Different organization of nif genes in nonheterocystous and heterocystous cyanobacteria

Summary Labeled probes carrying the Anabaena PCC 7120 nitrogenase (nifK and nifD) and nitrogenase reductase (nifH) genes were hybridized to Southern blots of DNA from diverse N₂-fixing cyanobacteria in order to test a previous observation of different nif gene organization in nonheterocystous and heterocystous strains. The nif probes showed no significant hybridization to DNA from a unicellular cyanobacterium incapable of N₂ fixation. All nonheterocystous cyanobacteria examined (unicellular and filamentous) had a contiguous nifKDH gene cluster whereas all of the heterocystous strains showed separation of nifK from contiguous nifDH genes. These findings suggest that nonheterocystous and heterocystous cyanobacteria have characteristic and fundamentally different nif gene arrangements. The noncontiguous nif gene pattern, as shown with two Het^- mutants, is independent of phenotypic expression of heterocyst differentiation and aerobic N₂-fixation. Thus nif arrangement could be a useful taxonomic marker to distinguish between phenotypically Het^- heterocystous cyanobacteria and phylogenetically unrelated nonheterocystous strains.     

Diversity of nifH Genotypes in Floating Periphyton Mats Along a Nutrient Gradient in the Florida Everglades

Periphyton mats are an important component of many wetland ecosystems, performing a range of vital ecosystem functions, including nitrogen fixation. The composition and integrity of these mats are affected by nutrient additions, which might result in changes in their function. The overall objective of this study was to investigate the distribution of nifH sequences in floating periphyton mats collected along a nutrient gradient in the Florida Everglades. Distribution of nifH clone libraries indicated nutrient enrichment selected primarily for sequences branching deeply within the heterocystous cyanobacteria and within a novel group of cyanobacteria; sequences from low-nutrient sites were broadly distributed, with no clear dominance of sequences associated with heterocystous and nonheterocystous cyanobacteria and alpha-, gamma-, and delta-proteobacteria. The dominance of heterocystous cyanobacteria in nutrient-enriched sites and the lack of clear dominance by heterocystous cyanobacteria is consistent with previously reported diurnal cycles of nitrogen fixation rates in these systems. Sequences clustering with those harbored by methanotrophs were also identified; sequences from nutrient-impacted and transition regions clustered with those characteristic of type II methanotrophs, and sequences from oligotrophic regions clustered with type I methanotrophs.     

Dinitrogen fixation by exposed communities on the rim of Tikehau atoll (Tuamotu Archipelago, French Polynesia)

Various types of sub-aerially exposed microbial mats, including emergent mats, beach sand, beach rock and Kopara mats, are widespread on the 78 km (25 km²) of rim surrounding the Tikehau atoll lagoon. These mats form laminated accretions or diffuse microbial communities growing under high insolation and temperatures, and are therefore subject to desiccation. Both heterocystous and non-heterocystous cyanobacteria occur in these mats. Using acetylene reduction techniques, nitrogenase activity was observed at all sites over a period of 5 years and was 3–17 times higher during daylight than at night in all communities except for beach rock. ¹⁵N₂ measurements indicated a molar ratio of acetylene reduction to N₂ fixed of 1.6 for all exposed communities. Estimated N₂ fixation ranged from 1.44 to 8.0 mg N m⁻² day⁻¹ in these exposed communities (mean of 4.66 mg N m⁻² day⁻¹) with beachrock showing the highest rates. For the whole reef rim, daily N₂ fixation amounted to 98.42 kg N day⁻¹ which represents 28% of the rate of fixation in the entire lagoon (area 400 km²).     

