Fermentation of cassava peels for the production of cellulolytic enzymes

Cassava peels were used as a substrate for the production of cellulolytic enzymes. Under solid substrate fermentation conditions and a Rhizopus sp., thermostable cellulolytic enzymes were produced. Optimal production temperature and pH were 45°C and 5.6 respectively. Kinetic studies of the enzymes showed that the cellulase C₁ activity was optimal at pH 5.0 and 50°C, whereas that of cellulase C_x was optimal at pH 7.0 and 60°C. The enzymes degraded ca 44% of sorghum grains in 6 h, thus suggesting a possible use in saccharification processes. The results also showed the possibility of re-cycling cassava peels as a cheap substrate for the enzyme industry. and accepted 6 June 1989     

Growth, respiration and survival of Legionella pneumophila at high temperatures

The optimum temperature for multiplication of legionella strains in culture media is around 37°C. The effect of high temperatures on the growth of strains isolated from various environments is poorly known. We studied the growth (cell multiplication, respiration) of clinical and environmental Legionella pneumophila strains in liquid media at intervals of 0.5°C in the temperature range from 41.6 to 51.6°C using a temperature gradient incubator. Cell multiplication and CO₂ production decreased markedly with all the strains at temperatures above 44–45°C. CO₂ continued to be produced up to 51.6C even if cell multiplication generally stopped at around 48.4–50.0C. Thus, legionella retained its metabolic activity beyond the maximum temperature for cell multiplication. The CO₂ production per bacterial cell (metabolic quotient, qCO₂) increased with increasing temperature up to 45°C, whereafter it decreased, the turning point being almost at the same at which the rate of cell multiplication decreased. The difference in qCO₂ between the strains may reflect their different physiological capacities for tolerating high temperatures.     

Purification and characterization of cellulolytic enzymes produced by Aspergillus nidulans

Three exo-glucanases, two endo-glucanases and two β-glucosidases were separated and purified from the culture medium of Aspergillus nidulans. The optimal assay conditions for all forms of cellulase components ranged from pH 5.0 to 6.0 and 50°C and 65°C for exo-glucanases and endo-glucanases but 35°C and 65°C for β-glucosidases. A close relation of enzyme stability to their optimal pH range was observed. All the cellulase components were stable for 10 min at 40–50°C. Exo-II and Exo-III ( K _m, 38.46 and 37.71 mg/ml) had greater affinity for the substrate than Exo-I ( K _m, 50.00 mg/ml). The K _m values of Endo-I and Endo-II (5.0 and 4.0 mg/ml) and their maximum reaction velocities ( V _max, 12.0 and 10.0 IU/mg protein) were comparable. β-Glucosidases exhibited K _m values of 0.24 and 0.12 mmol and V _max values of 8.00 and 0.67 IU/mg protein. The molecular weights recorded for various enzyme forms were: Exo-I, 29000; Exo-II, 72500; Exo-III, 138000; Endo-I, 25000; Endo-II, 32500; β-Gluco-I, 14000 and β-Gluco-II, 26000. Exo- and endo-glucanases were found to require some metal ions as co-factors for their catalytic activities whereas β-glucosidases did not. Hg²⁺ inhibited the activity of all the cellulase components. The saccharification studies demonstrated a high degree of synergism among all the three cellulase components for hydrolysis of dewaxed cotton.     

Cellulase production and activity by Trichoderma sp. A-001

Trichoderma species A-001 was grown on various carbon and nitrogen sources supplemented with surfactants on shake cultures. Although the degree of growth was variable, the organism grew on all carbon substrates. Large amounts of the cellulase enzyme components were released into the growth medium during growth on filter paper. In the filter paper containing medium, the organism produced 167 U/ml of carboxymethylcellulase (CMCase), 18 U/ml of filter paper activity (FPase) and 49 U/ml of beta-glucosidase activity (BGDase). Wheat straw and grass were better carbon sources than cotton or barley husks. Nitrogen in the form of KNO₃ was better than NH₄Cl or urea in facilitating the production of cellulase. Of the surfactants used, Tween-80 at 0.2% concentration in the medium increased the production of cellulase several-fold. All the cellulase components were optimally active in the assay at pH 5.5 and 60°C. CMCase and FPase lost 20–33% of their activities when kept at 60°C for 4 h before assaying. On the other hand, BGDase was moderately stable; it lost only 37% of its activity when maintained at 70°C for 4 h.     

