Isolation of natural red colorants from fermented broth using ionic liquid-based aqueous two-phase systems

There is a growing demand for natural colorants. This is prompting the search for new alternative and “benign” separation systems allowing higher recoveries, extraction yields, and selectivities. This work investigates the use of aqueous two-phase systems (ATPS) based on ionic liquids as extraction processes for the recovery of red colorants from the fermented broth of Penicillium purpurogenum DPUA 1275. Several ATPS based on quaternary ammonium and imidazolium were studied in this work aiming at separating the red colorants produced from the remaining colorants and contaminant proteins present in the fermented broth. The results suggest that the red colorants can be isolated by an appropriate manipulation of some of the process conditions, such as the use of quaternary ammonium with short alkyl chains, alkaline media, and short tie-line lengths (extraction point systems with lower concentrations of ionic liquid). These conditions allow large partition coefficients for the red colorants (K _red = 24.4 ± 2.3), high protein removal (60.7 ± 2.8 %) and selectivity parameters (S _red/prot = 10.05).     

Extraction of natural red colorants from the fermented broth of Penicillium purpurogenum using aqueous two‐phase polymer systems

Safety concerns related to the increasing and widespread application of synthetic coloring agents have increased the demand for natural colorants. Fungi have been employed in the production of novel and safer colorants. In order to obtain the colorants from fermented broth, suitable extraction systems must be developed. Aqueous two‐phase polymer systems (ATPPS) offer a favorable chemical environment and provide a promising alternative for extracting and solubilizing these molecules. The aim of this study was to investigate the partitioning of red colorants from the fermented broth of Penicillium purpurogenum using an ATPPS composed of poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA). Red colorants partitioned preferentially to the top (PEG‐rich phase). In systems composed of PEG 6,000 g/mol/NaPA 8,000 g/mol, optimum colorant partition coefficient (K_C) was obtained in the presence of NaCl 0.1 M (K_C = 10.30) while the PEG 10,000 g/mol/NaPA 8,000 g/mol system in the presence of Na₂SO₄ 0.5 M showed the highest K_C (14.78). For both polymers, the mass balance (%MB) and yield in the PEG phase (%η_TOP) were close to 100 and 79%, respectively. The protein selectivity in all conditions evaluated ranged from 2.0–3.0, which shows a suitable separation of the red colorants and proteins present in the fermented broth. The results suggest that the partitioning of the red colorants is dependent on both the PEG molecular size and salt type. Furthermore, the results obtained support the potential application of ATPPS as the first step of a purification process to recover colorants from fermented broth of microorganisms. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1295–1304, 2015     

Medium pH, carbon and nitrogen concentrations modulate the phosphate solubilization efficiency of Penicillium purpurogenum through organic acid production

Aims: To study phosphate solubilization in Penicillium purpurogenum as function of medium pH, and carbon and nitrogen concentrations. Methods and Results: Tricalcium phosphate (CP) solubilization efficiency of P. purpurogenum was evaluated at acid or alkaline pH using different C and N sources. Glucose‐ and (NH₄)₂SO₄‐based media showed the highest P solubilization values followed by fructose. P. purpurogenum solubilizing ability was higher in cultures grown at pH 6·5 than cultures at pH 8·5. Organic acids were detected in both alkaline and neutral media, but the relative percentages of each organic acid differed. Highest P release coincided with the highest organic acids production peak, especially gluconic acid. When P. purpurogenum grew in alkaline media, the nature and concentration of organic acids changed at different N and C concentrations. A factorial categorical experimental design showed that the highest P‐solubilizing activity, coinciding with the highest organic acid production, corresponded to the highest C concentration and lowest N concentration. Conclusions: The results described in the present study show that medium pH and carbon and nitrogen concentrations modulate the P solubilization efficiency of P. purpurogenum through the production of organic acids and particularly that of gluconic acid. In the P solubilization optimization studies, glucose and (NH₄)₂SO₄ as C and N sources allowed a higher solubilization efficiency at high pH. Significance and Impact of the Study: This organism is a potentially proficient soil inoculant, especially in P‐poor alkaline soils where other P solubilizers fail to release soluble P. Further work is necessary to elucidate whether these results can be extrapolated to natural soil ecosystems, where different pH values are present. Penicillium purpurogenum could be used to develop a bioprocess for the manufacture of phosphatic fertilizer with phosphate calcium minerals.     

