Hydride-mediated reduction of 2,4,6-trinitrotoluene by yeasts as the way to its deep degradation

Broad screening of microorganisms from natural and anthropogenic ecological niches has revealed strains Candida sp. AN-L15 and Geotrichum sp. AN-Z4 which transform, 2,4,6-trinitrotoluene (TNT) via alternative pathways (with the domination of hydride ion-mediated reduction of the aromatic ring) and produce relatively high amounts of nitrites. According to the spectrophotometry data, the hydride attack of TNT by Candida sp. AN-L15 and Geotrichum sp. AN-Z4 grown at pH 5.0–8.0 leads to the mono-and dihydride complexes of TNT (H^?-TNT and 2H^?-TNT, respectively) and to protonated forms of the latter. Analysis by HPLC, GC-mass spectrometry, and ion chromatography revealed the products of deep conversion of TNT. The growth of the yeast strains in a weakly acidic medium with TNT (440 μM) is accompanied by formation of 2,4-dinitrotoluene (2,4-DNT, up to 18.2 μM). Together with accumulation of nitrites (up to 76.0 μM, depending on pH of the medium), these findings demonstrate the capacity of both strains for TNT denitration. Formation of 2,4-DNT reflects the realization of one of the possible mechanisms of TNT ortho-nitro group elimination and switching over to the pathways of metabolism of dinitrotoluenes, which are much more easily biodegradable than TNT. Simultaneously with the dominating TNT hydride attack, the mechanism of 4-and 6-electron reduction of the nitro group also functions in Candida sp. AN-L15 and Geotrichum sp. AN-Z4. Realization of the studied mechanisms of TNT transformation under growth of Candida sp. AN-L15 on n-alkane is important for bioremediation in the cases of combined pollution by oil products and explosives.     

Hydride-mediated reduction of 2,4,6-trinitrotoluene by yeasts as the way to its deep degradation

Broad screening of microorganisms from natural and anthropogenic ecological niches has revealed strains Candida sp. AN-L15 and Geotrichum sp. AN-Z4 which transform 2,4,6-trinitrotoluene (TNT) via alternative pathways (with the domination of hydride ion-mediated reduction of the aromatic ring) and produce relatively high amounts of nitrites. According to the spectrophotometry data, the hydride attack of TNT by Candida sp. AN-L15 and Geotrichum sp. AN-Z4 grown at pH 5.0-8.0 leads to the mono- and dihydride complexes of TNT (H(-)-TNT and 2H(-)-TNT, respectively) and to protonated forms of the latter. Analysis by HPLC, GC-mass spectrometry, and ion chromatography revealed the products of deep conversion of TNT. The growth of the yeast strains in a weakly acidic medium with TNT (440 microM) is accompanied by formation of 2,4-dinitrotoluene (2,4-DNT, up to 18.2 microM). Together with accumulation of nitrites (up to 76.0 microM, depending on pH of the medium), these findings demonstrate the capacity of both strains for TNT denitration. Formation of 2,4-DNT reflects the realization of one of the possible mechanisms of TNT ortho-nitro group elimination and switching over to the pathways of metabolism of dinitrotoluenes, which are much more easily biodegradable than TNT. Simultaneously with the dominating TNT hydride attack, the mechanism of 4- and 6-electron reduction of the nitro group also functions in Candida sp. AN-L15 and Geotrichum sp. AN-Z4. Realization of the studied mechanisms of TNT transformation under growth of Candida sp. AN-L15 on n-alkane is important for bioremediation in the cases of combined pollution by oil products and explosives.     

Anaerobic transformation of 2,4,6-TNT and related nitroaromatic compounds by Clostridium acetobutylicum

The transformation of TNT and related aminated nitrotoluenes by Clostridium acetobutylicum was investigated. 2,4,6-trinitrotoluene (TNT) was rapidly reduced (537 nM min⁻¹ mg protein⁻¹) to undetermined end products via monohydroxylamino derivatives. TNT reduction was more rapid than that of 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene and 2,4-diamino-6-nitrotoluene. The metabolic phase of clostridial cultures affected rates and extents of transformation of TNT and its intermediates. Acidogenic cultures showed rapid transformation rates and the ability to transform TNT and its primary reduction products to below detection limits; solventogenic cultures did not transform TNT completely, and showed accumulation of its hydroxylamino derivatives. Carbon monoxide-induced solventogenesis was capable of slowing the transformation of TNT and intermediates. Studies employing [ring-U-¹⁴C]-TNT demonstrated that no significant mineralization occurred and that products of transformation were water-soluble. Received 06 November 1995/ Accepted in revised form 15 August 1996     

