Isolation and Identification of Camptothecin from Cells of Camptotheca acuminata Suspension Cultures

Ten strains of non-sulfur purple photosynthetic bacteria were isolated from soil and water samples gathered in Bangkok and its surrounding area. The isolated strains from Thailand were divided into two groups, Al to A4 and BI to B6. They were identified as Rhodopseudomonas gelatinosa and Rhodopseudomonas sphaeroides, respectively. All strains grew well either at 30°C or 40°C, but failed to grow at 45°C. Strains belonging to group A had weak activities of nitrogenase (acetylene reduction) and hydrogen production, while strains of group B showed much higher activities than group A. The activities of nitrogenase and hydrogen production of isolates in Thailand were compared with those of isolates in Japan. The activities of isolated strains in Thailand at 40°C were almost equal to those at 30°C or even higher. On the other hand, both hydrogen production and the nitrogenase activity of isolates in Japan decreased significantly at 40°C as compared to the activities at 30°C. These results suggest an intrinsic thermostability in hydrogen production by the non-sulfur purple photosynthetic bacteria of Thailand. Among isolated strains in Thailand, strain B5 was the most active in nitrogenase and hydrogen production, and its activity was significantly higher than strain TN3 at 40°C. TN3 had been selected as the most active strain among isolates in the Sendai area.     

Biomethanation: Anaerobic fermentation of CO2, H2 and CO to methane

Studies to examine the microbial fermentation of coal gasification products (CO₂, H₂ and CO) to methane have been done with a mixed culture of anaerobic bacteria selected from an anaerobic sewage digestor. The specific rate of methane production at 37°C reached 25 mmol/g cell hr. The stoichiometry for methane production was 4 mmol H₂/mol CO₂. Cell recycle was used to increase the cell concentration from 2.5 to 8.3 g/liter; the volumetric rate of methane production ran from 1.3 to 4 liter/liter hr. The biogasification was also examined at elevated pressure (450 psi) and temperature to facilitate interfacing with a coal gasifier. At 60°C, the specific rate of methane production reached 50 mmol/g cell hr. Carbon monoxide utilization by the mixed culture of anaerobes and by a Rhodopseudomonas species was examined. Both cultures are able to carry out the shift conversion of CO and water to CO₂ and hydrogen.     

Selection and characterization of a high-temperature tolerant strain of <Emphasis Type="Italic">Porphyra haitanensis</Emphasis> Chang et Zheng (Bangiales,Rhodophyta)

Porphyra haitanensis is one of the most economical nutritive marine algae; however, its production and quality are significantly jeopardized by high temperatures. Selection of heat-resistant strains will greatly reduce the economic risks and benefit to the nori industry. Three previously isolated and improved strains with a high yield were screened at 28°C and identified, of which one strain, ZS-1, showed significantly improved heat tolerance. Upon further characterizing of the cultures of the ZS-1 strain and the wild-type (WT) strain at 28°C and 30°C, the ZS-1 conchospore germlings survived at rates of 69.9% and 59.6%, while the WT conchospore germlings survived at significantly lower rates of 15.9% and 6.7%, respectively, over a period of 15 days. Furthermore, ZS-1 conchospore germlings divided at significantly higher rates of 100% and 88.6% compared to the WT conchospore germlings with 90.4% and 63.8%, respectively. When the 35-day-old conchospore germlings were transferred from the optimal temperature of 24°C to higher temperatures of 28°C and 30°C, the ZS-1 blades sustained growth over a 25-day period without decay and increase of blade lengths with a factor of 18.5 and 10.3 times, respectively. The blade lengths of the WT germlings only increased by a factor of 1.7 and 0.9 times and began to decay after being cultured for 15 days at 28°C and 30°C. At 24, 28, and 30°C, the ZS-1 blades grew 3.4, 8.6, and 8.0 times faster than those of the WT. Evidently, ZS-1 is a fast-growing and heat-resistant strain compared to the WT strain and may offer an alternative for the nori industry.     