Sequencing and phylogenetic analyses of talaromyces amestolkiae from amazon: A producer of natural colorants

The population interest in health products is increasing day-by-day. Thus, the demand for natural products to be added in food and pharmaceutical commodity is also rising. Among these additives, colorants, which provides color to products, can be produced by microorganism through bioprocess. Looking for new source of natural colorants, fungi have been employed to this purpose producing novel and safer natural colorants. So, the main goal of this study was to describe a Talaromyces species able to produce natural colorants and investigate nutritional parameters of colorants production using statistical tool. The taxonomy classified the microorganism as Talaromyces amestolkiae. The statistical design evaluated pH and glucose, meat extract and meat peptone concentration as independent variables, and red colorants production as main response. Under the best condition (g/L: glucose 30, meat extract 1, meat peptone 10, and initial pH of 7.0) an increase of 229% in the red colorant production was achieved as compared with the initial media used. The dried fermented broth containing red colorants showed low cytotoxicity against fibroblasts cells (IC₅₀ > 187.5 g/L) and effective antimicrobial activity against S. aureus (MIC of 2.5 g/L). Thus, T. amestolkiae colorants can be attractive to food and pharmaceutical applications as it does not produce toxic compounds and can promote protection against microorganism contaminants. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2684, 2019     

Production and characterization of thermostable alkaline phytase from <Emphasis Type="Italic">Bacillus laevolacticus</Emphasis> isolated from rhizosphere soil

A novel phytase producing thermophilic strain of Bacillus laevolacticus insensitive to inorganic phosphate was isolated from the rhizosphere soil of leguminous plant methi (Medicago falacata). The culture conditions for production of phytase by B. laevolacticus under shake flask culture were optimized to obtain high levels of phytase (2.957 ± 0.002 U/ml). The partially purified phytase from B. laevolacticus strain was optimally active at 70 °C and between pH 7.0 and pH 8.0. The enzyme exhibited thermostability with ∼80% activity at 70 °C and pH 8.0 for up to 3 h in the presence/absence of 5 mM CaCl₂. The phytase from B. laevolacticus showed high specificity for phytate salts of Ca⁺ > Na⁺. The enzyme showed an apparent K _m 0.526 mM and V _max 12.3 μmole/min/mg of activity against sodium phytate.     

Formation of radicals during heating lysine and glucose in solution with an intermediate water activity

Abstract

Heating glucose with lysine under alkaline conditions (pH 7.0–10.0) was found to take place with consumption of oxygen together with formation of brown-colored compounds. Highly reactive intermediary radicals were detected when lysine and glucose were heated at intermediate water activity at pH 7.0 and 8.0. The detection was based on initial trapping of highly reactive radicals by ethanol followed by spin trapping of 1-hydroxyethylradicals with α-(4-pyridyl N-oxide)-N-tert-butylnitrone (POBN) and Electron Spin Resonance (ESR) spectroscopy. The generation of reactive intermediary radicals from the Maillard reactions was favored by enhancing alkaline conditions (pH 8.0) and stimulated by presence of the transition metal ion Fe²⁺. The stability of the nitrone spin traps, N-tert-butyl-α-phenylnitrone and POBN was examined in buffered aqueous solutions within the pH range 1–12, and found to be less temperature dependent at acidic pH compared to alkaline conditions. A low rate (k_obs) of hydrolysis of POBN was found at the used experimental conditions of 70°C and pH 7.0 and 8.0, which made this spin trap method suitable for the detection of radicals in the Maillard reaction system.     

Optimization of cultural conditions for the production of antifungal chitinase by Streptomyces sporovirgulis

Actinomycetes were screened from soil in the centre of Poland on chitin medium. Amongst 30 isolated strains one with high activity of chitinase was selected. It was identified as Streptomyces sporovirgulis. Chitinase activity was detected from the second day of cultivation, then increased gradually and reached maximum after 4 days. The maximum chitinase production was observed at pH 8.0 and 25–30°C in the medium with sodium caseinate and asparagine as carbon and nitrogen sources and with shrimp shell waste as inducer of enzyme. Chitinase of S. sporovirgulis was purified from a culture medium by fractionation with ammonium sulphate as well as by chitin affinity chromatography. The molecular weight of the enzyme was 27 kDa. The optimum temperature and pH for the chitinase were 40°C and pH 8.0. The enzyme activity was characterised by high stability at the temperatures between 35 and 40°C after 240 min of preincubation. The activity of the enzyme was strongly inhibited in the presence of Pb²⁺, Hg²⁺ and stabilized by the ions Mg²⁺. Purified chitinase from S. sporovirgulis inhibited growth of fungal phytopathogen Alternaria alternata. Additionally, the crude chitinase inhibited the growth of potential phytopathogens such as Penicillium purpurogenum and Penillium sp.     

