Effect of nitrate feeding strategies on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana

Controlled nitrate feeding strategies for fed-batch cultures of microalgae were applied for the enhancement of lipid production and microalgal growth rates. In particular, in this study, the effect of nitrate feeding rates on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana were investigated using three feeding modes (i.e., pulse, continuous, and staged) and under two light variations on both lipid productivity and fatty acid compositions. Higher nitrate levels negatively affected lipid production in the study. Increasing the light intensity increased the lipid contents of the microalgae in all three fed-batch feeding modes. A maximum of 58.3% lipid- to dry weight ratio was achieved when using pulse-fed cultures at an illumination of 200 μmol photons m⁻² s⁻¹ and 10 mg/day of nitrate feeding. This condition also resulted in the maximum lipid productivity of 44.6 mg L⁻¹ day⁻¹. The fatty acid compositions of the lipids consisted predominantly of long-chain fatty acids (C:16 and C:18) and accounted for 70% of the overall fatty acid methyl esters. Pulse feeding mode was found to significantly enhance the biomass and lipid production. The other two feeding modes (continuous and staged) were not ideal for lipid and biomass production. This study demonstrates the applicability of pulse feeding strategies in fed-batch cultures as an appropriate cultivation strategy that can increase both lipid accumulation and biomass production.     

Differential responses in EPA and fucoxanthin production by the marine diatom Stauroneis sp. under varying cultivation conditions

There has been an increasing drive toward better valorising raw biological materials in the context of the sustainability of bio-based industries and the circular economy. As such, microalgae hold the ability to biosynthesise valuable metabolites, which are sought after within the bioenergy, pharmaceuticals, cosmetics or nutrition sectors. Owing to their bioactivities, the xanthophyll pigment fucoxanthin and the omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) have fostered increasing interests in terms of sustainably refining them from natural sources, such as microalgae. Together with the suitability of individual species to industrial cultivation, a key challenge resides in optimizing the yields of these compounds within the microalgal biomass they are retrieved from. The marine diatom Stauroneis sp. LACW24 was batch cultivated into its stationary phase of growth prior to being subjected at high cell density (1 × 10⁶ cells mL⁻¹) to seven different regimes of light exposure in replenished medium and under nutritional limitation (silica and nitrate) for 12 days. The highest EPA proportions and yields were obtained under blue LED in f/2 medium (16.5% and 4.8 mg g⁻¹, respectively), double the values obtained under red LED illumination. The fucoxanthin yield was the highest when cells were subjected to blue LEDs (5.9 mg g⁻¹), a fourfold increase compared to the nitrogen-limited treatment under white LEDs. These results indicate that a two-stage approach to the batch cultivation of this diatom can be used for enhancing the production of the high-value metabolites fucoxanthin and EPA post-stationary phase.     

Effect of nitrate on lipid production by T. suecica, M. contortum, and C. minutissima

Microalgae are an alternative and sustainable source of lipids that can be used as a feedstock for biodiesel production. Nitrate is a good nitrogen source for many microalgae and affects biomass and lipid yields of microalgae. In this study, the effect of nitrate on cell growth and lipid production and composition in Monoraphidium contortum, Tetraselmis suecica, and Chlorella minutissima was investigated. Nitrate affected the production of biomass and the production and composition of lipids of the three microalgae tested. Increasing the nitrate concentration in the culture medium resulted in increased biomass production and higher biomass productivity. Furthermore, increasing the nitrate concentration resulted in a reduction in lipid content and productivity in M. contortum; however, the opposite effect was observed in T. suecica and C. minutissima cultures. C. minutissima and M. contortum lipids contain high levels of oleic acid, with values ranging from 26 to 45.7% and 36.4 to 40.1%, respectively. The data suggest that because of its high lipid productivity (13.79 mg L^?1 d^?1) and high oleic acid productivity (3.78 mg L^?1 d^?1), Chlorella minutissima is a potential candidate for the production of high quality biodiesel.     