Effect of temperature on sulphate reduction, growth rate and growth yield in five psychrophilic sulphate-reducing bacteria from Arctic sediments

Five psychrophilic sulphate-reducing bacteria (strains ASv26, LSv21, PSv29, LSv54 and LSv514) isolated from Arctic sediments were examined for their adaptation to permanently low temperatures. All strains grew at −1.8°C, the freezing point of sea water, but their optimum temperature for growth ( T _opt) were 7°C (PSv29), 10°C (ASv26, LSv54) and 18°C (LSv21, LSv514). Although T _opt was considerably above the in situ temperatures of their habitats (−1.7°C and 2.6°C), relative growth rates were still high at 0°C, accounting for 25–41% of those at T _opt. Short-term incubations of exponentially growing cultures showed that the highest sulphate reduction rates occurred 2–9°C above T _opt. In contrast to growth and sulphate reduction rates, growth yields of strains ASv26, LSv54 and PSv29 were almost constant between −1.8°C and T _opt. For strains LSv21 and LSv514, however, growth yields were highest at the lowest temperatures, around 0°C. The results indicate that psychrophilic sulphate-reducing bacteria are specially adapted to permanently low temperatures by high relative growth rates and high growth yields at in situ conditions.     

Bacterial cellulases and saccharification of lignocellulosic materials

Five locally isolated bacterial strains produced extracellular cellulase enzymes, primarily CMCase, when grown on different natural and commercial cellulosic substrates. Extracellular CMCase and avicelase activity was higher with the strain CLS-32, a Cytophaga sp., compared to four other strains. The whole-cell preparations of these isolates were found to saccharify cellulosic substrates to reducing sugars. Maximum release of reducing sugar (5.75 mg ml⁻¹) was obtained with CLS-32 using sugar cane bagasse as growth and hydrolysis substrates.     

Location of tyrosine phenol-lyase in some Gram-negative bacteria

Abstract From various habitats (plant material, fruits, soil), yeasts belonging to the species of Pichia kluyveri and Hanseniaspora uvarum were isolated that showed killer activity. According to the activity spectrum against other yeasts these strains belonged to 11 different groups that were distinguishable from the killer strains K₁-K₁₀. The isoelectric points of the killer proteins were in the range of pH 3.5–3.9, the activity optimum was observed at pH 4.2–4.6. Above pH 5 and above a temperature of 25–35°C the killer proteins were inactivated.     

Keratinolytic activity of Streptomyces sp. S.K1–02: a new isolated strain

A Streptomyces sp. producing a high keratinolytic activity when cultured on feather meal medium was isolated from a naturally degraded feather. Maximal keratin degradation using supernatant fluid obtained from batch culture of this organism was observed at 70 °C and pH 10. Keratinolytic activity was only partially inhibited by EDTA or PMSF, suggesting that the overall keratinolytic activity was supported by different proteases. Comparisons between proteolytic activities derived from this new strain ( S .K_1–02) and commercial proteases indicated that S .K_1–02 could be a useful biotechnological tool in valorization of keratin-containing wastes, or in the depilation process in the leather industry.     

Effect of various gas atmospheres on the growth and survival of Campylobacter jejuni on beef

Pieces of fresh beef were inoculated with three strains of Campylobacter jejuni . The meat was then allocated to three treatments: (a) vacuum packaged, (b) packaged in an atmosphere of 20% CO₂+ 80% N₂, and (c) packaged into sterile Petri dishes in anaerobic cultivation boxes, which were filled with a gas mixture of 5% O₂+ 10% CO₂+ 85% N₂. The packaging material in the first two treatments was PA 80/PE 100–PE 100/PA 80/PE 100. The survival of Campylobacter cells was followed at 37°C, 20°C and 4°C for 48 h, 4 days and 25 days, respectively. At 37°C the counts of two Campylobacter strains increased in each package treatment for 48 h. At 20°C and at 4°C the counts of the same two strains decreased by 1 to 2 log units and 0.5 to 1 log unit, respectively, during storage. The survival of the two strains was about the same in all package treatments. The third strain was the most sensitive of the strains studied. At 37°C its numbers increased only in the optimal gas atmosphere; at 20°C the strain was not detectable after 24 to 48 h storage and at 4°C after 4 days storage. The aerobic plate counts were determined for all samples at the same time as Campylobacter counts. The high indigenous bacterial numbers of the meat samples did not appear to have a great effect on the survival or growth of campylobacters.     