Characterization of β-glucosidase from a strain of Penicillium purpurogenum KJS506

A novel β-glucosidase (BGL)-producing strain was isolated and identified as Penicillium purpurogenum KJS506 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. When rice straw and corn steep powder were used as carbon and nitrogen sources, respectively, the maximal BGL activity of 12.3 U ml⁻¹, one of the highest levels among BGL-producing microorganisms was observed. The optimum temperature and pH for BGL production were 32 °C and 4, respectively. An extracellular BGL was purified to homogeneity by sequential chromatography of P. purpurogenum culture supernatants, and the purified BGL showed higher activity (V _max = 934 U mg protein^–1) than most BGLs from other sources. The complete ORF of bgl3 was cloned from P. purpurogenum by a modified thermal asymmetric interlaced polymerase chain reaction. The bgl3 gene consists of a 2,571-bp ORF and encodes a putative protein containing 856 amino acids with a calculated molecular mass of 89,624 Da. The putative gene product was identified as a member of glycoside hydrolase family 3. The present results should contribute to improved industrial production of BGL by P. purpurogenum KJS506.     

The effect of pH on hydrogen production with Citrobacter intermedius

Summary Citrobacter intermedius was grown in a 14-liter fermenter under batch anaerobic conditions at the following controlled pH values: 5, 5.75, 6.0, 6.5, 7.0, 7.5, and 8.0. The growth medium was a glucose mineral salts medium with 0.1% ammonium sulfate as the source of sulfur. The optimum pH for H₂ production was 5.75 and 6.0 which gave a yield of 1.1 moles H₂/mole glucose. The optimum H₂-productivity was 144 moles H₂ per hour at pH 6.0.     

Characterization of a thermostable and solvent-tolerant laccase produced by Streptomyces sp. LAO

Objectives

Decaying wood samples were collected, and actinomycetes were isolated and screened for laccase production. The identity of the efficient laccase-producing isolate was confirmed by using a molecular approach. Fermentation conditions for laccase production were optimized, and laccase biochemical properties were studied.

Results

Based on the 16S rRNA gene sequencing and phylogenetic analysis, the isolate coded as HWP₃ was identified as Streptomyces sp. LAO. The time-course study showed that the isolate optimally produced laccase at 84 h with 40.58?±?2.35 U/mL activity. The optimized physicochemical conditions consisted of pH 5.0, ferulic acid (0.04%; v/v), pine back (0.2 g/L), urea (1.0 g/L), and lactose (1 g/L). Streptomyces sp. LAO laccase was optimally active at pH and temperature of 8.0 and 90 °C, respectively, with remarkable pH and thermal stability. Furthermore, the enzyme had a sufficient tolerance for organic solvents after 16 h of preincubation, with laccase activity?>?70%. Additionally, the laccase maintained considerable residual activity after pretreatment with 100 mM of chemical agents, including sodium dodecyl sulphate (69.93?±?0.89%), ethylenediaminetetraacetic acid (93.1?±?7.85%), NaN₃ (96.28?±?3.34%) and urea (106.03?±?10.72%).

Conclusion

The laccase's pH and thermal stability; and robust catalytic efficiency in the presence of organic solvents suggest its industrial and biotechnological application potentials for the sustainable development of green chemistry.

