Marine microalgae as a potential source of single cell protein (SCP)

Summary The marine microalgaeTetraselmis suecica, Isochrysis galbana, Dunaliella tertiolecta andChlorella stigmatophora are good biological sources of single cell protein (SCP). Protein content accounts for 39.12%–54.20% of the dry matter,D. tertiolecta having the highest. Lysine values are between 3.67 and 4.52 g/100 g of protein, and thus are higher than those for freshwater species. The total nucleic acid content is less than 7% of the dry matter; this value is definitely lower than that for yeasts or bacteria, commonly used as SCP sources. Amino acid profiles of the four species are very similar and comparable to the FAO reference protein, buth with a low content of methionine and cystine and a high content of lysine. The MEAA indices are between 81 and 84.98, without significant differences among the four species. Marine microalgae can be used as a potential SCP source.     

The relationship of substrate, growth rate, and maintenance coefficient to single cell protein production

Various organic compounds were assessed as potential substrates for single cell protein production. Substrate evaluation was based on the costs associated with the substrate, oxygen, and heat yield coefficients: Y_sub, Y_o, and Y_kcal, respectively. Y_o, and Y_kcal, were calculated from experimental values of Y_sub, and from the elemental composition of bacterial cells. The dependence of the yield coefficients on the specific growth rate (μ) and maintenance coefficient (m) also was assessed. The analysis disclosed that m caused two- to threefold variations in the yield coefficients as μ was increased from 10% to 100% of μ_max. The effect of different m values at constant specific growth rates also was determined. The value of m had a significant effect on the yield coefficients even at high specific growth rates. Assignment of cost factors to the yield coefficients provided an estimation of the impact m and μ on biomass production costs.     

Wheat straw,a potential substrate for cellulase production usingTrichoderma reesei

Wheat straw, pre-treated with either alkali or steam, or both together, was used 46% more efficiently byTrichoderma reesei than untreated straw. Carboxymethyl cellulase and filter paper cellulase were higher by 52% and 74%, respectively, in the treated substrate compared with the untreated one.     

Filamentous fungi as cell factories for heterologous protein production

Filamentous fungi have been used as sources of metabolites and enzymes for centuries. For about two decades, molecular genetic tools have enabled us to use these organisms to express extra copies of both endogenous and exogenous genes. This review of current practice reveals that molecular tools have enabled several new developments. But it has been process development that has driven the final breakthrough to achieving commercially relevant quantities of protein. Recent research into gene expression in filamentous fungi has explored their wealth of genetic diversity with a view to exploiting them as expression hosts and as a source of new genes. Inevitably, the progress in the 'genomics' technology will further develop high-throughput technologies for these organisms.     

Ribosomal protein as substrate for a GTP-dependent protein kinase from yeast

A protein kinase specific for casein and acidic ribosomal proteins was isolated and partly characterized.It was found that the enzyme utilizes GTP and ATP as phosphoryl donors. Its affinity for ATP was considerably higher than for GTP with the km values of 7.6 × 10^-6M and 5.5 × 10^-5M, respectively.Two-dimensional acrylamide gel electrophoresis revealed the phosphorylation of the same ribosomal proteins with either of the [-³²P] nucleotides used. It was also shown that one acidic protein (S1 or S2) of 40 S and two acidic proteins (L2 and L3) of 60 S ribosomal subunits were predominantly phosphorylated in vitro. The phosphorylated proteins: L2 and L3 seem to correspond to the proteins of L7 and L12 of E. coli ribosomes. The isolated kinase phosphorylated several basic ribosomal proteins though to a lower extent than the acidic ones.     

Acid hydrolysis of sunflower seed husks for production of single cell protein

Summary Sunflower seed husks were chosen as a typical lignocellulosic waste product of low value. This model substrate was hydrolyzed with sulphuric acid at 120°C. The hydrolysis was carried out in two steps: hydrolysis of the pentosan fraction and subsequent hydrolysis of the cellulose fraction. The pentosan fraction was nearly quantitatively hydrolyzed. For the cellulose hydrolysis the yield was 79% of the theoretical yield. The hydrolyzates were neutralized to pH 5 with solid calcium hydroxide and used for preparation of growth media forCandida yeasts andPaecilomyces variotii. For the pentosan hydrolyzates the yields of yeast biomass were 35–36 g per 100 g available reducing sugars (supplied to the medium). In cellulose hydrolyzates the corresponding yields were 45–48 g withCandida utilis andC. tropicalis and about 30 g withC. pseudotropicalis. P. variotii was noticeably superior to the yeasts. In pentosan hydrolyzates it produced 63 g dry mycelium from 100 g reducing sugars supplied; in cellulose hydrolyzates, 94 g. This suggests that it must be an effective utilizer of a wide range of compounds, for example, organic acids in the medium.     