Modification of matrix polysaccharides during avocado (Persea americana) fruit ripening: an assessment of the role of Cx-cellulase

The role of C_x-cellulase (EC 3.2.1.4) in fruit ripening and softening is unknown. In the present study, avocado ( Persea americana ) fruit, a rich source of C_x-cellulase, were examined to determine if the enzyme plays a role in ripening-related hemicellulose metabolism. Hemicelluloses (4 M alkali-soluble) from avocado fruit exhibited a very broad distribution of polymer sizes and an overall decrease in M_r during ripening. Polymers affected were primarily those of large M_r (relative molecular mass). The characteristic total hemicellulose M_r distribution and changes with ripening were also evident for xyloglucan (XG), a putative substrate for avocado C_x-cellulase. Hydrolytic activity toward hemicelluloses from preripe fruit was detected in crude buffer-soluble protein extracts derived from ripe avocado mesocarp tissue. XG was also degraded, and in a pattern similar to that observed during ripening. Purified C_x-cellulase also exhibited activity against specific components of isolated hemicelluloses; however, in contrast to the crude protein. C_x-cellulase alone was without influence on the M_r distribution of avocado XG. Protein depleted of C_x-cellulase was capable of moderate XG depolymerization. We conclude from the present studies that the enzyme C_x-cellulase is not involved in the ripening-related depolymerization of XG in avocado fruit.     

Partial purification, characterization and regulation of cellulolytic enzymes from Trichoderma longibrachiatum

A net purification of 9·46-, 18·6- and 16·7-fold for filter paper (FP) hydrolytic activity, carboxymethyl (CM) cellulase and β-glucosidase, respectively was achieved through ion exchange and gel chromatographies. The purified enzyme preparation showed an optimal pH of 5·0 for CM cellulase and 5·5 for the other two components. The enzyme activities increased up to 60°–65°C for the three enzyme components and they were stable at 30° or 40°C and pH 4·5 to 5·0 after 20–30 min treatment. The four enzyme components, that is, two FP activities (unadsorbed and adsorbed), a CM cellulase and a β-glucosidase, had K_m values of 47·6 mg, 33·3 mg, 4·0 mg and 0·18 mmol/l with V _max of 4, 1·28, 66·5 and 1·28 units per mg protein. The molecular weights as determined with SDS-PAGE were found to be 44000, 38000, 55000 and 63000 for the above four enzyme components in the same sequence. A distinct type of synergistic action was observed between these components by their action on dewaxed cotton. Glycerol at 1% strongly repressed the formation of all the cellulolytic enzymes. The role of proteolytic enzymes in in vitro inactivation of cellulases was not apparent.     

CMC-liquefying enzyme, a low molecular mass initial cellulose-decomposing cellulase responsible for fragmentation from Streptomyces sp. LX

Streptomyces sp. LX, newly isolated from soil, was shown to secrete a carboxylmethylcellulose (CMC)-liquefying enzyme that cleaves the CMC chains, releasing negligible reducing terminals. The new enzyme, named component C₂, was purified to homogeneity by dialysation. It has a molecular mass of 9·8 kDa. The pH optimum of the enzyme activity is 6·4 and its temperature optimum is 50°C. It retains full activity at pH 4–6·4 upon incubation at 50°C for 30 min. The enzyme has significant fragmentation activity on filter paper despite the absence of weight loss, release of reducing sugars and depolymerization during incubation with filter paper. The one-electron oxidative reaction is shown not to participate in the fragmentation of filter paper by enzyme C₂.     

Effect of temperature and water activity on in vitro germination of Monilinia spp.