     

Production and characterization of keratinase from<Emphasis Type="Italic">Paracoccus</Emphasis> sp. WJ-98

A bacterial strain WJ-98 found to produce active extracellular keratinase was isolated from the soil of a poultry factory. It was identified asParacoccus sp. based on its 16S rRNA sequence analysis, morphological and physiological characteristics. The optimal culture conditions for the production of keratinase byParacoccus sp. WJ-98 were investigated. The optimal medium composition for keratinase production was determined to be 1.0% keratin, 0.05% urea and NaCl, 0.03% K₂HPO₄, 0.04% KH₂PO₄, and 0.01% MgCl₂·6H₂O. Optimal initial pH and temperature for the production of keratinase were 7.5 and 37°C, respectively. The maximum keratinase production of 90 U/mL was reached after 84 h of cultivation under the optimal culturing conditions. The keratinase fromParacoccus sp. WJ-98 was partially purified from a culture broth by using ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, followed by gel filtration chromatography on Sephadex G-75. Optimum pH and temperature for the enzyme reaction were pH 6.8 and 50°C, respectively and the enzymes were stable in the pH range from 6.0 to 8.0 and below 50°C. The enzyme activity was significantly inhibited by EDTA, Zn²⁺ and Hg²⁺. Inquiry into the characteristics of keratinase production from these bacteria may yield useful agricultural feed processing applications.     

A reduction in temperature induces bioactive red pigment production in a psychrotolerant Penicillium sp. GEU_37 isolated from Himalayan soil

Filamentous fungi are being globally explored for the production of industrially important bioactive compounds including pigments. In the present study, a cold and pH tolerant fungus strain Penicillium sp (GEU_37), isolated from the soil of Indian Himalaya, is characterized for the production of natural pigments as influenced by varying temperature conditions. The fungal strain produces a higher sporulation, exudation, and red diffusible pigment in Potato Dextrose (PD) at 15 °C as compared to 25 °C. In PD broth, a yellow pigment was observed at 25 °C. While measuring the effect of temperature and pH on red pigment production by GEU_37, 15 °C and pH 5, respectively, were observed to be the optimum conditions. Similarly, the effect of exogenous carbon and nitrogen sources and mineral salts on pigment production by GEU_37 was assessed in PD broth. However, no significant enhancement in pigmentation was observed. Chloroform extracted pigment was separated using thin layer chromatography (TLC) and column chromatography. The two separated fractions i.e., fractions I and II with Rf values 0.82 and 0.73, exhibited maximum light absorption, λ_max, at 360 nm and 510 nm, respectively. Characterization of pigments using GC–MS showed the presence of the compounds such as phenol, 2,4-bis (1,1-dimethylethyl) and eicosene from fraction I and derivatives of coumarine, friedooleanan, and stigmasterole in fraction II. However, LC-MS analysis detected the presence of derivatives of compound carotenoids from fraction II as well as derivative of chromenone and hydroxyquinoline as major compounds from both the fractions along with other numerous important bioactive compounds. The production of such bioactive pigments under low temperature conditions suggest their strategic role in ecological resilience by the fungal strain and may have biotechnological applications.     

Properties of Geobacillus stearothermophilus levansucrase as potential biocatalyst for the synthesis of levan and fructooligosaccharides

The production of levansucrase (LS) by thermophilic Geobacillus stearothermophilus was investigated. LS production was more effective in the presence of sucrose (1%, w/v) than fructose, glucose, glycerol or raffinose. The results (T_op 57°C; stable for 6 h at 47°C) indicate the high stability of the transfructosylation activity of G. stearothermophilus LS as compared with LSs from other microbial sources. Contrary to temperature, the pH had a significant effect on the selectivity of G. stearothermophilus LS‐catalyzed reaction, favoring the transfructosylation reaction in the pH range of 6.0–6.5. The kinetic parameter study revealed that the catalytic efficiency of transfructosylation activity was higher as compared with the hydrolytic one. In addition to levan, G. stearothermophilus LS synthesized fructooligosaccharides in the presence of sucrose as the sole substrate. The results also demonstrated the wide acceptor specificity of G. stearothermophilus LS with maltose being the best fructosyl acceptor. This study is the first on the catalytic properties and the acceptor specificity of LS from G. stearothermophilus. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1405–1415, 2013     

Microbial Production of Glucose Oxidase

Productivity of extracellular glucose oxidase was examined for various microorganisms and it was found in strains belonging to genus Penicillium except one species of Tallalomyces.

As the best glucose oxidase producer, Penicillium purpurogenum No. 778 was isolated from natural source. This microorganism produced 32,000 units per ml broth of glucose oxidase in a simple medium containing beet molasses, NaNO³ and KH²PO⁴ by submerged culture for 3 days. That value was about 10-times of that of Penicillium amagasakiense which has been known as an excellent glucose oxidase producer.

