In vitro production of microtubers for conservation of potato germplasm: Effect of genotype,abscisic acid,and sucrose

Summary With the objective of using microtubers for conservation of potato germplasm, the main effects of genotype, abscisic acid (ABA), and sucrose level, and of their interactions on biomass production, microtuberization, microtuber dormancy, and dry matter content, were studied. ABA decreased both microtuber production and microtuber dormancy, whereas higher concentrations (60–80 gl⁻¹) of sucrose promoted biomass production, microtuber production as well as microtuber dry matter content. Microtubers stored under diffused light had longer dormancy than those kept continuously in the dark. Interactions among various factors conditioned the main effects for some characters. In vitro performance of the genotypes studied was related to their known performance under in vivo conditions for most of the characters. Microtubers produced on media devoid of ABA and containing high sucrose concentrations and N⁶-benzyladenine (44.38 μM) could be stored for 12 mo. under diffused light at 6±1°C.     

Effects of photoperiod,mineral medium strength,inorganic ammonium,sucrose and cytokinin on root,shoot and microtuber development in shoot cultures of Dioscorea alata L. and D. bulbifera L. yams

The effects of photoperiod (8, 12 or 16 h), mineral medium strength (dilutions of a tuberization medium, the T medium), sucrose (0, 2, 4, 8% w/v) and kinetin (0, 2.5μM) on the development of roots, shoots and microtubers in shoot cultures of Dioscorea alata L. and D. bulbifera L. yams were evaluated. All of the factors were found to have substantial effects on microtuber induction in these two species. The effects of high and low inorganic ammonium containing media on microtuberization of yam shoot cultures indicated that ammonium ions inhibited microtuber induction in D. alata but not in D. bulbifera. Microtubers of D. alata were only formed on shoot cultures if these were held under 8-h days. D. bulbifera cultures on the other hand produced microtubers under this photoperiod treatment as well as under 16-h photoperiods provided that kinetin was present in media at 2.5μM. Most microtuberization in D. alata shoot cultures occurred on full-strength T medium supplemented with 2% sucrose, 2.5μM kinetin held under 8-h photoperiod at 25°C, whereas most microtuberization in D. bulbifera shoot cultures occurred on full-strength MS medium supplemented with 4% sucrose, 2.5μM kinetin held under 8-h photoperiods at 25°C. Under these two sets of conditions, yam shoot cultures consistently produced microtubers with individual weights in excess of 100 mg which were large enough to be capable of direct planting and subsequent growth in unsterilized soils.     

Effect of salinity on physiological characteristics,yield and quality of microtubers <Emphasis Type="Italic">in vitro</Emphasis> in potato

In vitro microtuberization provides an adequate experimental model for the physiological and metabolic studies of tuberization and the preliminary screenings of potential potato genotypes. The effects of saline stress at 0–80 mmol concentration on in vitro tuberization of two potato cultivars were investigated in this study. With an increase in the salt concentration, the microtuberization of potato was either delayed by 5–10 days (20 and 40 mmol NaCl) or inhibited completely (80 mmol NaCl) in addition to the reduction in microtuber yields. The two potato genotypes studied showed different trends in total soluble sugars, sucrose and starch contents of microtubers under NaCl stress, while glucose and fructose levels remained unchanged. The vitamin C content in microtubers of two potato genotypes was reduced by salt stress. Salinity applied from 20 to 60 mmol progressively increased proline and malondialdehyde (MDA) levels in microtubers of both the potato cultivars. In genotype Zihuabai, NaCl at a low concentration (20 mmol) led to a significant increase in peroxidase (POD) and polyphenoloxiadase (PPO) activities, while in Jingshi-2, the PPO activity decreased progressively with an increase in NaCl concentrations. Genotype Zihuabai exhibited higher tolerance to salt stress than Jingshi-2 under in vitro conditions. These results could be used for preliminary selections of salt tolerance in potato breeding programmes.     