Paddy straw as substrate for ethanol production

Pretreatment of paddy straw with 2% sodium hydroxide at 15 psi for 1 h resulted in 83% delignification. The hydrolysis of alkali treated paddy straw with a commercial preparation of cellulase for 2 h at 50°C resulted in release of 65% total reducing sugars. Maximum sugars were released at enzyme loading of 1.5% (v/v). The fermentation of hydrolysate supplemented with nutrients by S. cerevisiae resulted in the production of 20–30 g L⁻¹ ethanol after 48 h incubation which was further improved with addition of yeast nitrogen base and inoculated with 1% (w/v) yeast cells.     

Thermotolerant single cell protein production byKluyveromyces marxianus var.marxianus

Summary Amino acid analyses were undertaken on single cell protein (SCP) produced by thermotolerant strains ofKluyveromyces marxianus var.marxianus grown on sugar cane molasses at 40°C. The maximum conversion of available sugars to biomass at 45°C was only 10.8% (g dry wt.·g^–1 total sugars). The amino acid composition of the SCP did not differ markedly from that reported for other yeast species.     

Methane as a carbon substrate for the production of microbial cells

The cultivation of aerobic, methane-utilizing, microbial cells by submerged culture techniques, in an entirely mineral salts medium, with a view to their use as an edible protein source is discussed. Particular emphasis is placed on the potentially explosive nature of gaseous mixtures containing methane and oxygen. The experiments described investigate if fully safe operation at all times, by oxygen concentration control, is possible in agitated and sparged batch fermentors. Appreciable wastage of methane is prevented by gaseous-phase recirculation. It is concluded that fully safe operation is possible, cultures being able to grow exponentially without substrate limitation by the gaseous-phase nutrients.     

The marine microalgaChlorella stigmatophora as a potential source of single cell protein: Enhancement of the protein content in response to nutrient enrichment

Summary Mass cultures ofChlorella stigmatophora were carried out in order to obtain maximum protein production and to study the chemical variations in function of the nutrient concentration. Cultures reached maximum cellular densities of 2.2·10⁸ cells/ml, with a growth velocity between 0.49 and 0.55 doublings/day. Carbohydrate content in the stationary phase ranged between 2.23 and 2.74 pg/cell, RNA between 0.78 and 1.36 pg/cell and DNA between 0.013 and 0.016 pg/cell. The maximum value for chlorophylla was 0.13 pg/cell. Maximum protein content was obtained with a nutrient concentration of 16 mM of NaNO₃, giving 4.85 pg/cell and a protein concentration of 0.7 g/l. The protein content can be manipulated by changes in the nutrient concentration, showing differences up to a 9.2-fold increase. This characteristic makesChlorella stigmatophora a suitable source of single cell protein.     

Coffee oil as a potential feedstock for biodiesel production

A preliminary evaluation of the feasibility of producing biodiesel using oil extracted from defective coffee beans was conducted as an alternative means of utilizing these beans instead of roasting for consumption of beverage with depreciated quality. Direct transesterifications of triglycerides from refined soybean oil (reference) and from oils extracted from healthy and defective coffee beans were performed. Type of alcohol employed and time were the reaction parameters studied. Sodium methoxide was used as alkaline catalyst. There was optimal phase separation after reactions using both soybean and healthy coffee beans oils when methanol was used. This was not observed when using the oil from defective beans which required further processing to obtain purified alkyl esters. Nevertheless, coffee oil was demonstrated to be a potential feedstock for biodiesel production, both from healthy and defective beans, since the corresponding oils were successfully converted to fatty acid methyl and ethyl esters.     

Wild rice as fermentation substrate for mycotoxin production

Many cereal grains have been studied for their suitability as substrates for the fermentative production of mycotoxins. However, except for aflatoxin, wild rice has not been investigated. Hence, five mold cultures known to produce the mycotoxins ochratoxin-A, penicillic acid, patulin, vomitoxin, and zearalenone were grown on wild rice under varying conditions of moisture and temperature to determine whether this grain would serve as a suitable substrate for toxin production. Under appropriate fermentation conditions, good yields of ochratoxin-A and moderate amounts of patulin were obtained, but only small amounts of penicillic acid, vomitoxin, and zearalenone were elaborated. An extract from a sample of naturally molded wild rice contained 0.8 microgram of patulin per g of rice. The predominating mold was identified as Aspergillus clavatus. Under identical cultural conditions, this isolate and a known patulin-producing strain of A. clavatus yielded approximately equivalent amounts of the mycotoxin.     