Use of cheese whey to enhance <Emphasis Type="Italic">Geotrichum candidum</Emphasis> biomass production in olive mill wastewater

Geotrichum candidum is a yeast-like filamentous fungus that has attracted industrial interest. The present work investigated G. candidum biomass production in agro-industrial wastewaters (olive mill wastewater (OMW) and cheese whey (CW)) as the only substrate. Different solid media (Sabouraud dextrose agar (SDA), CW, OMW, and OMW/CW mixtures in different proportions) were tested. OMW/CW mixtures proved to be suitable for optimal mycelia growth of G. candidum with a very high hyphae density. The highest fungal and expansion rate growth of 83 ± 1 mm and 12.4 day⁻¹, respectively, were obtained on a 20:80 mixture of OMW/CW, which was incubated for 7 days. This optimal mixture was used to study the biomass production and the OMW decolorization ability of G. candidum in the presence of CW in liquid medium. Liquid cultures were also conducted in OMW and CW separately. After 5 days of incubation, fungal biomass reached 9.26 g l⁻¹ in the OMW/CW mixture and only 2.83 g l⁻¹ in CW, while no biomass production was observed in OMW alone. OMW decolorization and dephenolization by G. candidum also improved in the presence of CW with a decolorization efficiency of 54.5% and a total phenolic reduction of 55.3%, compared with the control which yielded values of about 10% and 15%, respectively. These results suggested that OMW/CW—as the only substrate—could be used as a cost-effective medium to produce G. candidum biomass, without the need for water dilution or supplementation with other nutriments.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the chinese medicinal plant, <Emphasis Type="Italic">Dendrobium candidum</Emphasis> wall. ex lindl., from axenic nodal segments

Summary Dendrobium candidum Wall. Ex Lindl. is an important species used in the formulation of Shih-hu, a Chinese traditional medicine. An efficient protocol for in vitro propagation of D. candidum using the axenic nodal segments of the shoots, originated from the in vitro germinated seedlings, was developed. The seeds from 120-d-old capsules after pollination were first germinated on half-strength Murashige and Skoog (MS) basal medium supplemented with 30 g l⁻¹ sucrose. After 4 mo., the seedlings were subcultured on a similar medium supplemented with 1 ml l⁻¹ HYPONeX, 80 g l⁻¹ potato homogenate and 2 g l⁻¹ activated charcoal for further growth. Axenic nodal segments excised from 9-mo.-old seedlings were cultured on the medium in the presence of 2 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ naphthaleneacetic acid (NAA). After 75 d, 73.2% of the explants gave rise to buds/shoots. The elongated shoots were rooted on the medium containing 0.2 mg l⁻¹ NAA and the plantlets were successfully acclimatized in soil.     

Decolorization of dye and molasses by continuous and semi-continuous jar-fermentor cultures ofGeotrichum candidum Dec 1

Two culture modes, continuous and semi-continuous, of the decolorization fungus,Geotrichum candidum Dec 1, were compared to obtain a high treatment efficiency of molasses decolorization and a large productivity of peroxidase (DyP) to simultaneously decolorize dyes and molasses. The continuous culture ofG. candidum Dec 1 using a 5-l jar-fermentor showed high DyP activity at a low dilution ratio of 0.005h⁻¹, and decolorization ratio of molasses of 80% was obtained concomitantly. Therefore, a semi-continuous culture was performed by repeated refill and draw. In this mode, approximately 1.5 liters of the culture broth was replaced per cycle when the decolorization ratio of molasses was near 80%. The molasses medium (1.0 liter per day) was treated and the peroxidase productivity in the drawn culture broth was 26.6 U/day, whereas the peroxidase productivity was 17.9 U/day in the continuous culture with a dilution rate of 0.005 h⁻¹. The semi-continuous treatment system was an efficient decolorization method for the strain,G. candidum Dec 1.     