Studies on α-amylase production by Bacillus licheniformis MIR-61

An acid α-amylase hyperproducing strain, designated as MIR-61, was isolated in a screening procedure from South American soil samples. MIR-61, a 60°C thermoresistant strain, was identified using 98 biochemical and morphological tests and characterized as Bacillus licheniformis by numerical taxonomy. Batch cultures of B. licheniformis MIR-61 showed extracellular α-amylase and α-glucosidase activities during the exponential growth phase. The production of α-amylase was studied at free and constant pH values at 37 and 45°C. Maximum α-amylase activity (4,767 kU/dm³ in a liquid medium) was detected at 45°C at a constant pH (7.0) in the late exponential phase. The α-amylase production by B. licheniformis MIR-61 is 10 to 300 times higher than the enzyme production reported in strains of the same species. Optimum α-amylase activity was found at 50 to 67°C in an acid pH range from 5.5 to 6.0. These properties would allow its use in starch industry processes.     

Isolation and Identification of Acetic Acid Bacteria for Submerged Acetic Acid Fermentation at High Temperature

Industrial vinegar production by submerged acetic acid fermentation has been carried out using Acetobacter strains at about 30°C. To obtain strains suitable for acetic acid fermentation at higher temperature, about 1,100 strains of acetic acid bacteria were isolated from vinegar mash, soils in vinegar factories and fruits, and their activities to oxidize ethanol at high temperature were examined. One of these strains, No. 1023, identified as Acetobacter aceti, retained full activity to produce acetic acid in continuous submerged culture at 35°C and produced 45% of activity at 38°C, while the usual strain of A. aceti completely lost its activity at 35°C. Thus the use of this strain may reduce the cooling costs of industrial vinegar production.     

Utilization of protein-hydrolyzed cheese whey for production of β-galactosidase by Kluyveromyces marxianus

We studied the utilization of protein-hydrolyzed sweet cheese whey as a medium for the production of β-galactosidase by the yeasts Kluyveromyces marxianus CBS 712 and CBS 6556. The conditions for growth were determined in shake cultures. The best growth occurred at pH 5.5 and 37°C. Strain CBS 6556 grew in cheese whey in natura, while strain CBS 712 needed cheese whey supplemented with yeast extract. Each yeast was grown in a bioreactor under these conditions. The strains produced equivalent amounts of β-galactosidase. To optimize the process, strain CBS 6556 was grown in concentrated cheese whey, resulting in a higher β-galactosidase production. The β-galactosidase produced by strain CBS 6556 produced maximum activity at 37°C, and had low stability at room temperature (30°C) as well as at a storage temperature of 4°C. At −4°C and −18°C, the enzyme maintained its activity for over 9 weeks. Received 20 January 1999/ Accepted in revised form 30 April 1999     

Fermentation production of keratinase from Bacillus licheniformisPWD-1 and a recombinant B. subtilis FDB-29

Bacillus licheniformis PWD-1, the parent strain, and B. subtilis FDB-29, a recombinant strain. In both strains, keratinase was induced by proteinaceous media, and repressed by carbohydrates. A seed culture of B. licheniformis PWD-1 at early age, 6–10 h, is crucial to keratinase production during fermentation, but B. subtilis FDB-29 is insensitive to the seed culture age. During the batch fermentation by both strains, the pH changed from 7.0 to 8.5 while the keratinase activity and productivity stayed at high levels. Control of pH, therefore, is not necessary. The temperature for maximum keratinase production is 37°C for both strains, though B. licheniformis is thermophilic and grows best at 50°C. Optimal levels of dissolved oxygen are 10% and 20% for B. licheniformis and B. subtilis respectively. A scale-up procedure using constant temperature at 37°C was adopted for B. subtilis. On the other hand, a temperature-shift procedure by which an 8-h fermentation at 50°C for growth followed by a shift to 37°C for enzyme production was used for B. licheniformis to shorten the fermentation time and increase enzyme productivity. Production of keratinase by B. licheniformis increased by ten-fold following this new procedure. After respective optimization of fermentation conditions, keratinase production by B. licheniformis PWD-1 is approximately 40% higher than that by B. subtilis FDB-29. Received 16 July 1998/ Accepted in revised form 07 March 1999     

Glutamic Acid Production in Biotin-rich Media by Temperature-sensitive Mutants of Brevibacterium lactofermentum,a Novel Fermentation Process

Temperature-sensitive mutants were derived from Brevibacterium lactofermentum strain 2256 in a search for mutants which would produce a large amount of L-glutamic acid in biotin- rich media at the nonpermissive temperature. A total of 159 mutant strains was selected which showed adequate growth at 30°C but showed little or no growth at 37°C on minimal medium. Twenty of these were found to produce glutamic acid in a biotin-rich medium after a temperature shift from 30°C to 37°C, while the wild-type strain 2256 did not produce it under the same cultural condition.