Effect of 5-Aminolevulinic Acid on Development and Salt Tolerance of Potato (Solanum tuberosum L.) Microtubers in vitro

The effects of 5-aminolevulinic acid (ALA), a key precursor in the biosynthesis of porphyrins such as chlorophyll and heme, on development and salt tolerance of microtubers of two potato (Solanum tuberosum L.) cultivars Jingshi-2 and Zihuabai were examined under in vitro conditions. ALA at 0.3–3 mg/l promoted microtuber formation by increasing the average number, diameter, and fresh weight of microtubers especially under 0.5% NaCl stress conditions, but further increase in ALA concentration resulted in a reduction of microtuber yield irrespective of NaCl stress. Under 1.0% NaCl stress conditions, microtuberization was seriously repressed and could not be restored by the addition of ALA. The accumulation of malondialdehyde in the microtubers treated with 30 mg/l ALA increased by 22% compared to the controls (no salinity), while only a 7% increase was observed when the microtubers were exposed to 0.5% NaCl, indicating that ALA functions as a protectant against oxidative damages of membranes. Under 0.5% NaCl stress conditions, the highest activities of peroxidase and polyphenoloxidase were detected in microtubers treated with ALA at 0.3 and 3 mg/l, being by 73% and by 28% greater than those in the untreated controls, respectively. These results demonstrate that ALA at lower concentrations of 0.3–3 mg/l promotes development and growth of potato microtubers in vitro and enhances protective functions against oxidative stresses, but ALA at 30 mg/l and higher concentrations seems to induce oxidative damage probably through formation and accumulation of photooxidative porphyrins.     

Carboxylic acids affect induction,development and quality of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) microtubers grown <Emphasis Type="Italic">in vitro</Emphasis> from single-node explants

The role of three carboxylic acids with increasing alkyl-chain length, viz., formic, acetic and propionic acids in microtuberization was investigated in three potato (Solanum tuberosum L.) genotypes in vitro. Different concentrations of these carboxylic acids (0.0, 1.5, 3.0, 4.5 and 6.0 mM) were supplemented in microtuber induction medium, which was based on MS medium containing 8% sucrose, and their efficacy for induction, development and quality of microtubers was studied using single-node explants under continuous darkness at 20 °C. The carboxylic acids exhibited a strong stolon- and root-inhibiting effect on single-node explants with their increasing concentrations as well as alkyl-chain length (i.e., formic < acetic < propionic acids), and their mode of action was synonymous with antigibberellin substances. However, they did not have any significant inductive effect on microtuberization as compared to that under 8% sucrose medium. Rather they did show a detrimental effect on microtuber development in terms of average microtuber fresh weight with increasing concentrations as well as alkyl-chain length; both acetic and propionic acids at 6.0 mM induced the smallest microtubers in vitro. The carboxylic acids could, however, significantly increase the harvest indices suggesting their possible role in the regulation of source-sink co-ordination during microtuberization from single-node explants. But the most favourable effect of carboxylic acids on microtubers was apparent in terms of dry matter concomitant with higher starch synthesis and enhanced accumulation of reducing and total sugars. Acetic acid was the most effective in increasing the percentage of microtuber dry matter. The higher percentage of dry matter with higher carbohydrate reserves in microtubers induced by the carboxylic acids could be assumed to affect the quality of microtubers for subsequent storage, dormancy release and sprout growth.     

In vitro shoot culture and microtuber induction in the steroid yam Dioscorea composita Hemsl

The individual effects of sucrose, plant growth regulators and basal salt media formulations were investigated on microtuber induction and development in shoot cultures of the steroid yam Dioscorea composita. Sucrose at 8% (w/v) was the single most significant medium constituent for microtuber induction. Of the four cytokinins tested, 6-benzyladenine at 1.25 and 2.5 μM showed strong inhibitory effects on microtuber induction. By contrast, the auxins α-naphthaleneacetic acid and indole-3-butyric acid at 5.0 μM showed striking promotive effects on microtuber induction and growth. In the presence of either one of these auxins at 5.0 μM shoot cultures produced microtubers weighing 300–400 mg fresh weight whilst kinetin, 6-(γ,γ-dimethylallylamino)-purine, 6-benzyladenine and abscisic acid failed to promote microtuber growth (microtubers weighed generally <200 mg). Media formulations Lloyd and MacCown and White supported the lowest frequencies of microtuber induction when kinetin was present at 2.5 μM. Anderson Rhododendron was as effective as Murashige and Skoog overall in promoting both microtuber induction and growth. When removed from cultures and planted in sterilized moist sand, microtubers sprouted readily (60–87% within 2 weeks) and produced vigorous shoot growth and after 5–7 months minitubers of sizes (30–80 g) suitable for direct field planting. This revised version was published online in June 2006 with corrections to the Cover Date.     