Initial Reductive Reactions in Aerobic Microbial Metabolism of 2,4,6-Trinitrotoluene

Because of its high electron deficiency, initial microbial transformations of 2,4,6-trinitrotoluene (TNT) are characterized by reductive rather than oxidation reactions. The reduction of the nitro groups seems to be the dominating mechanism, whereas hydrogenation of the aromatic ring, as described for picric acid, appears to be of minor importance. Thus, two bacterial strains enriched with TNT as a sole source of nitrogen under aerobic conditions, a gram-negative strain called TNT-8 and a gram-positive strain called TNT-32, carried out nitro-group reduction. In contrast, both a picric acid-utilizing Rhodococcus erythropolis strain, HL PM-1, and a 4-nitrotoluene-utilizing Mycobacterium sp. strain, HL 4-NT-1, possessed reductive enzyme systems, which catalyze ring hydrogenation, i.e., the addition of a hydride ion to the aromatic ring of TNT. The hydride-Meisenheimer complex thus formed (H⁻-TNT) was further converted to a yellow metabolite, which by electrospray mass and nuclear magnetic resonance spectral analyses was established as the protonated dihydride-Meisenheimer complex of TNT (2H⁻-TNT). Formation of hydride complexes could not be identified with the TNT-enriched strains TNT-8 and TNT-32, or with Pseudomonas sp. clone A (2NT⁻), for which such a mechanism has been proposed. Correspondingly, reductive denitration of TNT did not occur.     

Bio-hydrogen production from cellulose by sequential co-culture of cellulosic hydrogen bacteria of <Emphasis Type="Italic">Enterococcus gallinarum</Emphasis> G1 and <Emphasis Type="Italic">Ethanoigenens harbinense</Emphasis> B49

Microbial conversion of lignocellulose to hydrogen is a fascinating way to provide a renewable energy source. A mesophilic bacterium strain G1 that had high cellulose degradation and hydrogen production activity (2.38 mmol H₂ g⁻¹ cellulose) was isolated from rumen fluid and identified as the Enterococcus gallinarum. Hydrogen production from cellulose by using sequential co-cultures of a cellulosic-hydrolysis bacterium G1 and Ethanoigenens harbinense B49 was investigated. With an initial Avicel concentration of 5 g l^−l, the sequential co-culture with G1 and strain Ethanoigenens harbinense B49 produced H₂ yield approximately 2.97 mmol H₂ g⁻¹ cellulose for the co-culture system.     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

TNT Biotransformation and Detoxification by a Pseudomonas Aeruginosa Strain

Successful microbial-mediated remediation requires transformationpathways that maximize metabolism and minimize the accumulation of toxic products. Pseudomonas aeruginosa strain MX, isolated from munitions-contaminated soil, degraded 100 mg TNT L^-1 in culture medium within 10 h under aerobic conditions. The major TNT products were 2-amino-4,6-dinitrotoluene (2ADNT, primarily in the supernatant) and 2,2'-azoxytoluene (2,2'AZT, primarily in the cell fraction), which accumulated as major products via the intermediate2-hydroxylamino-4,6-dinitrotoluene (2HADNT). The 2HADNT and2,2'AZT were relatively less toxic to the strain than TNT and 2ADNT. Aminodinitrotoluene (ADNT) production increased when yeast extract was added to the medium. While TNT transformation rate was not affected by pH, more HADNTs accumulated at pH 5.0 than at pH 8.0 and AZTs did not accumulate at the lower pH. The appearance of 2,6-diamino-4-nitrotoluene (2,6DANT) and 2,4-diamino-6-nitrotoluene (2,4DANT); dinitrotoluene (DNT) and nitrotoluene (NT); and 3,5-dinitroaniline (3,5DNA) indicated various routes of TNT metabolism and detoxification by P. aeruginosa strain MX.     