Aims:  This study evaluated the effect of temperature (0–38°C) and water activity ( a _w: 0·87–0·99) on the lag phase prior to germination and the percentage of germination over time for Monilinia laxa , Monilinia fructicola and Monilinia fructigena .
Methods and Results:  More than 80% of viable conidia germinated at 25°C and 0·99 a _w within 2 h for M. fructicola and M. fructigena and 4 h for M. laxa . There was no germination at 38°C, and all three Monilinia spp. germinated at 0°C. At the lowest a _w (0·87), none of the Monilinia spp. was able to germinate at any of the incubation temperatures studied. Whereas at 0·90 a _w, conidia were only able to germinate at 15, 25 and 30°C for the three species studied, except for M. fructicola at 15°C. In contrast, at 0·95, 0·97 and 0·99 a _w, germination occurred at all studied temperatures less 38°C. Generally, the lag phase was longer at low levels of a _w (0·90–095), and differences were more evident as temperatures were far from the optimum (0–5°C).
Conclusions:  Germination and lag phase period were markedly influenced by temperature and a _w, and in general when conditions of temperature and a _w were suboptimal, the lag phase was longer and the percentage of germination was lower.
Significance and Impact of the Study:  Knowledge of the germination requirements of this fungus is important in order to understand their behaviour in natural situations and to provide baseline data required for the construction of new prediction models. Our study might be used to develop a predictive model to understand and control the disease caused by Monilinia spp.     

Reappraisal of the effect of temperature on the growth kinetics of Aeromonas salmonicida

The effect of temperature (1–34 °C ) on the maximum specific growth rate of Aeromonas salmonicida could not be described by the classical growth models ; for some strains, two optimal temperatures at 23 °C and 30 °C were observed, as well as an unexpected increase in the pseudolag time above 27 °C. This could be explained by the presence of two subsets, notably S-layer⁺ and S-layer⁻ sub-populations. The A⁻ cells had higher growth parameters (T_opt and μ_opt) than the A⁺ cells and were selected by subcultures above 30 °C. Yet the relative proportion of A⁺ cells did not explain all the variation of μ_max versus temperature, and the growth kinetics of an Aer. salmonicida isolate remained unpredictable.     

A note on Aeromonas spp. from chickens as possible food-borne pathogens

The possible role of Aeromonas spp. as potential food-borne psychrotrophic pathogens was investigated by examining organisms isolated from processed raw chicken for their biochemical characteristics, ability to produce exotoxins and to grow at chill temperatures. These strains, in particular A. sobria , with identical characteristics to human diarrhoea-associated aeromonads were readily found. Chicken, and human and environmental (water) strains characterized in a previous study, were investigated for their ability to grow at refrigeration temperatures (5 ± 2°C) and, for selected strains, the theoretical minimum temperature for growth ( T _min) was determined from the growth pattern in a temperature gradient incubator. All enterotoxigenic chicken strains tested were typical mesophiles, with an optimal growth temperature of ˜37°C and T _min values ˜4.5°C. They were rapidly outgrown by a psychrotrophic Pseudomonas sp. typical of spoilage biota found on food. Enterotoxin was not produced below 15°C by any of the toxigenic food strains tested. The Aeromonas strains isolated from chickens in this study seem unlikely therefore to be a significant health risk, provided the chickens are properly stored and cooked. This would appear to be substantiated by the lack of reports of food-associated outbreaks of illness from these sources.     

Regional and interannual variations in spawning activity of Pacific saury Cololabis saira during northward migration in spring in the north-western Pacific

Regional and interannual variations in spawning activity of Pacific saury Cololabis saira during the northward migration in spring were investigated for 1995–1997 in the north-western Pacific. Only females with a large body size (≥270–290 mm knob length) appeared to spawn. Almost all females in this cohort were actively spawning in a region of sea surface temperature ( T _SS) >18° C, the proportion of active spawners appeared positively related to T _SS in a region of 13° C < T _SS≤ 18° C, and no females were spawning in the region of T _SS≤13° C. Although this relationship between spawning activity and T _SS was common in all years, the distribution of females in the large-size cohort among the regions varied interannually. Consequently, the proportion of active spawners among adult females showed considerable interannual variations.     