Hypocrellin-B acetate as a fluorogenic substrate for enzyme-assisted cell photosensitization

Photosensitizing molecules (PSs) undergo chemico-physical changes upon addition of suitable substituents, influencing both their photophysical properties and their ability to accumulate into cells. Once inside the cells, the modified PS acts as a fluorogenic substrate: the added substituent is removed by a specific enzyme, restoring the native PS in subcellular sensitive sites. We investigated the photophysical properties and interaction with HeLa cells of Hypocrellin-B (HypB), as native molecule and upon acetate-group addition (HypB-Ac). Chemical modification alters both absorption and fluorescence features of HypB; consequently, the dynamics of the enzyme hydrolysis of HypB-Ac can be monitored through restoring the native HypB spectral properties. At the cellular level, only the HypB emission signal was detected within 5 min of incubation with either HypB or HypB-Ac, allowing a direct comparison of the time courses of their intracellular accumulation. Plateau values were reached within 15 min of incubation with both compounds, the emission signals being significantly higher in HypB-Ac than in HypB treated cells. Consistently, imaging showed a rapid appearance of red fluorescence in the cytoplasm, with more abundant bright spots in HypB-Ac treated cells. Both compounds did not induce dark toxicity at concentrations up to 1 × 10(-6) M, while upon irradiation at 480 nm phototoxicity was significantly higher for cells exposed to HypB-Ac than for HypB-loaded cells. These findings suggest an improved efficacy of acetylated HypB to be internalized by cells through membrane trafficking, with a preferential interaction of the photoactive molecules on sensitive intracellular sites. After irradiation, in HypB-Ac treated cells, prominent disorganization of several cytoplasmic organelles such as the endoplasmic reticulum, Golgi apparatus, lysosomes, microfilaments and microtubules were observed.     

Utilization of by-products from ethanol production as substrate for biogas production

The aims of this work were to determine the specific biogas yields of steam-exploded sugarcane straw and bagasse as well as to estimate their energy potential under Brazilian conditions. Steam-explosion was carried out under different time and temperature conditions. The specific biogas yields were analyzed in batch-tests according to VDI 4630.Results have shown that steam-explosion pre-treatment increased the specific biogas yields of straw and bagasse significantly compared to the untreated material. The utilization of these by-products can contribute to 5% of the total energy consumption and thereby higher energy independence in Brazil. Further efforts in defining the optimum pretreatment conditions with steam-explosion as well as implementing this technology in large scale plants should be made.     

Holocellular retinol binding protein as a substrate for microsomal retinal synthesis

Holocellular retinol binding protein (holo-CRBP) was substrate for retinal synthesis at physiological pH with microsomes prepared from rat liver, kidney, lung, and testes. Four observations indicated that retinal synthesis was supported by holo-CRBP directly, rather than by the unbound retinol in equilibrium with CRBP. First, the rate of retinal synthesis with holo-CRBP exceeded the rate that was observed from the concentration of unbound retinol in equilibrium with CRBP. Second, NADP was the preferred cofactor only with holo-CRBP, supporting a rate about 3-fold greater than that of NAD. In contrast, with unbound retinol as substrate, similar rates of retinal formation were supported by either NAD or NADP. Third, the rate of retinal synthesis was not related to the decrease in the concentration of unbound retinol in equilibrium with holo-CRBP caused by increasing the concentration of apo-CRBP. Fourth, the rate of retinal synthesis increased with increases in the concentration of holo-CRBP as a fixed concentration of unbound retinol was maintained. This was achieved by increasing both apo-CRBP and holo-CRBP, but keeping constant the ratio apo-CRBP/holo-CRBP. Retinal formation from holo-CRBP displayed typical Michaelis-Menten kinetics with a Km about 1.6 microM, less than the physiological retinal concentration of 4-10 microM in the livers of rats fed diets with recommended vitamin A levels. The Vmax for retinal formation from holo-CRBP was 14-17 pmol min-1 (mg of protein)-1, a rate sufficiently high to generate adequate retinal to contribute significantly to retinoic acid synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic modelling of continuous submerged fermentation of cheese whey for single cell protein production

A mathematical model describing the kinetics of continuous production of single cell protein from cheese whey using Kluyveromyces fragilis was developed from the basic principles of mass balance. The model takes into account the substrate utilization for growth and maintenance and the effect of substrate concentration and cell death rate on the net cell growth and substrate utilization during the fermentation process. A lactose concentration below 1.91 g/L limited growth of yeast cells whereas a lactose concentration above 75 g/L inhibited the growth of the yeast. The model was tested using experimental data obtained from a continuous system operated at various retention times (12, 18 and 24 h), mixing speeds (200, 400 and 600 rpm) and air flow rates (1 and 3 vvm). The model was capable of predicting the effluent cell and substrate concentrations with R2 ranging from 0.95 to 0.99. The viable cell mass and lactose consumption ranged from 1.3 to 34.3 g/L and from 74.31% to 99.02%, respectively. A cell yield of 0.74 g cell/g lactose (close to the stoichiometric value of 0.79 g cell/g lactose) was achieved at the 12 h retention time-3 vvm air flow rate-600 rpm mixing speed combination. The total biomass output (viable and dead cells) at this combination was 37 g/L.