Increased astaxanthin production by a Phaffia rhodozyma mutant grown on date juice from Yucca fillifera

The wild strain and the astaxanthin-overproducing mutant strain 25–2 of Phaffia rhodozyma were analyzed in order to assess their ability to grow and synthesize astaxanthin in a minimal medium composed of g L⁻¹: KH₂PO₄ 2.0; MgSO₄ 0.5; CaCl₂ 0.1; urea 1.0 and supplemented with date juice of Yucca fillifera as a carbon source (yuca medium). The highest astaxanthin production (6170 μg L⁻¹) was obtained at 22.5 g L⁻¹ of reducing sugars. The addition of yeast extract to the yuca medium at concentrations of 0.5–3.0 g L⁻¹ inhibited astaxanthin synthesis. The yuca medium supported a higher production of astaxanthin, 2.5-fold more than that observed in the YM medium. Journal of Industrial Microbiology & Biotechnology (2000) 24, 187–190. Received 14 July 1999/ Accepted in revised form 02 December 1999     

Evaluation of different culture conditions ofClostridium bifermentans DPH-1 for cost effective PCE degradation

Clostridium bifermentans strain DPH-1 has already been found to dechlorinate perchloroethylene (PCE) tocis-dichloroethylene (cis-DCE)via trichloroethylene (TCE). In this study, our investigation on different culture conditions of this DPH-1 strain was extended to find a more efficient and cost effective growth medium composition for this DPH-1 strain in bioremediation practices. Temperature dependency of strain DPH-1 showed that the growth starting time and PCE degradation at 15°C was very slow compared to that of 30°C, but complete PCE degradation occurred in both cases. For the proper utilization of strain DPH-1 in more cost effective bioremediation practices, a simpler composition of an effective media was studied. One component of the culture medium, yeast extract, had been substituted by molasses, which served as a good source of electron donor. The DPH-1 strain in the medium containing molasses, in the presence of K₂HPO₄ and KH₂PO₄, showed identical bacterial multiplication (0.135 mg protein mL⁻¹h⁻¹) and PCE degradation rates (0.38 μM/h) to those of the yeast extract containing medium.     

Transformation of 2,4,6-trinitrotoluene (TNT) by <Emphasis Type="Italic">Raoultella</Emphasis> <Emphasis Type="Italic">terrigena</Emphasis>

Manufacture of nitroorganic explosives generates toxic wastes leading to contamination of soils and waters, especially groundwater. For that reason bacteria living in environments highly contaminated with 2,4,6-trinitrotoluene (TNT) and other nitroorganic compounds were investigated for their capacity for TNT degradation. One isolate, Raoultella terrigena strain HB, removed TNT at concentrations between 10 and 100 mg l⁻¹ completely from culture supernatants under optimum aerobic conditions within several hours. Only low concentrations of nutrient supplements were needed for the cometabolic transformation process. Radioactivity measurements with ring-labelled ¹⁴C–TNT detected about 10–20% of the initial radioactivity in the culture supernatant and the residual 80–90% as water-insoluble organic compounds in the cellular pellet. HPLC analysis identified aminodinitrotoluenes (2-ADNT, 4-ADNT) and diaminonitrotoluenes (2,4-DANT) as the metabolites which remained soluble in the culture medium and azoxy-dimers as the main products in the cell extracts. Hence, the new isolate could be useful for the removal of TNT from contaminated waters.     

Astaxanthin production by <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis> and <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>: a comparative study

Phaffia rhodozyma (now Xanthophyllomyces dendrorhous) and Haematococcus pluvialis are known as the major prominent microorganisms able to synthesize astaxanthin natural pigment. Important research efforts have been made to determine optimal conditions for astaxanthin synthesis. When the focus is on astaxanthin production, the maximal reported value of 9.2 mg/g cell is obtained within H. pluvialis grown on BAR medium, under continuous illumination (345 μmol photon m⁻² s⁻¹) and without aeration. Whereas fermentation by mutated R1 yeast grown on coconut milk produced 1,850 μg/g yeast. However, when looking at astaxanthin productivity, the picture is slightly different. The figures obtained with P. rhodozyma are rather similar to those of H. pluvialis. Maximal reported values are 170 μg/g yeast per day with a wild yeast strain and 370 μg/g yeast per day with mutated R1 yeast. In the case of H. pluvialis, maximal values ranged from 290 to 428 μg/g cell per day depending on the media (BG-11 or BAR), light intensity (177 μmol photon m⁻² s⁻¹), aeration, etc. The main aim of this work was to examine how astaxanthin synthesis, by P. rhodozyma and H. pluvialis, could be compared. The study is based on previous works by the authors where pigment productions have been reported.     

