize n-alkanes at room temperature and P. moriformis ATCC 50081, which does not assimilate n-alkanes, are closely related to RND16.

Optimization of Heterotrophic Culture Conditions for n-Alkane Utilization and Phylogenetic Position Based on the 18S rDNA Sequence of a Thermotolerant Prototheca zopfii Strain

This study reports on the optimization of the culture conditions for a thermotolerant eukaryotic algal strain, Prototheca zopfii RND16, which can effectively degrade and assimilate n-alkanes at elevated temperatures. RND16 was able to grow on 1% (v/v) n-alkanes (C14-C17) at temperatures up to 38 degrees C. This ability differs from a previous finding that P. zopfii did not grow on hydrocarbons under temperature conditions above 25 degrees C. Increasing the temperature from 25 degrees C to 30-35 degrees C resulted in an increase in the rate of n-alkanes consumption during growth of RND16 in quiescent culture. In shaking culture at 35 degrees C, RND16 removed a 1% n-alkanes mixture almost completely at the basal medium salinity within 8 d. However, an appreciable decrease in the extent of hydrocarbon utilization was observed with increasing salinity and substrate concentration in the medium. The slow consumption of the n-alkanes in the early stage of degradation at 25 degrees C was improved by supplementing 0.1% glucose. A comparative study on the nuclear small subunit rDNA (18S rDNA) sequences of three strains within the genus Prototheca revealed that both P. zopfii ATCC 30253, which utilize n-alkanes at room temperature and P. moriformis ATCC 50081, which does not assimilate n-alkanes, are closely related to RND16.

High photosynthetic photon flux and high CO2 concentration under increased number of air exchanges promote growth and photosynthesis of four kinds of orchid plantlets in vitro

In vitro plantlets of Phalaenopsis ‘Happy Valentine’, Neofinetia falcate Hu, Cymbidium kanran Makino, and Cymbidium goeringii Reichb. f. were grown under photoautotrophic [high photosynthetic photon flux (PPF), high CO2 concentration, and increased number of air exchanges] and heterotrophic (low PPF, low CO2 concentration, no air exchanges) culture conditions. After 40 d of culture, a significant difference in plantlet growth was observed between the two cultures. Total fresh and dry mass were on average 1.5 times greater in photoautotrophic culture than in heterotrophic culture. Higher net photosynthetic rates were also observed for Phalaenopsis in photoautotrophic culture. In photoautotrophic culture, little difference was observed in air temperature between the inside and outside of the culture vessel, whereas in heterotrophic culture, air temperature inside the culture vessel was 1–2°C higher than that outside the culture vessel. Relative humidity inside the culture vessel was remarkably different between the two cultures: 83–85% in photoautotrophic culture and 97–99% in heterotrophic culture. These results indicated that growth and net photosynthetic rate of in vitro orchid plantlets were susceptible to the culture environments such as PPF, CO2 concentration, relative humidity (RH), and the number of air exchanges, which would allow a more efficient micropropagation system for these orchid plants.

Screening of diatoms for heterotrophic eicosapentaenoic acid production

Nine strains of diatoms (representing four species) were screened for their ability to produce eicosapentaenoic acid (EPA) when cultured heterotrophically on glucose. Four strains were able to produce EPA heterotrophically using glucose as its carbon and energy source. Of the four,Nitzschia laevis was the best EPA producer, yielding 0.017 g g−1 dry cell weight.N. laevis was the only species tested which synthesised more EPA heterotrophically than photosynthetically. This study shows thatN. laevis is a potential source of EPA production using heterotrophic culture conditions with glucose as the carbon and energy substrate.

Tolerance of Cultured Amaranthus retroflexus Cells to Atrazine

Resistance to s-triazine-herbicides in weeds is the most widespread and extensively studied of all intraspecific herbicide-resistance. It is of interest that the resistant biotype appears in some limited genera such as Amaranthus spp. and Chenopodium spp. much more frequently than in many other significant weeds. We examined the response of cultured Amaranthus retroflexus cells to atrazine in comparison with those of several other plant species to understand what causes this differentially inter-specific response. Atrazine scarcely inhibited the cell growth of either atrazine-resistant and susceptible-Amaranthus cells. Tobacco cells, however, could not grow as cultured Amaranthus cells in high concentrations of atrazine even under heterotrophic culture conditions. Atrazine-resistant tobacco cells were also sensitive to high concentrations of atrazine. The inhibition of cell growth by this secondary effect of atrazine was also observed in cultured wheat and rice cells. Atrazine-sensitive Chenopodium cells are relatively more resistant to high concentrations of atrazine. The importance of potential tolerance to the secondary effects of atrazine is discussed with respect to the frequent occurrence of triazine- resistant biotypes in limited plant species.

Photosynthetic growth of tobacco cells in liquid suspension

Two clones of Nicotiana tabacum in liquid suspension culture were grown photosynthetically using air plus 2% CO2 as carbon source, and agitation. In general, the transfer from heterotrophic to autotrophic growth in these cells was accompanied by an increase in the concentration of photosynthetic pigments, an increase in the N, C, and H contents and in the N:C ratio, an increase in the photosynthetic oxygen evolution capacity, and a decrease in the respiratory rate. The limiting factors are discussed.Several clones were also grown photosynthetically on agar slants and the development of their photosynthetic pigments studied. Results showed that one cannot accurately predict the photosynthetic capability of tobacco cells from their appearance in heterotrophic culture conditions.