Novel bioreactor technology for mass propagation of potato microtubers

Potato (Solanum tuberosum L. ssp. tuberosum) microtubers were produced in vitro with Liquid Lab™ Rocker system. A thin-layer liquid culture was applied together with a regular pitch in autoclavable simple plastic vessels. All cultures were carried out at room temperature without contamination problems. Each cultivar tested (Asterix, Timo, Van Gogh, Velox) formed microtubers in the Liquid Lab system. The mean number of microtubers per vessel (50 explants) varied between 30 (cv. Asterix in 8 weeks tuber induction) and 75 microtubers (cv. Velox in 11 weeks tuber induction). Majority (63%) of the microtubers was sufficient by size and weight (above 200 mg) for further storage at dormancy (4°C). The cv. Velox yielded the highest number of microtubers with cultivation capacity. As a result of prolonged microtuber induction of 2–3 weeks, more microtubers with competence for cultivation were obtained per cultivar, except for cv. Van Gogh. Still, the mean weight of Van Gogh microtubers was significantly higher after prolonged microtuber induction (0.67 g) than after short induction (0.51 g). In conclusion, Liquid Lab™ Rocker system is a novel, efficient and rapid system for mass propagation of potato.     

Effect of paclobutrazol onin vitro formation of potato microtubers and their sprouting after storage

Paclobutrazol, the gibberellin biosynthesis inhibitor, acceleratedin vitro tuber initiation of potato cv. Rema and increased the uniformity of tuberization period. However, the high concentrations (10–1000 mg l^?1) of this retardant, strongly decreased mass and/or number of microtubers. The microtubers were harvested and stored in darkness (22±2°C) for 250 d. After this period both sprouting and growth of sprouts were affected by previous paclobutrazol treatment.     

Sucrose utilization during potato microtuber growth in bioreactors

 Potato microtubers are used as pathogen-tested in vitro stocks for certified seed potato production. Microtubers grown in a rotating bioreactor grew at a faster rate when the medium was replaced frequently. Although the total microtuber number was not affected, the number of microtubers over 1 g quadrupled when 75% of the medium was replaced every 2 weeks when compared with no medium refreshment. Significantly slower microtuber growth rates resulted when a lower sugar concentration (40 g 1⁻¹ instead of 80 g 1⁻¹) was used or when a mixture of glucose and fructose replaced sucrose. Although high sucrose levels are necessary for optimal microtuber production, the sucrose supplied was rapidly hydrolyzed into glucose and fructose, making the long-term maintenance of desirable sucrose levels difficult. These results indicate that successful strategies to reduce sucrose hydrolysis without inhibiting microtuber growth will improve the efficiency of sucrose utilization in potato microtuber bioreactors. Received: 1 December 1998 / Revision received: 6 May 1999 · Accepted: 19 May 1999     

<Emphasis Type="Italic">In vitro</Emphasis> propagation,microtuberization, and molecular characterization of three potato cultivars

Sprouts of potato tubers were excised from the three potato cultivars Agria, Hermes, and Spunta, sterilized and subjected to shoot formation and propagation on Murashige and Skoog (MS) medium supplemented with 1 mg dm^-3 6-benzylaminopurine (BAP) + 0.5 mg dm^-3 gibberellic acid. Shoots were rooted on MS medium supplemented with 1 mg dm^-3 indole-3-butyric acid. To increase shoot vigour prior tuber formation, shoots were subcultured on MS medium supplemented with 0.56 mg dm^-3 BAP, 0.11 mg dm^-3 2,4-dichlorophenoxyacetic acid, and 0.96 mg dm^-3 naphthaleneacetic acid. Under dark, microtuberization on MS media supplemented with 4 mg dm^-3 of both BAP and kinetin was better than 4 mg dm^-3 BAP alone, where they induced higher number of microtubers per shoot and/or the percentage of shoots that formed microtubers. The highest frequency of microtuber formation was achieved when sucrose at high concentration (8 %) was used as carbon source in culture media. Glucose ranked at the second position whereas fructose reduced the microtuber formation frequency when it was used alone or in combination with glucose. Under the applied culture conditions, cvs. Agria and Hermes showed better micropropagation and microtuberization in comparison to cv. Spunta. In addition, isozyme and RAPD techniques revealed that Agria and Hermes are closer to each other when compared with the third cultivar.     