Harvesting <Emphasis Type="Italic">Chlorella minutissima</Emphasis> using cell coagulants

Twelve salts were tested for their ability to coagulate microalgae cells in cultures of Chlorella minutissima. The final aim was to develop an easy and efficient approach for harvesting microalgae biomass in dense cultures. Aluminum, ferric, and zinc salts coagulated C. minutissima cultures, while optimum concentration was 0.75 and 0.5 g L⁻¹ for sulfate and chloride salts, respectively. Aluminum salts were most efficient, but caused some cell lysis, which may render this approach inappropriate in some cases. Ferric and zinc salts were ranked second and third, respectively, according to their culture cell-coagulation efficiency. Ferric salts caused a change in the color of the cells, mainly at concentrations higher than 1 g L⁻¹. Zinc salts were less harmful for the microalgal cells, but an additional problem was observed with cell aggregates adhering to the walls of the glass test tubes. Selection of the appropriate coagulant is related to the purpose of the coagulation process.     

Optimization of pH induced flocculation of marine and freshwater microalgae via central composite design

Microalgae harvesting via pH induced flocculation along with utilization of recovered medium after flocculation is one of the most economical methods for separating the microalgal biomass in order to reduce the dewatering cost. In this study, optimization of marine and freshwater microalgae flocculation by pH adjustment was investigated via central composite design methodology. One molar of KOH and NaOH solutions were used to increase the pH level of the microalgal culture. Increasing pH value of the medium provided the highest flocculation efficiency up to 92.63 and 86.18% with pH adjusted to 10.5 with KOH and NaOH solutions for marine microalgae Nannochloropsis oculata and freshwater microalgae Chlorella minutissima, respectively. Also, it was revealed that microalgae cells were still alive after flocculation process and their biochemical composition was not changed, and flocculated medium can be used again for the next microalgal production. According to the results, it can be said that this method is cheap and effective, simple to operate and provides the utilization of flocculated medium again.     

Mixed microalgae culture for ammonium removal in the absence of phosphorus: Effect of phosphorus supplementation and process modeling

Microalgal growth and ammonium removal in a P-free medium have been studied in two batch photobioreactors seeded with a mixed microalgal culture and operated for 46 days. A significant amount of ammonium (106 mg NH₄-Nl⁻¹) was removed in a P-free medium, showing that microalgal growth and phosphorus uptake are independent processes. The ammonium removal rate decreased during the experiment, partly due to a decrease in the cellular phosphorus content. After a single phosphate addition in the medium of one of the reactors, intracellular phosphorus content of the corresponding microalgal culture rapidly increased, and so did the ammonium removal rate. These results show how the amount of phosphorus internally stored affects the ammonium removal rate. A mathematical model was proposed to reproduce these observations. The kinetic expression for microalgae growth includes a Monod term and a Hill's function to represent the effect of ammonium and stored polyphosphate concentrations, respectively. The proposed model accurately reproduced the experimental data (r = 0.952, P-value <0.01).     

Ability of marine eukaryotic red tide microalgae to utilize insoluble iron

Iron is an essential trace metal and a limiting factor for microalgal growth, but bioavailable dissolved iron concentrations in seawater are low. Microalgal blooms have frequently occurred in coastal areas under such iron limitation accompanied by mass mortalities of fish and bivalves. Their massive growth despite physiological iron-deficiency has long been an enigma, because most of them cannot grow in chemically defined artificial media. We developed a feasible artificial medium for the culture of many species of red tide microalgae modified for investigation of iron utilization. Here, we report on the ability of marine eukaryotic red tide microalgae to utilize insoluble iron. Some microalgal species could utilize particulate FePO₄ and FeS for growth. Particulate FePO₄ was available for the growth of the raphidophyte Heterosigma akashiwo, the dinoflagellate Heterocapsa triquetra and the diatom Ditylum brightwellii. The dinoflagellates Heterocapsa circularisquama and Karenia mikimotoi, and the cryptophyte Rhodomonas ovalis utilized both particulate FePO₄ and particulate FeS for growth. In contrast, particulate FeO(OH) and Fe₂O₃ did not support the growth of any of the red tide microalgae examined. Except for Chattonella species (Raphidophyceae), the growth of red tide microalgae were confirmed in the medium with very easily soluble FeCl₃ added. The order of bioavailability of tested iron-source species were Fe–EDTA > FeCl₃ > FePO₄ > FeS > FeO(OH), Fe₂O₃ for most of microalgae examined, although for H. circularisquama the utilization of FeCl₃ was higher than that of Fe–EDTA. The results suggest that red tide microalgae show different patterns of specific strategies for the utilization of various iron sources. The occurrence of red tides in coastal areas may depend on the combination of microalgal species and insoluble iron species present.     