One of the typical mutant strains, Ts-88, produced approximately 2g/dl of glutamic acid from beet molasses (the yield > 55%) in the presence of 33 µg/liter of biotin when tempera- , ture was shifted from 30°C to 40°C during the cultivation. It was concluded that, by controlling only temperature during fermentation, glutamic acid production could be realized in media containing biotin-rich natural carbon sources, without any chemical control such as the addition of expensive surface-active agents or antibiotics. Characteristics and merits of the novel fermentation process are discussed.     

Growth,trichome size and akinete production of Cylindrospermopsis raciborskii (cyanobacteria) under different temperatures: Comparison of two strains isolated from the same pond

Coiled morphotype Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju that forms a water bloom in a shallow pond in northern Taiwan exhibits a negative correlation between trichome size and temperature. To investigate how temperature influences the growth and trichome size of C. raciborskii, two C. raciborskii strains isolated from the pond in August and February were grown at three temperatures (18, 24 and 30°C). Both strains exhibited the lowest and highest specific growth rates at 18°C and 24°C, respectively, and the trichomes became the largest at 18°C. However, specific growth rates of the strain isolated in August exceeded those of the strain isolated in February, and the trichomes of the strain isolated in February were larger than those of the strain isolated in August regardless of temperature. Moreover, although both strains produced larger numbers of akinetes at higher temperatures, the strain isolated in August produced many more akinetes than did the other. These findings suggest that the two strains are not identical, leading to the conclusion that the C. raciborskii population in the pond consists of at least two ecotypes. Large trichome formation and akinete production are thought to be different types of countermeasure against cold of C. raciborskii, and the patterns of investment in developing these functions seemed to vary between the strains.     

Isolation and Characterization of a Novel Ice-nucleating Bacterium,Pseudomonas sp. KUIN-4, Which Has Stable Activity in Acidic Solution

A novel ice-nucleating bacterium, KUIN-4 was isolated from a cherry leaf, which was unsusceptible to frost injury. Strain KUIN-4 was identified as Pseudomonas sp. from its characteristics and taxonomies; the optimum temperature and pH for its growth were 18°C and 4.5, respectively. When strain KUIN-4 was cultured aerobically in CYE medium (pH 4.5) for 48 h at 18°C, the ice-nucleating activities of strain KUIN-4 cells and culture broth (extracellular ice-nucleating matter) after removal of the cell were obtained. The ice-nucleating temperature, T₅₀ (°C), was indicated to be –2.8°C in cell suspensions (4.25 x 10⁷ cell/ml) of strain KUIN-4. Also, it had become apparent that the ice-nucleating activity involved class A, B, and C structures as judged by its freezing difference spectrum in D₂O versus H₂O. The ice-nucleating activity of this strain as well as other ice-nucleating bacteria was significantly decreased by heat treatment (40°C, 30 min). The ice-nucleating activity from this strain had unique features, which was stable under acidic conditions (pH 3.5–5.0) and was weakly inhibited by denaturants and protein-modifying reagents.     

Induction of Freeze-sensitive Mutants from a Freeze-tolerant Yeast,Torulaspora delbrueckii

Freeze-sensitive strains of yeast were induced from a freeze-tolerant yeast Torulaspora delbrueckii by incubation with ethyl-methane sulfonate as a mutagen. A maximum ratio of mutation was attained by the incubation at 30°C for 75min. One-hundred and fifty strains of freeze-sensitive yeast were selected by plating-culture for the first screening. The freeze-tolerance ratio of each strain was examined based on the fermentative activity before and after freezing in liquid medium and dough. Strain 60B3 showed the highest freeze-sensitivity in a pre-fermented frozen dough (pre-fermented at 30°C for 2h, and frozen at ?20°C for 7 days) among eight strains finally selected.     