An unusual cyanobacterium from saline thermal waters with relatives from unexpected habitats

Cyanobacteria that grow above seawater salinity at temperatures above 45°C have rarely been studied. Cyanobacteria of this type of thermo-halophilic extremophile were isolated from siliceous crusts at 40–45°C in a geothermal seawater lagoon in southwest Iceland. Iceland Clone 2e, a Leptolyngbya morphotype, was selected for further study. This culture grew only at 45–50°C, in medium ranging from 28 to 94 g L⁻¹ TDS, It showed 3 doublings 24 h⁻¹ under continuous illumination. This rate at 54°C was somewhat reduced, and death occurred at 58°C. A comparison of the 16S rDNA sequence with all others in the NCBI database revealed 2 related Leptolyngbya isolates from a Greenland hot spring (13–16 g L⁻¹ TDS). Three other similar sequences were from Leptolyngbya isolates from dry, endolithic habitats in Yellowstone National Park. All 6 formed a phylogenetic clade, suggesting common ancestry. These strains shared many similarities to Iceland Clone 2e with respect to temperature and salinity ranges and optima. Two endolithic Leptolyngbya isolates, grown previously at 23°C in freshwater medium, grew well at 50°C but only in saline medium. This study shows that limited genotypic similarity may reveal some salient phenotypic similarities, even when the related cyanobacteria are from vastly different and remote habitats.     

Oxygenic Photosynthesis and Respiratory Activity in Microbial Mats of the Ebro Delta, Spain, by Oxygen Exchange Method

Photosynthetic and respiratory activities at low light intensities (300 μE m⁻² s⁻¹) in the microbial mats of the Ebro Delta were measured by the oxygen exchange method in the laboratory. The response to H₂S concentration, a significant factor in the dynamics of that ecosystem, was assessed. Total photosynthesis reached 23.78–28.17 μg O₂ cm⁻² h⁻¹. Photosynthetic activity was not significantly different at the two temperatures tested. Respiratory activity reached a consumption of 6.95–8.56 μg O₂ cm⁻² h⁻¹ at 25°C and 11.42–11.70 μg O₂ cm⁻² h⁻¹ at 35°C. The Q₁₀ value for respiration was 1.37–1.64. Oxygen production in Microcoleus chthonoplastes, the most abundant cyanobacterium in those microbial mats, was highly resistant to sulfide inhibition. Concentrations less than 0.02 mM sulfide did not affect the rate of photosynthesis. Concentrations up to 0.1 mM sulfide caused different degrees of partially reversible inhibition, with a maximum of 67% at 0.78 mM sulfide. Primary production (g C assimilated/m²/year) in those microbial mats was also assessed and compared with data from other ecosystems. Received: 24 October 1997 / Accepted: 18 December 1997     

Benthic cyanobacterial diversity of Iles Eparses (Scattered Islands) in the Mozambique Channel

The marine benthic cyanobacteria of the Iles Eparses, Mozambique Channel, were surveyed for the first time. A total of 39 species are reported: 29 from Europa, 17 from Glorioso and 23 from Juan de Nova Islands. The higher biodiversity in Europa is explained by greater habitat diversity on this Island with unique ecosystems (mangroves, fossil reefs, pools). Average species richness varied between the geomorphological habitat types with higher diversity in shallow environments (fossil reef pools, mangroves, reef flats), which are characterized by high temperatures and high irradiances. The most common species observed on the three islands were Hydrocoleum coccineum, Hydrocoleum glutinosum, Hydrocoleum lyngbyaceum, Phormidium laysanense, Lyngbya sordida, and Symploca hydnoides; which are also the dominant species observed in the Southwest Indian Ocean region. The most frequent species was Phormidium laysanense with extensive cover observed in the northwest of Juan de Nova Island. Our study provided a comparison between the cyanobacterial flora of Iles Eparses and the recorded surveys in the Southwest Indian Ocean region. The low similarity observed between these species lists could be explained by differences in sampling strategies and efforts, as well as by different taxonomic approaches employed in past regional studies.     