Energy Demands of Nitrogen Supply in Mass Cultivation of Two Commercially Important Microalgal Species, <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> and <Emphasis Type="Italic">Dunaliella tertiolecta</Emphasis>

Mass culture of microalgae is a potential alternative to cultivation of terrestrial crops for bioenergy production. However, microalgae require nitrogen fertiliser in quantities much higher than plants, and this has important consequences for the energy balance of these systems. The effect of nitrogen fertiliser supplied to microalgal bubble-column photobioreactor cultures was investigated using different nitrogen sources (nitrate, urea, ammonium) and culture conditions (air, 12% CO₂). In 20 L cultivations, maximum biomass productivity for Chlorella vulgaris cultivated using nitrate and urea was 0.046 and 0.053 g L⁻¹ day⁻¹, respectively. Maximum biomass productivity for Dunaliella tertiolecta cultivated using nitrate, urea and ammonium was 0.033, 0.038 and 0.038 g L⁻¹ day⁻¹, respectively. In intensive bubble-column photobioreactors using 12% CO₂, maximum productivity reached 0.60 and 0.83 g L⁻¹ day⁻¹ for C. vulgaris and D. tertiolecta, respectively. Recycling of nitrogen within the photobioreactor system via algal exudation of nitrogenous compounds and bacterial activity was identified as a potentially important process. The energetic penalty incurred by supply of artificial nitrogen fertilisers, phosphorus, power and CO₂ to microalgal photobioreactors was investigated, although analysis of all energy burdens from biomass production to usable energy carriers was not conducted. After subtraction of the power, nitrogen and phosphorus energy burdens, maximum net energy ratios for C. vulgaris and D. tertiolecta cultivated in bubble columns were 1.82 and 2.10. Assuming CO₂ was also required from a manufactured source, the net energy ratio decreased to 0.09 and 0.11 for C. vulgaris and D. tertiolecta, so that biomass production in this scenario was unsustainable. Although supply of nitrogen is unlikely to be the most energetically costly factor in sparged photobioreactor designs, it is still a very significant penalty. There is a need to optimise both cultivation strategies and recycling of nitrogen in order to improve performance. Data are supported by measurements including biochemical properties (lipid, protein, heating value) and bacterial number by epifluorescence microscopy.     

Growth and biomass productivity of Scenedesmus vacuolatus on a twin layer system and a comparison with other types of cultivations

Scenedesmus is a genus of microalgae employed for several industrial uses. Industrial cultivations are performed in open ponds or in closed photobioreactors (PBRs). In the last years, a novel type of PBR based on immobilized microalgae has been developed termed porous substrate photobioreactors (PSBR) to achieve significant higher biomass density during cultivation in comparison to classical PBRs. This work presents a study of the growth of Scenedesmus vacuolatus in a Twin Layer System PSBR at different light intensities (600 μmol photons m⁻² s⁻¹ or 1000 μmol photons m⁻² s⁻¹), different types and concentrations of the nitrogen sources (nitrate or urea), and at two CO₂ levels in the gas phase (2% or 0.04% v/v). The microalgal growth was followed by monitoring the attached biomass density as dry weight, the specific growth rate and pigment accumulation. The highest productivity (29 g m⁻² d⁻¹) was observed at a light intensity of 600 μmol photons m⁻² s⁻¹ and 2% CO₂. The types and concentrations of nitrogen sources did not influence the biomass productivity. Instead, the higher light intensity of 1000 μmol photons m⁻² s⁻¹ and an ambient CO₂ concentration (0.04%) resulted in a significant decrease of productivity to 18 and 10–12 g m⁻² d⁻¹, respectively. When compared to the performance of similar cultivation systems (15–30 g m⁻² d⁻¹), these results indicate that the Twin Layer cultivation System is a competitive technique for intensified microalgal cultivation in terms of productivity and, at the same time, biomass density.