Feeding of a Freshwater Flagellate,Bodo saltans,on Diverse Bacteria1

ABSTRACT Bodo saltans was isolated from a chalk stream and fed with pure cultures of seven bacteria obtained from the same river. The flagellates were allowed to migrate into suspensions of either of two bacterial species in a T-maze at 20–22°C. There was a significant difference (P < 0.01) between the numbers of flagellates which migrated into suspensions of different bacteria, which were subsequently arranged in an order of “attractiveness” to the flagellate. Bodo saltans grew successfully in monoxenic suspensions of all seven bacterial strains, but more rapid growth occurred with non-flagellated than with flagellated bacteria; this may be because while feeding, B. saltans tends to associate with surfaces where non-flagellated bacteria may also congregate. The efficiency with which B. saltans is able to utilize different bacteria may be influenced by the motility or secretory activities of the bacteria. There was no incontrovertible evidence that B. saltans responds to specific bacterial attractants.     

Growth and sporulation of entomopathogenic Beauveria bassiana,Metarhizium anisopliae,Isaria farinosa and Isaria fumosorosea strains in relation to water activity and temperature interactions

This study examined six strains of Beauveria bassiana s.l. and Isaria farinosa, one strain of Isaria fumosorosea and five strains of Metarhizium anisopliae s.l. to identify the ability for (1) growth and (2) sporulation under interacting environmental factors of water activity (a_w) and temperature stress. Growth on Sabouraud Dextrose Agar (SDA; water activity, a_w = 0.995) or SDA modified with glycerol to 0.98, 0.96 and 0.94 a_w was measured at four different temperatures (25, 30, 35 and 37°C). All M. anisopliae strains grew at 25–35°C and 0.995 a_w while only two strains tolerated extreme water stress at 0.94 a_w.Three strains of B. bassiana were able to grow at 25–37°C and 0.995 a_w. Only one strain of I. farinosa was able to grow at 25–37°C and 0.995 a_w. A_w and temperature interactions resulted in different strain-dependent responses, in terms of growth and sporulation. Only one strain of I. farinosa and three of M. anisopliae grew at 0.94 a_w and none of the B. bassiana strains tolerated such water stress. At 0.96 and 0.94 a_w and 35–37°C, sporulation by all the strains of the three species were significantly affected. Under elevated temperatures and drought stress, very few of these strains of entomopathogenic fungi are able to grow and sporulate. Indeed, the B. bassiana strains were unable to tolerate the extreme conditions examined. Resilience to such abiotic interactions is critical for selecting strains for formulations. Tolerance to water and temperature stress could be good criteria for selection of strains with secondary spread potential for use as part of an integrated pest management system where secondary cycling may be important, especially in sub-tropical and tropical environments.     

Activation of an Aquareovirus,Chum Salmon Reovirus (CSV), by the Ciliates Tetrahymena thermophila and T. canadensis

For the first time, ciliates have been found to activate rather than inactivate a virus, chum salmon reovirus (CSV). Activation was seen as an increase in viral titre upon incubation of CSV at 22 °C with Tetrahymena canadenesis and two strains of T. thermophila: wild type (B1975) and a temperature conditional mutant for phagocytosis (NP1). The titre increase was not likely due to replication because CSV had no visible effects on the ciliates and no vertebrate virus has ever been shown unequivocally to replicate in ciliates. When incubated with B1975 and NP1 at 30 °C, CSV was activated only by B1975. Therefore, activation required CSV internalization because at 30 °C only B1975 exhibited phagocytosis. CSV replicated in fish cells at 18 to 26 °C but not at 30 °C. Collectively, these observations point to CSV activation being distinct from replication. Activation is attributed to the CSV capsid being modified in the ciliate phagosomal‐lysosomal system and released in a more infectious form. When allowed to swim in CSV‐infected fish cell cultures, collected, washed, and transferred to uninfected cultures, T. canadensis caused a CSV infection. Overall the results suggest that ciliates could have roles in the environmental dissemination of some fish viral diseases.     