Microtuber formation in micropropagated Jerusalem artichoke (Helianthus tuberosus)

Clonal micropropagation of Jerusalem artichoke (Helianthus tuberosus L.) was initiated from axillary meristems of lateral shoots of field-grown plants on medium with MS salts, 2% sucrose, 1 mg l-1 thiamine-HCl, 1 mg l-1 IAA and 0.6% agar. Plantlets were cut into nodal sections and used for subsequent subcultures and for microtuber induction. Microtubers were induced from axillary meristems on medium with half-strength MS salts, 8% sucrose and 0.5 mg l-1 BA in darkness at 18 °C. They had near to 30% of dry matter. Microtubers resumed growth in light room at 23 °C after 4–6 months of cold storage. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of jasmonic acid on in vitro explant growth and microtuberization in potato

The shoot fresh mass, root length and root numbers of two potato (Solanum tuberosum L.) cultivars Favorita and Helanwuhua were increased significantly by the application of 0.2 – 2 mg dm⁻³ jasmonic acid (JA) in the Murashige and Skoog medium. However, the growth of potato explants was inhibited by JA at high concentrations (20 – 50 mg dm⁻³). Chlorophyll content in explant leaves decreased with an increase in the concentration of JA. In leaves treated with 0.2 mg dm⁻³ JA acid peroxidase activity increased, while in the leaves treated with more than 2 mg dm⁻³ JA peroxidase activity decreased. Under the dark, the microtuber numbers, fresh mass and percentage of big microtubers of two potato cultivars were not promoted by the application of 0.2 – 50 mg dm⁻³ JA.     

Effect of desiccation to low moisture content on germination,synchronization of root emergence,and plantlet regeneration of black spruce somatic embryos

A desiccation protocol was developed to evaluate the effect of different levels of desiccation on germination and plantlet regeneration of black spruce somatic embryos. Large desiccation chambers (80 l) with four liters of saturated salt solutions provided constant relative humidities (RH) of 63, 79, 88, and 97% (± 2%). Under these conditions, an embryo mass of 10 mg always dried fast even at 97% RH. In contrast, an embryo mass of 80 mg generated different kinetics of water loss, from fast drying at 63% RH to slow drying at 97% RH. Drying rates similar to those obtained with 80 mg embryos were also generated by combining 40 mg embryos with 40 mg water. The effects of drying rate and embryo MC on germination rate, root elongation, and plantlet regeneration were examined. A fast drying rate to 4–5% embryo MC, obtained under 63% RH, was detrimental to germination and plantlet development. However slower drying rates, obtained under 79–97% RH and generating 7–19% MC in the embryos, gave developmental responses similar to the control. Synchronization of root emergence was improved only for embryos desiccated to approx. 16% MC under 97% RH. The optimal desiccation protocol using large desiccation chamber at 97% RH and a constant embryo mass of 40 mg embryos plus 40 mg water was applied to five genotypes of black spruce. For all genotypes, desiccated embryos gave plantlet regeneration rates similar to the control undesiccated embryos. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of photoperiod and thermoperiod on microtuberization and carbohydrate levels in Cocoyam (Xanthosoma sagittifolium L. Schott)

Cocoyam (Xanthosoma sagittifolium L. Schott) is an important staple tuber crop for tropic populations and consists of three cultivars with different productivity. In vitro tuber induction of a broad range of genotypes could be a very useful way to propagate and increase valuable cocoyam. Shoot tips were cultured on a multiplication medium containing MS mineral salts, Morel and Wetmore (Ann J Bot 38:141–143, 1951) vitamins, 3% sucrose and 6% agar. Multiple shoots induced with 6-benzylaminopurine (BAP) were allowed to develop on the basal medium without plant growth regulators (PGRs). The subsequent explants obtained were used for the production of microtubers on MS-based PGRs free medium supplemented with 8% sucrose. Microtuberization was evaluated after 60 days of culture under different photo- and thermoperiod regimes. Changes in carbohydrates were also examined enzymatically during this process. Microtuberization response of three cocoyam cultivars (White, Red and Yellow) was influenced by photoperiod and thermoperiod regimes. A short photoperiod (8 h lighting), combined with an application of a day/night temperature regime 25/20°C for 10 days, followed by a continuous darkness at 20°C for 50 days, was the most effective treatment. The best response was observed with White followed by Red and Yellow cultivar. The sprouting frequency of microtubers varied significantly with cvs. after 15 days of culture (56% for White, 50% for Red and 40% for Yellow). Under tuber promoting condition, the onset of the process was characterized by an accumulation of starch and hexoses in leaves. The glucose:fructose ratio changed in favour of glucose earlier in White cultivar leaves where it doubled in 10 days. Moreover, in young developing tubers, the sucrose content increased concomitantly with starch.     