     

Evaluation of electro‐coagulation–flocculation for harvesting marine and freshwater microalgae

Although microalgae are considered as a promising feedstock for biofuels, the energy efficiency of the production process needs to be significantly improved. Due to their small size and low concentration in the culture medium, cost‐efficient harvesting of microalgae is a major challenge. In this study, the use of electro‐coagulation–flocculation (ECF) as a method for harvesting a freshwater (Chlorella vulgaris) and a marine (Phaeodactylum tricornutum) microalgal species is evaluated. ECF was shown to be more efficient using an aluminum anode than using an iron anode. Furthermore, it could be concluded that the efficiency of the ECF process can be substantially improved by reducing the initial pH and by increasing the turbulence in the microalgal suspension. Although higher current densities resulted in a more rapid flocculation of the microalgal suspension, power consumption, expressed per kg of microalgae harvested, and release of aluminum were lower when a lower current density was used. The aluminum content of the harvested microalgal biomass was less than 1% while the aluminum concentration in the process water was below 2 mg L⁻¹. Under optimal conditions, power consumption of the ECF process was around 2 kWh kg⁻¹ of microalgal biomass harvested for Chlorella vulgaris and ca. 0.3 kWh kg⁻¹ for Phaeodactylum tricornutum. Compared to centrifugation, ECF is thus more energy efficient. Because of the lower power consumption of ECF in seawater, ECF is a particularly attractive method for harvesting marine microalgae. Biotechnol. Bioeng. 2011;108: 2320–2329. © 2011 Wiley Periodicals, Inc.     

Determination of pioglitazone hydrochloride by flow injection chemiluminescence tris(2,2′-bipyridyl)ruthenium(II)–silver(III) complex system

A novel flow injection-chemiluminescence (FI–CL) approach is proposed for the assay of pioglitazone hydrochloride (PG-HCl) based on its enhancing influence on the tris(2,2′-bipyridyl)ruthenium(II)–silver(III) complex (Ru(bipy)₃²⁺-DPA) CL system in sulfuric acid medium. The possible CL reaction mechanism is discussed with CL and ultraviolet (UV) spectra. The optimum experimental conditions were found as: Ru(bipy)₃²⁺, 5.0 × 10⁻⁵ M; sulfuric acid, 1.0 × 10⁻³ M; diperiodatoargentate(III) (DPA), 1.0 × 10⁻⁴ M; potassium hydroxide, 1.0 × 10⁻³ M; flow rate 4.0 ml min⁻¹ for each flow stream and sample loop volume, 180 μl. The CL intensity of PG-HCl was linear in the range of 1.0 × 10⁻³ to 5.0 mg L⁻¹ (R² = 0.9998, n = 10) with limit of detection [LOD, signal-to-noise ratio (S/N) = 3] of 2.2 × 10⁻⁴ mg L⁻¹, limit of quantification (LOQ, S/N = 10) of 6.7 × 10⁻⁴ mg L⁻¹, relative standard deviation (RSD) of 1.0 to 3.3% and sampling rate of 106 h⁻¹. The methodology was satisfactorily used to quantify PG-HCl in pharmaceutical tablets with recoveries ranging from 93.17 to 102.77 and RSD from 1.9 to 2.8%.     

Effects of green LED light and three stresses on biomass and lipid accumulation with two-phase culture of microalgae

The effects of wavelengths of light-emitting diode (LED), nitrate concentration, and salt concentration were evaluated for the two-phase culture of the microalgal species Phaeodactylum tricornutum, Dunaliella tertiolecta, and Isochrysis galbana on cell growth and lipid production. Blue LEDs produced the highest biomass of P. tricornutum at a nitrate concentration of 8 mg/L, reaching 0.97 g dcw/L with a specific growth rate (μ) of 0.047 h⁻¹, followed by I. galbana with 0.79 g dcw/L and μ = 0.040 h⁻¹ and D. tertiolecta with 0.55 g dcw/L and μ = 0.028 h⁻¹. Of the three microalgae, P. tricornutum had the highest specific growth rate of μ_max = 0.070 h⁻¹ and lowest saturation constant of K_s = 4.18 mg/L, resulting in fast cell growth. The highest lipid production was obtained under green LED wavelength stress on day 14, reaching 60.6% (w/w) of the dry cell weight among the three microalgae. The main fatty acids produced by the three microalgae were myristic acid (C14:0), palmitic acid (C16:0), oleic acid (C18:1), and arachidic acid (C20:0), which comprised 72.68%–84.16% (w/w) of the total fatty acids content under three stresses.     