Culture conditions for glucose oxidase production were examined, which were extremely different among microbial species. In the case of Penicillium chrysogenum AJ 7007 and Penicillium purpurogenum No. 778, the effects of aeration and carbon sources were remarkably different from each other.

Penicillium purpurogenum No. 778 produces catalase sufficiently in a culture broth for glucose oxidase application in food industry.

Glucose oxidase was purified about 25-fold from culture supernatants of Penicillium purpurogenum No. 778, and some properties of the enzyme were examined. The optimum temperature and pH for the activity were 35°C and 5.0, respectively. The enzyme was stable at pH 5.0 to 7.0 when it was incubated at 40°C for 2 hr, while it was stable at temperature lower than 50°C when incubated at pH 5.6 for 15 min. The enzyme was specific for d-glucose and apparent Michaelis constant for d-glucose was 12.5 mm. The enzyme was inhibited by 1 mm of HgCl², CuSO⁴, NaHSO⁴ and phenylhydrazine, but not inhibited by 1 mm of p-hydroxy-mercuribenzoate, EDTA, hydroxylamine and dimedone. Four percents NaCl inhibited the activity about 50%, while the addition of ethanol (from 0 to 16%) increased oxygen uptake more than that expected from the peroxidase activity of catalase.     

Effect of pH on growth and ethanol production byZymomonas

Summary In a mineral salts medium containing yeast extract, NH₄Cl and glucose (50g/L), the pH range producing the fastest growth ofZ. mobilis was 5.5–6.5 with an apparent optimum at 6.5. At constant growth rate of 0.15hr^–1, the specific rates of glucose utilization (q_s) and ethanol production (q_p) were relatively unaffected by pH over the range 7.0–5.5 but increased sharply as the pH was further decreased below 5.5 to 4.0. Under these conditions the ethanol yield was unaffected by pH over the range 4.0–6.5 but decreased markedly at pH of 7.     

Production and Properties of Alkaline Dextranase from a Newly Isolated Brevibacterium

About 500 strains of dextranase producing microorganisms were examined in detail for pH- activity and enzyme stability. A gram positive bacterium identified as belonging to the genus Brevibacterium was found to produce alkaline dextranase. Maximal dextranase synthesis was obtained when grown aerobically at 26°C for 3 days in a medium containing 1 % dextran, 2% ethanol, 1 % polypeptone and 0.05 % yeast extract together with trace amounts of inorganic salts.

Brevibacterium dextranase had an optimum pH of 8.0 for activity at 37°C and an optimal temperature at 53°C at pH 7.5. The enzyme was quite stable over the range of pH 5.0 to 10.5 on 24 hr incubation at 37°C, especially on alkaline pH. The enzyme was also heat stable at 60°C for 10 min.     

Production,purification, characterization and usage of a detergent additive of endoglucanase from isolated halotolerant Amycolatopsis cihanbeyliensis mutated strain Mut43

The Amycolatopsis cihanbeyliensis Mut43, which is obtained by UV radiation, exhibited endoglucanase activity of 5.21?U/mL, which was ～2.3-fold higher than that of the wild strain (2.04?U/mL). The highest enzyme activity was obtained after 3 days of incubation at 32?°C, pH 7.0, 150?rpm, and 6% NaCl in a liquid medium containing 1.5% (w/v) wheat straw (0.25?mm of particle size) and 0.6% (w/v) yeast extract. Enzyme activity was eluted as a single peak (gel filtration chromatography), and Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of the corresponding peak revealed a molar mass of 30?kDa. Zymogram analysis confirmed the presence of a single active endoglucanase component. The enzyme was purified to ～21-fold, and the mean overall yield was ～6%. The purified endoglucanase was active up to 80?°C and showed a half-life of 214?min at 60?°C in the absence of substrate at pH 8.0. The apparent K_m value for the purified endoglucanase was 0.70?mg/mL, while the V_max value was 6.20 Units/μg. Endoglucanase activity was reduced (25%) by treatment with 30?U of proteinase K/mg. The addition of Mg⁺² and Ca⁺² (5?mM) enhanced endoglucanase activity. Additionally, endoglucanase activity in the presence of 5?mM SDS or organic solvents was 75 and 50% of maximum activity, respectively. The high levels of enzyme production from A. cihanbeyliensis Mut43 achieved under batch conditions, coupled with the temperature stability, activity over a broad pH range, relatively high stability (70–80%) in the presence of industrial laundry detergents and storage half-lives of 45 days at +4?°C and 75 days at ?20?°C signify the suitability of this enzyme for industrial applications as detergent additive.     