The role of GA,IAA and BAP in the regulation of <Emphasis Type="Italic">in vitro</Emphasis> shoot growth and microtuberization in potato

The effect of auxin, GA and BAP on potato shoot growth and tuberization was investigated under in vitro condition. The shoot length of potato explants increased with the increasing of concentrations (0.5 – 10 mg dm⁻³) of IAA treatment especially with the addition of GA₃ (0.5 mg dm⁻³), but was inhibited by BAP (5 mg dm⁻³). The root number and root fresh weight of potato explants increased with the increasing of IAA levels either in the presence of GA₃ (treatment IAA+GA) or not (IAA alone). However, no root was observed in the treatment IAA+BAP, instead there were brown swollen calli formed around the basal cut surface of the explants. The addition of GA₃ remarkably increased the fresh weight and diameter of calli. Microtubers were formed in the treatments of IAA+BAP and IAA + GA + BAP but not observed in the treatments of IAA alone or IAA + GA. IAA of higher concentrations (2.5 – 10 mg dm⁻³) was helpful to form sessile tubers. With the increasing of IAA levels, the fresh weight and diameter of microtubers increased progressively. At 10 mg/L IAA, the fresh weight and diameter of microtubers in the treatment of IAA + GA + BAP were 409.6 % and 184.4 % of that in the treatment of IAA + BAP respectively, indicating the interaction effect of GA and IAA in potato microtuberization.     

Methods of short-term storage of cultures of the blowfly <Emphasis Type="Italic">Calliphora Vicina</Emphasis> R.-D. (Diptera,Calliphoridae)

Three methods of short-term storage of the blowfly Calliphora vicina strains are considered based on the experimental study of 21 strains originating from different parts of the species range. The colony can be preserved as diapausing adults at 6° and darkness for 2–3 months or more, depending on the geographical origin of the population. During the first five days of adult life the flies should be kept at 12° and short day on a sugar diet, after which they should be transferred into a refrigerator. During artificial hibernation the flies also require periodic sugar feeding every 20 days (3–4 h at 20°C) to maintain their vital functions. The combination of temperatures of 20–23°C and a protein diet terminates reproductive diapause, and oviposition starts in 10–17 days. The fly strain may be preserved as reproductive females at 6°C and darkness with sugar feeding. Flies also require periodic sugar feeding at 20°C (3–4 hours). In this case the flies start laying eggs 2–3 days after being transferred to 20–23°C. The preservation of diapausing larvae is a more reliable method of prolonged strain storage. In this case the flies of maternal generation are maintained at 20–23°C on sugar and protein diet. The egg rafts laid during 5–6 hours are then transferred into 12°C and short day until hatchment. The hatched larvae should be immediately placed into a refrigerator (2–3 or 5–6°C), where they feed during 1–1.5 months and enter diapause. For strain restoration, the diapausing larvae should be transferred into 20–25°C, where they pupariate in 3–5 days and the flies emerge in nearly 10 days.     

Studies of the effect of environmental factors on the rotifer predator–prey system in freshwater