REVERSAL OF HELIX ORIENTATION IN THE CYANOBACTERIUM ARTHROSPIRA1

A survey of the morphological characters of 36 clonal axenic strains of Arthrospira showed that 34 had helical and 2 had straight trichomes. Of those with helical trichomes, five were right‐handed and 29 left‐handed. After repeated subculture for 1 year, the orientation of one helical strain (D893) had changed from right‐ to left‐handed, suggesting a probable genetic shift. The influence of environmental factors on helix orientation was tested on a subset of 10 strains. A temperature upshift from 30 to 32–34° C for 7 days led to a change in orientation in three strains (D918/H, D923, D925). Incubation at 32° C (D918/H, D923) or 34° C (D925) for periods less than needed for the morphological change to show still permitted the change to take place subsequently, when the temperature was reduced to 30° C; however, further subculture at 30° C led to the orientation reverting to its original state. In strain D925, but not the other nine strains, continuous shaking at 30° C also led to a change in helix orientation. In this case, some trichomes showed both orientations in a single trichome, with a snag at the point of reversion. A repeat survey of the stock cultures of all 34 strains after 2 years showed that another strain (D918/H) had now changed orientation from right‐handed to left‐handed. These observations are compared with the behavior of other helical structures in the literature, including filamentous Bacillus subtilis mutants and helix reversal in tendrils of climbing plants.     

Obligately and facultatively autotrophic,sulfur- and hydrogen-oxidizing thermophilic bacteria isolated from hot composts

A variety of autotrophic, sulfur- and hydrogen-oxidizing thermophilic bacteria were isolated from thermogenic composts at temperatures of 60–80° C. All were penicillin G sensitive, which proves that they belong to the Bacteria domain. The obligately autotrophic, non-spore-forming strains were gram-negative rods growing at 60–80°C, with an optimum at 70–75°C, but only under microaerophilic conditions (5 kPa oxygen). These strains had similar DNA G+C content (34.7–37.6 mol%) and showed a high DNA:DNA homology (70–87%) with Hydrogenobacter strains isolated from geothermal areas. The facultatively autotrophic strains isolated from hot composts were gram-variable rods that formed spherical and terminal endospores, except for one strain. The strains grew at 55–75° C, with an optimum at 65–70° C. These bacteria were able to grow heterotrophically, or autotrophically with hydrogen; however, they oxidized thiosulfate under mixotrophic growth conditions (e.g. pyruvate or hydrogen plus thiosulfate). These strains had similar DNA G+C content (60–64 mol%) to and high DNA:DNA homology (> 75%) with the reference strain of Bacillus schlegelii. This is the first report of thermogenic composts as habitats of thermophilic sulfur- and hydrogen-oxidizing bacteria, which to date have been known only from geothermal manifestations. This contrasts with the generally held belief that thermogenic composts at temperatures above 60° C support only a very low diversity of obligatory heterotrophic thermophiles related to Bacillus stearothermophilus. Received: 20 July 1995 / Accepted: 25 September 1995     

Comparative laboratory studies on three fungal pathogens of the elm bark beetle Scolytus scolytus: Effect of temperature and humidity on infection by Beauveria bassiana,Metarhizium anisopliae, and Paecilomyces farinosus

Larvae of the elm bark beetle, Scolytus scolytus, were inoculated with conidia of the entomogenous fungi Beauveria bassiana (two strains), Metarhizium anisopliae (two strains), and Paecilomyces farinosus (two strains) and incubated over a range of temperatures (2°, 6°, 10°, 15°, and 20°C). One strain each of B. bassiana and P. farinosus caused infection even at 2°C, whereas the two strains of M. anisopliae caused no infection below 10°C. Infection of adult beetles by B. bassiana (one strain) and M. anisopliae (one strain) was tested at 15°, 20°, and 25°C (B. bassiana) and at 15° and 20°C (M. anisopliae). Fungal infection occurred at all three temperatures, but at 25°C beetles tended to succumb to bacterial infection. The effect of relative humidity on infection of larvae by B. bassiana (one strain), M. anisopliae (one strain), and P. farinosus (one strain) was tested at 51, 74, 86, 90, 95, 97.5, and 100% relative humidity. B. bassiana and M. anisopliae caused some infection at all humidities: with P. farinosus there was no infection at the two lowest humidities. Mortality due to infection by these fungi was most rapid at the highest humidities.     