Use of response surface methodology for optimizing process parameters for high inulinase production by the marine yeast <Emphasis Type="Italic">Cryptococcus aureus</Emphasis> G7a in solid-state fermentation and hydrolysis of inulin

The optimization of process parameters for high inulinase production by the marine yeast strain Cryptococcus aureus G7a in solid-state fermentation (SSF) was carried out using central composite design (CCD), one of the response surface methodologies (RSMs). We found that moisture, inoculation size, the amount ratio of wheat bran to rice husk, temperature and pH had great influence on inulinase production by strain G7a. Therefore, the CCD was used to evaluate the influence of the five factors on the inulinase production by strain G7a. Then, five levels of the five factors above were further optimized using the CCD. Finally, the optimal parameters obtained with the RSM were the initial moisture 61.5%, inoculum 2.75%, the amount ratio of wheat bran to rice husk 0.42, temperature 29 °C, pH 5.5. Under the optimized conditions, 420.9 U g⁻¹ of dry substrate of inulinase activity was reached in the solid-state fermentation culture of strain G7a within 120 h whereas the predicted maximum inulinase activity of 436.2 U g⁻¹ of inulinase activity of 436.2 U g⁻¹ of dry weight was derived from the RSM regression. This is the highest inulinase activity produced by the yeast strain reported so far. A large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase.     

High-frequency shoot regeneration through transverse thin cell layer culture in <Emphasis Type="Italic">Dendrobium Candidum</Emphasis> Wall Ex Lindl.

An efficient in vitro propagation protocol for Dendrobium candidum Wall ex Lindl. using transverse thin cell layer (tTCL) culture system was established. The frequency of shoot regeneration and the number of adventitious buds produced from the regenerated shoots significantly relied on the concentration of plant growth regulators, and the position and orientation of the explant. Murashige and Skoog (MS) medium with half-strength macronutrients and 2% sucrose, supplemented with 1.2 mg l⁻¹ naphthaleneacetic acid (NAA) and 1.2 mg l⁻¹ 6-benzyladenine (6-BA), was optimal for shoot regeneration. Upon this medium, the youngest explant inoculated in the upright orientation exhibited a high frequency of shoot regeneration (92%), and the highest number of adventitious buds (an average of 24.5) per explant. Rooting of shoots and adventitious buds was achieved on MS medium with half-strength macronutrients and 2% sucrose with 1.0 mg l⁻¹ NAA and 1.0 mg l⁻¹ indole-3-acetic acid (IAA). Plantlets were transplanted into vermiculite with a 95% survival rate in a greenhouse. Ontogenetic studies revealed that the shoots originated from the stem vascular bundles.     

Gas phase H<Subscript>2</Subscript>S product recovery in a packed bed bioreactor with immobilized sulfate-reducing bacteria

An N₂ strip gas was used in a packed bed sulfate-reducing bioreactor to recover the dissolved sulfide product and improve sulfate conversion. The highest volumetric productivity obtained was 261 mol H₂S m⁻³ d⁻¹. Lowering the initial pH of the medium from 7 to 6 increased the H₂S content of the strip gas from 3.6 to 5.8 mol%. The ratio of strip gas to liquid flow rates (G/L) was found be to a suitable basis for scaling the process. Calculations indicated that modest G/L values (<10²) were required to recover the residual dissolved sulfide in a downstream stripping column.     