The aim of this work is to evaluate the effect of environmental factors: temperature and photoperiod on the zooplankton predator–prey system. Rotifers, an important and cosmopolitan group of zooplankton in freshwater, were used in our study. We investigated the effect of temperature (20, 23, and 30°C) and of photoperiod (L:D = 12:0 and 0:12) on the predatory rotifer Asplanchna brightwelli consuming rotifer Brachionus calyciflorus as prey. Under A. brightwelli predation, populations of B. calyciflorus prey were consumed more slowly at 20 ± 1 and 30 ± 1°C as compared to 23 ± 1°C. Prey consumption by A. brightwelli increased from 0.63 ± 0.09 ind. predator⁻¹ at 20°C to a peak of 1.22 ± 0.12 ind. predator⁻¹ at 23°C, then decreased significantly to 0.93 ± 0.14 ind. predator⁻¹ at 30 ± 1°C. In addition, predation responded to temperature changing sensitively and rapidly. Statistical analysis showed that the prey consumption were significant different under altered temperature periods during 12 h. Photoperiod also significantly influenced the rate of A. brighwelli predation. B. calyciflorus suffered less predation in darkness than in light. The rate of prey consumption in light (1.06 ind. predator⁻¹) was twice the average of that in darkness (0.51 ind. predator⁻¹). Furthermore, predation rate varied under changing photoperiod but predators moved back into the light did not resume their original consumption rate. Our results demonstrate that whether the predation in rotifer successfully or not is strongly influenced by temperature and photoperiod.     

Kinetic comparisons of mesophilic and thermophilic aerobic biomass

Kinetic parameters describing growth and decay of mesophilic (30°C) and thermophilic (55°C) aerobic biomass were determined in continuous and batch experiments by using oxygen uptake rate measurements. Biomass was cultivated on a single soluble substrate (acetate) in a mineral medium. The intrinsic maximum growth rate (μ _max) at 55°C was 0.71±0.09 h⁻¹, which is 1.5 times higher than the μ _max at 30°C (0.48±0.11 h⁻¹). The biomass decay rates increased from 0.004 h⁻¹ at 30°C to 0.017 h⁻¹ at 55°C. Monod constants were very low for both types of biomass: 9±2 mg chemical oxygen demand (COD) l⁻¹at 30°C and 3±2 mg COD l⁻¹at 55°C. Theoretical biomass yields were similar at 30 and 55°C: 0.5 g biomass COD (g acetate COD)⁻¹. The observed biomass yields decreased under both temperature conditions as a function of the cell residence time. Under thermophilic conditions, this effect was more pronounced due to the higher decay rates, resulting in lower biomass production at 55°C compared to 30°C. Electronic Publication     

Optimization of immersion frequency and medium substitution on microtuberization of Chlorophytum borivilianum in RITA system on production of saponins

In this study, different immersion times were compared for optimization of Chlorophytum borivilianum microtuber production in a RITA (Récipient à Immersion Temporaire Automatique) system. Significantly higher mean number and length of microtubers and growth index were obtained (16, 52 mm and 16, respectively) with 15 min immersion in microtuberization medium (MM) for every 60 min rest period, and could eliminate the occurrence of hyperhydricity. The substitution of MM with liquid hormone-free MS (MSO) medium on week 6 of culture differed significantly in terms of mean number and length of microtubers and growth index compared to earlier substitution at week 3 with MSO but did not differ significantly in comparison to cultures maintained throughout the 9 weeks in MM. Comparison of total saponin of TIS-derived microtubers with tubers of field grown mother plant showed a 1.3-fold increase of saponin (≈21% diosgenin) in the microtubers. The above results hold a promising application of TIS for large scale production of microtubers and accumulated saponin in C. borivilianum.     

Influence of temperature on process efficiency and microbial community response during the biological removal of chlorophenols in a packed-bed bioreactor

Two reactors, initially operated at 14 and 23±1°C (RA and RB, respectively), were inoculated with a bacterial consortium enriched and acclimatized to the respective temperatures over 4 months. The biofilms, formed in the reactors, were studied using scanning electron microscopy, cultivation of the biofilm microflora, and physiological analysis of the isolates. Two bacteria able to mineralize chlorophenols under a large range of temperature (10–30°C) were isolated from the biofilm communities of reactors RA and RB and characterized as Alcaligenaceae bacterium R14C4 and Cupriavidus basilensis R25C6, respectively. When temperature was decreased by 10°C, the chlorophenols removal capacity was reduced from 51.6 to 22.8 mg l⁻¹ h⁻¹ in bioreactor RA (from 14 to 4°C) and from 59.3 to 34.7 mg l⁻¹ h⁻¹ in bioreactor RB (from 23±1 to 14°C). Fluorescence in situ hybridization (FISH) of the biofilm communities showed that, in all temperatures tested, the β-proteobacteria were the major bacterial community (35–47%) followed by the γ-proteobacteria (12.0–6.5%). When the temperature was decreased by 10°C, the proportions of γ-proteobacteria and Pseudomonas species increased significantly in both microbial communities.