Hydrogen production of a salt tolerant strain Bacillus sp. B2 from marine intertidal sludge

To isolate a salt tolerant hydrogen-producing bacterium, we used the sludge from the intertidal zone of a bathing beach in Tianjin as inoculum to enrich hydrogen-producing bacteria. The sludge was treated by heat-shock pretreatment with three different temperature (80, 100 and 121°C) respectively. A hydrogen-producing bacterium was isolated from the sludge pretreated at 80°C by sandwich plate technique and identified using microscopic examination and 16S rDNA gene sequence analysis. The isolated bacterium was named as Bacillus sp. B2. The present study examined the hydrogen-producing ability of Bacillus sp. B2. The strain was able to produce hydrogen over a wide range of initial pH from 5.0 to 10.0, with an optimum at pH 7.0. The level of hydrogen production was also affected by the salt concentration. Strain B2 has unique capability to adapt high salt concentration. It could produce hydrogen at the salt concentration from 4 to 60‰. The maximum of hydrogen-producing yield of strain B2 was 1.65 ± 0.04 mol H₂/mol glucose (mean ± SE) at an initial pH value of 7.0 in marine culture conditions. Hydrogen production under fresh culture conditions reached a higher level than that in marine ones. As a result, it is likely that Bacillus sp. B2 could be applied to biohydrogen production using both marine and fresh organic waste.     

Temperature and pH affect the production of bacterial biofilm

The effect of different cultivation temperatures (30 and 37 °C) and pH of the media (5.5, 7.5, 8.5) on the biofilm production was compared in Pseudomonas aeruginosa, Klebsiella pneumoniae, and Vibrio cholerae non-O1 and O1 using the crystal-violet test for estimation of quantitative production of the biofilm. Decrease (46.4–98.4 %) in the biofilm production was observed at 37 °C in 8 of the tested strains (P. aeruginosa three strains, K pneumoniae two, V. cholerae non-O1 two, and V. cholerae O1 one strain) compared with the production at 30 °C. On the other hand, five strains (P. aeruginosa 1, K. pneumoniae 3, V. cholerae non-O1 1) exhibited under these conditions a higher biofilm production (103–143 %). However, this difference was not significant (p = 0.196). Increased pH lead to a higher biofilm production using all media tested. In P. aeruginosa the biofilm production at pH 8.5 was 139–244 %, at pH 7.5 136–164 % in comparison with pH 5.5. Similarly, in K. pneumoniae the biofilm production increased to 151–319 % at pH 8.5 while with the drop of pH to 7.5 the biofilm production was 113–177 % compared with pH 5.5. In V. cholerae non-O1 and O1 the biofilm production reached 204–329 % at pH 8.5, and 123–316 % at pH 7.5 (compared with the production at pH 5.5). An increase in biofilm production represented an average of 169 % (p = 0.001) at pH change from 5.5 to 7.5, with the rise of pH from 5.5 to 8.5 caused an average difference of 229 % (p = 0.001).     

Exposure of Fischerella [Mastigocladus] to high and low temperature extremes: strain evaluation for a thermal mitigation process

In conjunction with a proposed algal cultivation scheme utilizing thermal effluent, twelve Fischerella strains were tested for tolerance to temperatures above and below their growth range. Exposure to 65 °C or 70 °C for 30 min caused bleaching and death of most or all cells. Effects of 60 °C exposure for periods of up to 2 h ranged from undetectable to severe for the various strains. Chlorophyll a content typically decreased 21–22% immediately following 60 °C or 65 °C (1 h) exposure. However, the 60 °C-shocked cultures regained normal Chl a content after 24 h at 45 °C, whereas Chl a in 65 °C-shocked cultures immediately lost visible autofluorescence and was later degraded. Exposure to 15 °C virtually stopped growth of all strains during a 48 h exposure period. Most strains grew as rapidly as 45 °C controls when restored to 45 °C, while a few strains recovered more slowly. Comparison with dark-incubated controls indicated that photooxidative damage did not occur during cold shock. Certain strains exhibited relatively rapid recovery from both heat and cold exposure, thus meeting the temperature tolerance criteria for the proposed algal cultivation process.