Comparison of β-glucosidase activities in different Debaryomyces strains

Summary β-Glucosidase production by Debaryomyces vanrigii and Debaryomyces hansenii was studied using two media. Cellobiose was found to stimulate the biosynthesis of the enzyme, while NH₄NO₃ (1.0 g/l) and NH₄Cl (1.26 g/l) were the best nitrogen sources for D. hansenii and D. vanrigii respectively. Optimal conditions for enzyme activity were established in relation to pH, temperature and enzyme stability. Thermal and pH stability studies show that β-glucosidase from D. vanrigii was more stable at pH 4.5–5.0 at 50°C, while that enzyme from D. hansenii was stable at pH 6.5 at 35°C. This feature may be advantageous in the commercial application by hydrolysing cellobiose, the potent inhibitor of cellulose solubilizing enzymes.     

Production and characterization of a collagenolytic serine proteinase by <Emphasis Type="Italic">Penicillium aurantiogriseum</Emphasis> URM 4622: A factorial study

A 2⁴ full factorial design was used to identify the main effects and interactions of the initial medium pH, soybean flour concentration, temperature and orbital agitation speed on extracellular collagenase production by Penicillium aurantiogriseum URM4622. The most significant variables for collagenase production were soybean flour concentration and initial medium pH that had positive main effects, and temperature that had a negative one. Protein concentration in soybean flour revealed to be a significant factor for the production of a collagenase serine proteinase. The most favorable production conditions were found to be 0.75% soybean flour, pH 8.0, 200 rpm, and 28°C, which led to a collagenase activity of 164 U. The enzyme showed an optimum activity at 37°C and pH 9.0, was stable over wide ranges of pH and temperature (6.0 ∼ 10.0 and 25 ∼ 45°C, respectively) and was strongly inhibited by 10 mM phenylmethylsulphonylfluoride. The firstorder rate constants for collagenase inactivation in the crude extract, calculated from semi-log plots of the residual activity versus time, were used in Arrhenius and Eyring plots to estimate the main thermodynamic parameters of thermoinactivation (E* _d = 107.4 kJ/mol and ΔH* _d = 104.7 kJ/mol). The enzyme is probably an extracellular neutral serine collagenase effective on azocoll, gelatin and collagen decomposition.     

Effect of salinity and pH on growth and agar yield of Gracilaria tenuistipitata var. liui in laboratory and outdoor cultivation

Acclimation responses of the red alga Gracilaria tenuistipitata var. liui collected on the northwest coast of Philippines were determined in laboratory setups and outdoor cultivation tanks in Haifa, Israel. Growth under laboratory conditions was influenced by all three variables studied, namely, temperature (20 or 30 ^°C), salinity (20, 30 or39‰) and seawater pH (6.5, 7.0, 8.0 or ≥ 9.0). In 250 mL flasks lacking pH control growth was influenced by temperature only at 20 ‰, whereas at 39 ‰, growth rates were similar at 20 or 30 ^°C. In 500 mL cylinders in which pH was controlled, growth rates were significantly different at a pH of 6.5 and 7.0 for all salinities, with maximal rates occurring in 39 ‰. At pH 8.0, and above, growth rates between salinities were similar and reduced to approximately 50% at a pH of 9.0 compared to rates at a pH of 6.5. Photosynthesis responses generally resembled growth responses both, in 250 mL and 500 mL cultures. In 40-L outdoor tanks, weekly growth and agar yields were apparently enhanced by increasing light intensities (up to full sunlight) and nutrient concentrations (up to 0.2 mM PO₃ ^2- and 2.0 mM NH₄ ⁺), and rates averaged four times higher than rates determined in the smaller flask cultures. This study shows broad salinity tolerance of G. tenuistipitata var. liui and its ability to sustain growth rates that are among the highest measured for Gracilaria spp. in outdoor cultures. This revised version was published online in August 2006 with corrections to the Cover Date.