Nitrogenase activity (acetylene reduction) in root-associated, cold-climate species of Azospirillum, Enterobacter, Klebsiella and Pseudomonas growing at various temperatures

Abstract 23 Strains of diazotrophic root-associated bacteria isolated from various parts of Finland were tested for nitrogenase activity during growth at various temperatures. Nitrogenase activity was optimal at 20–37°C in cultures of Klebsiella pneumoniae , and at 14–20°C in cultures of Klebsiella terrigena and Enterobacter agglomerans . Strains of K. terrigena and E. agglomerans showed no activity at 37°C, and K. pneumoniae only minimal or no activity at 14°C. Azospirillum lipoferum exhibited high nitrogenase activity at both 28–37°C, but less than 25% of optimal activity at 20°C and no activity at 14°C. Pseudomonas sp. expressed nitrogenase activity at 14–28°C. None of the strains manifested nitrogenase activity at 4 or 42°C. There were only small local variations within a species between strains isolated at different locations.     

Effect of Some Environmental Factors on Psychrophilic Microbacteria

S ummary . The growth of three psychrophilic strains of Microbacterium isolated from meat was studied at 10 temperatures between 0° and 35° and at 6 water activity (a_w) levels between 0.99 and 0.94. The temperature and water relations of the three strains were similar. For all strains the rate of growth was fastest at 25° and a_w 0.99, and growth did not occur at 35°.
Further experiments on one strain showed that under aerobic conditions the range of water activities permitting growth was independent of temperature, but under anaerobic conditions the minimum water activity increased from about 0.94—0.96 as the temperature was reduced from 25° to 0°. Growth was inhibited by low concentrations of undissociated nitrous acid, and inhibition was greater at 0° than at 10° or 25°.     

Temperature profile of oxygen activation and defense against oxygen toxicity in a psychrophilic member of the Bacteroidaceae

Abstract The temperature profiles have been determined for O₂ reduction by activating substrates for whole cells and cell extracts of the psychrophilic, obligately anaerobic bacterium, strain B6, belonging to the Bacteroidaceae. The profiles were similar whether the cells were grown at 15 or 1°C, and also for cells harvested in the exponential or stationary phase. The H₂O producing pyruvate oxidase displayed in cell-free extracts a considerably higher activity than the H₂O₂ producing NADH and NADPH oxidases at all temperatures in the range 30–1°C, and characteristically makes up a larger proportion of the total O₂ reduction capacity the lower the temperature. It thus seems that the O₂ scavenging property of the pyruvate oxidase, postulated to be utilized in a defense mechanism against the detrimental effects of the H₂O₂ producing pyridine nucleotide oxidases, is particularly well adapted to function at the low temperatures of the Barents Sea, from which this obligately anaerobic organism originates.     

Neural function in an alpine grylloblattid: a comparison with the house cricket, Acheta domesticus

ABSTRACT. The effect of temperature on evoked activity in giant interneurons was compared in Grylloblatta sp. (an alpine insect with a temperature preference close to 0°C) and Acheta domesticus. The response to sound and air-puff stimulation of the abdominal cerci was monitored with extracellular electrodes placed under the ventral nerve cord. Responses to air puffs were elicited in Grylloblatta while the animal was supercooled to -6°C. The supercooling point was reached and freezing occurred in the range -5.5 to 8.5°C. The upper extinction temperature could not be determined due to massive spontaneous giant fibre activity in the 12–15°C range masking responses to air puffs. Lethal heat coma occurred at 15–20°C, although most animals did not recover from the spontaneous CNS activity which preceded it. The extinction ranges for Acheta giant neurons were 3.5-4.0°C and 42–44°C. Spike number peaked at 33–36 C and amplitude levels diminished as heat or cold coma was approached; -7.5 C was the mean supercooling point. Both species recovered from cold coma if supercooling was avoided; in Acheta , heat coma was also reversible. Giant fibre latencies in Acheta displayed a Q₁₀ of 1.2 above 11–14°C, below which a break occurred in the curve and delays increased sharply. Latencies for the grylloblattid exhibited a Q₁₀ of 2 but no break in the curve was observed until 0° C was approached. Spike number and amplitude increased with temperature until spontaneous CNS activity obliterated responses to air puffs. The potential role of unsaturated fatty acids in low temperature adaptation in Grylloblatta is discussed.