Enhanced CO2 biofixation and protein production by microalgae biofilm attached on modified surface of nickel foam

In this work, a photobioreactor with microalgae biofilm was proposed to enhance CO₂ biofixation and protein production using nickel foam with the modified surface as the carrier for immobilizing microalgae cells. The results demonstrated that, compared with microalgae suspension, microalgae biofilm lowered mass transfer resistance and promoted mass transfer efficiency of CO₂ from the bubbles into the immobilized microalgae cells, enhancing CO₂ biofixation and protein production. Moreover, parametric studies on the performance of the photobioreactor with microalgae biofilm were also conducted. The results showed that the photobioreactor with microalgae biofilm yielded a good performance with the CO₂ biofixation rate of 4465.6 µmol m⁻³ s⁻¹, the protein concentration of effluent liquid of 0.892 g L⁻¹, and the protein synthesis rate of 43.11 g m⁻³ h⁻¹. This work will be conducive to the optimization design of microalgae culture system for improving the performance of the photobioreactor.

     

Tris not only controls the pH in microalgal cultures,but also feeds bacteria

Tris (Tris(hydroxymethyl)amino methane), a compound often used as a buffer in microalgal culture media, sustains active bacterial growth in non-axenic microalgal cultures when sodium phosphate is present. The low pH levels caused by bacterial growth and probably the depletion of phosphorus in the medium caused the collapse ofPhaeodactylum tricornutum cultures resulting in a reduction of microalgal growth from 32 x 10⁶ to 1.1 x 10⁶ cells ml^–1. This emphasizes the need for care when interpreting the results of non-axenic microalgae cultures in which Tris or other organic buffer is added.     

Anaerobic digestion of microalgal biomass with ultrasonic disintegration

The use of photosynthetic microalgae for nutrient removal and biofuel production has been widely discussed. Anaerobic digestion of waste microalgal biomass to produce biogas is a promising technology for bioenergy production. However, the methane yield from this anaerobic process was limited because of the hard cell wall of Chlorella vulgaris. The use of ultrasound has proven to be successful at improving the disintegration and anaerobic biodegradability of Chlorella vulgaris. Ultrasonic pretreatment in the range of 5–200 J ml⁻¹ was applied to waste microalgal biomass, which was then used for batch digestion. Ultrasound techniques were successful and showed higher soluble COD at higher applied energy. During batch digestion, cell disintegration due to ultrasound increased in terms of specific biogas production and the degradation rate. Compared to the untreated sample, the specific biogas production was increased in the ultrasound-treated sample by 90% at an energy dose of 200 J ml⁻¹. For the disintegrated samples, volatile solids reduction was also increased according to the energy input and degradation. These results indicate that the hydrolysis of microalgal cells is the rate-limiting step in the anaerobic digestion of microalgal biomass.     

Evaluation of electrocoagulation,flocculation, and sedimentation harvesting methods on microalgae consortium grown in anaerobically digested abattoir effluent

Microalgae dewatering is a major bottleneck for biomass production in a large-scale microalgal production system which accounts for 20–60% of production cost. In this study, three dewatering systems of electrocoagulation, flocculation, and pH-induced flocculation were evaluated for microalgal consortium grown in anaerobically digested abattoir effluent at pH 6.5 and 9.5. At the shortest time (15 min) and the highest current density (0.08 A cm^?2), the highest microalgae recoveries of 78 and 84% were obtained with the corresponding power consumptions of 1.25 and 1.07 kWh kg^?1 for cultures at pH 6.5 and 9.5. For microalgae suspension at pH 6.5, the highest biomass recovery of 77% was obtained when 100 mg L^?1 of FeCl₃·6H₂O (after 15 min) or 100 mg L^?1 of Al₂(SO₄)₃·18H₂O (after 30 min) was added. However, microalgal recoveries significantly increased when FeCl₃·6H₂O or Al₂(SO₄)₃·18H₂O was used with the culture at pH 9.5. pH-Induced experiments showed that cultures adjusted at pH 10.5 had 36% higher biomass recovery compared to that in cultures at pH 8.5 after 2 h. The results of this study showed that cultures at higher pH (9.5) had a better microalgae recovery in all dewatering systems than cultures at lower pH (6.5).