Optimization of polyhydroxybutyrate production by <Emphasis Type="Italic">Bacillus</Emphasis> sp. CFR 256 with corn steep liquor as a nitrogen source

Polyhydroxyalkanotes (PHAs), the eco-friendly biopolymers produced by many bacteria, are gaining importance in curtailing the environmental pollution by replacing the non-biodegradable plastics derived from petroleum. The present study was carried out to economize the polyhydroxybutyrate (PHB) production by optimizing the fermentation medium using corn steep liquor (CSL), a by-product of starch processing industry, as a cheap nitrogen source, by Bacillus sp. CFR 256. Response surface methodology (RSM) was used to optimize the fermentation medium using the variables such as corn steep liquor (5–25 g l⁻¹), Na₂HPO₄ 2H₂O (2.2–6.2 g l⁻¹), KH₂PO₄ (0.5–2.5 g l⁻¹), sucrose (5–55 g l⁻¹) and inoculum concentration (1–25 ml l⁻¹). Central composite rotatable design (CCRD) experiments were carried out to study the complex interactions of the variables. The optimum conditions for maximum PHB production were (g l⁻¹): CSL-25, Na₂HPO₄ 2H₂O-2.2, KH₂PO₄ − 0.5, sucrose − 55 and inoculum − 10 (ml l⁻¹). After 72 h of fermentation, the amount of PHA produced was 8.20 g l⁻¹ (51.20% of dry cell biomass). It is the first report on optimization of fermentation medium using CSL as a nitrogen source, for PHB production by Bacillus sp.     

Physiological alterations and regulation of heterocyst and nitrogenase formation in Het(-) Fix(-) mutant strain of Anabaena variabilis

Physiological alterations and regulation of heterocyst and nitrogenase formation have been studied in Het⁻ Fix⁻ mutant strain of diazotrophic cyanobacterium Anabaena variabilis. Het⁻ Fix⁻ mutant strain of A. variabilis has been isolated by N-methyl-N′-nitro-N″-nitrosoguanidine (NTG) mutagenesis and was screened with the penicillin enrichment (500 μg ml⁻¹). Growth, heterocyst differentiation, nitrogenase and glutamine synthetase (biosynthetic and transferase), ¹⁴CO₂-fixation, nitrate reductase (NR), nitrite reductase (NiR), glucose-6-phosphate dehydrogenase (G6PDH), and isocitrate dehydrogenase (IDH) activities, and NO₃ ⁻, NO₂ ⁻, and NH₄ ⁺ uptake and whole cell protein profile in different metabolic conditions were studied in the Het⁻ Fix⁻ mutant strain taking wild-type A. variabilis as reference. Het⁻ Fix⁻ mutant strain was incapable of assimilating elemental nitrogen (N₂) due to its inability to form heterocysts and nitrogenase and this was the reason for its inability to grow in BG-11₀ medium (free from combined nitrogen). In contrast, wild-type strain grew reasonably well in the absence of combined nitrogen sources and also showed heterocyst differentiation (8.5%) and nitrogenase activity (10.8 ηmol C₂H₄ formed μg⁻¹ Chl a h⁻¹) in N₂-medium. Wild-type strain also exhibited higher NR, NiR, and GS activities compared to its Het⁻ Fix⁻ mutant strain, which may presumably be due to acquisition of high uptake of NO₃ ⁻, NO₂ ⁻, and NH₂ ⁺. Wild-type strain in contrast to its Het⁻ Fix⁻ mutant strain also exhibited high level of G6PDH, IDH, and ¹⁴CO₂ fixation activities. Low levels of G6PDH and IDH activities in Het⁻ Fix⁻ mutant strain further confirmed the lack of heterocyst differentiation and nitrogenase activity in the Het⁻ Fix⁻ mutant strain. NR, NiR, and GS activities in both the strains were energy-dependent and the energy required is mainly derived from photophosphorylation. Furthermore, it was found that de novo protein synthesis is necessarily required for the activities of NR, NiR, and GS in both wild-type and its Het⁻ Fix⁻ mutant strain. Received: 21 December 2001 / Accepted: 28 January 2002     

Fungal autolysate as a nutrient supplement for ethanol and chitosan production by <Emphasis Type="Italic">Mucor indicus</Emphasis>

Mucor indicus can be used to produce ethanol from a variety of sugars, including pentose’s. An extract of it, produced by autolysis, could replace yeast extract in culture medium with improved production of ethanol. At 10 g l⁻¹, the extract gave a higher ethanol yield (0.47 g g⁻¹) and productivity (0.71 g l⁻¹ h⁻¹) compared to medium containing yeast extract (yield 0.45 g g⁻¹; productivity 0.67 g l⁻¹ h⁻¹).