     

Evaluation of <Emphasis Type="Italic">Isochrysis galbana</Emphasis> (clone T-ISO) cell numbers by digital image analysis of color intensity

In microalgal cultivation, measuring cell numbers as a means to monitor growth rates is a long-standing problem. Many automated counting systems and schemes have been developed; among these are image analysis systems. However, such imaging systems have presented difficulties in dealing with the complexities of computer recognition of individual microscopic cells. It is known that the coloration of microalgae suspension is species specific and that color intensity increases are typically associated with increasing numbers. Using this qualitative insight, the present work describes the design, construction, and comparative performance of an inexpensive digital imaging system optimized for counting microalgal cells. The system circumvents the need to count individual cells and extracts cell numbers directly from the macroscopic color intensity of a microalgal suspension. The results suggest, using Isochrysis galbana (T-ISO) as an illustrative example, that this scheme is potentially useful for inexpensive and automated biomonitoring of microalgal cell numbers. Percentage difference comparisons with a standard Coulter Counter indicated that the three algorithms tested provided better than 10% accuracy over density thresholds of 1.52 × 10⁶ to 8.10 × 10⁶ cells mL⁻¹ with precision of 4% attainable at high density concentrations.     

Optimization of a chemically defined medium for mycelial growth and polysaccharide production by medicinal mushroom <Emphasis Type="Italic">Phellinus igniarius</Emphasis>

A chemically defined medium for mycelial growth and exopolysaccharide (EPS) production by submerged culture of Phellinus igniarius was investigated. The mainly defined medium compositions were optimized by using orthogonal matrix method. The optimal defined medium (per liter) was 40.0 g glucose, 4.0 g. glutamic acid, 4.0 g (NH₄)₂SO₄, and initial pH 6.0. Under the optimal medium, the maximal mycelial biomass and EPS production were 12.33 ± 0.89 and 1.21 ± 0.08 g l⁻¹ at 192 h in shake flask, while the maximal mycelial biomass and EPS production reached 13.86 ± 0.52 and 1.92 ± 0.07 g l⁻¹ at 168 h in 3 l fermenter, respectively. The molecular weights (g mol⁻¹) of four fractions isolated from EPS by gel permeation were about 6.4 × 10⁶, 3.3 × 10⁵, 2.7 × 10⁵ and 2.9 × 10³. This study should be widely applied to other secondary metabolites production from higher fungus in a chemically defined medium and quantitative regulation of the metabolic flux in polysaccharide biosynthesis.     

Microalgal system for treatment of effluent from poultry litter anaerobic digestion

The potential of mixotrophic microalgae to utilize poultry litter anaerobic digester (AD) effluent (PLDE) as nutritional growth medium was evaluated. Three algal strains viz. Chlorella minutissima, Chlorella sorokiniana and Scenedesmus bijuga and their consortium showed significant biomass productivity in 6% (v/v) concentration of PLDE in deionized water. Multiple booster dosage of PLDE supported better growth relative to a single dose PLDE. The maximum biomass productivity of 76 mg L⁻¹ d⁻¹ was recorded. The biomass was rich in protein (39% w/w) and carbohydrates (22%) while lipids (<10%) were low, making it most suitable as an animal feed supplement. The mixotrophic algae showed sustainable growth against variations in PLDE composition in different AD batches, thus proving to be a suitable candidate for large scale wastewater treatment with concomitant production of renewable biomass feedstock for animal feed and bioenergy applications.     

Prophylactic effect of L-cysteine to acute and subclinical nitrate toxicity in sheep

Four rumen fistulated Suffolk wethers were allocated to a 4×4 Latin square designed experiment. High nitrate (1.5 g NaNO₃ kg^−0.75 body weight), high nitrate with high L-cysteine (0.55 g sulphur equivalent kg^−0.75 body weight), low nitrate (0.75 g NaNO₃ kg^−0.75 body weight), and low nitrate with low L-cysteine (0.275 g sulphur equivalent kg^−0.75 body weight) were administered into the rumen through fistulae as a single dose after a morning meal. Gaseous exchanges were monitored by an open circuit respiratory system using a hood over the animal's head. High or low L-cysteine remarkably decreased nitrite production from ruminal reduction of high or low nitrate. Consequently, methaemoglobin formation was suppressed by L-cysteine in both levels of nitrate. Oxygen consumption, carbon dioxide production and metabolic rate were depressed as methaemoglobin was formed. L-cysteine suppressed the pulmonary dysfunction induced by methaemoglobin. L-cysteine equivalent to 60% of the upper allowance of dietary sulphur appeared to be useful as a prophylactic for acute poisoning of nitrate. Thus, dosage of L-cysteine can be adjusted to correspond with the nitrate content in feeds.