Primary production dynamics of dominant hydrophytes in Lake Provala (Serbia)

The objective of this investigation was to analyze the primary production of the dominant hydrophytes by monitoring levels of organic matter and organic carbon and estimating photosynthetic potential via the total chlorophyll content. The survey was conducted in Lake Provala (Serbia) throughout the peak vegetation period of the year 2000. The contents of organic matter and organic carbon for Myriophyllum spicatum L. were 105.11 g m⁻² and 73.66 g m⁻², Nymphoides peltata (Gmel.) Kunt. were 95.51 g m⁻² and 45.26 g m⁻² and Ceratophyllum demersum L. were 52.17 g m⁻² and 29.75 g m⁻². Chlorophyll A (Chl a) and chlorophyll A+B (Chl a+b) pigments ranged from 1.54 mg g⁻¹(Chl a) and 2.1 mg g⁻¹(Chl a+b) in M. spicatum to 5.27 mg g⁻¹(Chl a) and 7.53 mg g⁻¹(Chl a+b) in C. demersum. At full leaf out, the latter aquatic plants exceeded 50% cover of the open water surface. All species achieved maximum growth in June, but significant differences in growth dynamics were observed. At the end of the vegetation period, these plants sink to the bottom and decompose     

Actual and potential rates of hydrogen photoproduction by continuous culture of the purple non-sulphur bacterium Rhodobacter capsulatus

The influence of (NH₄)₂SO₄ concentration and dilution rate (D) on actual and potential H₂ photoproduction has been studied in ammonium-limited chemostat cultures of Rhodobacter capsulatus B10. The actual H₂ production in a photobioreactor was maximal (approx. 80 ml h⁻¹l⁻¹) at D = 0.06 h⁻¹ and 4 mM (NH₄)₂SO₄. However, it was lower than the potential H₂ evolution (calculated from hydrogen evolution rates in incubation vials), which amounted to 100–120 ml h⁻¹ l⁻¹ at D = 0.03–0.08 h⁻¹. Taking into account the fact that H₂ production in the photobioreactor under these conditions was not limited by light or lactate, another limiting (inhibiting) factor should be sought. One possibility is an inhibition of H₂ production by the H₂ accumulated in the gas phase. This is apparent from the non-linear kinetics of H₂ evolution in the vials or from its inhibition by the addition of H₂; initial rates were restored in both cases after the vials had been refilled with argon. The actual H₂ production in the photobioreactor at D = 0.06 h⁻¹ was shown to increase from approximately 80 ml h⁻¹ l⁻¹ to approximately 100 ml h⁻¹ l⁻¹ under an argon flow at 100 ml min⁻¹. Under maximal H₂ production rates in the photobioreactor, up to 30% of the lactate feedstock was utilised for H₂ production and 50% for biomass synthesis. Received: 22 April 1997 / Received revision: 14 July 1997 / Accepted: 27 July 1997     

Effects of bacillamide and newly synthesized derivatives on the growth of cyanobacteria and microalgae cultures

The antialgal activity of newly synthesized bacillamides against several cyanobacteria and microalgae isolates was screened using a rapid 96-well microplate bioassay. Cultures were exposed to serial dilutions of each bacillamide derivative (0–160 μg mL⁻¹) in the microplate wells and daily optical measurements were used to estimate growth over a 216 h period. Inhibition values (%) were calculated from the estimated growth curves and inhibitory concentrations (IC₅₀-216 h) were obtained from the sigmoidal inhibition curves fitted by regression analysis. The effects of bacillamides on cell morphology and ultrastructure were also analysed by light and transmission electron microscopy. In general, the toxic cyanobacteria Microcystis aeruginosa, Aphanizomenon gracile, Anabaena circinalis and Anabaenopsis circularis were much more sensitive to bacillamides then the chlorophytes Ankistrodesmus falcatus and Scenedesmus obliquus. However, clear signs of morphological and ultrastructural changes induced by bacillamide were observed on both cyanobacteria and chlorophytes. Other cyanobacteria, namely the nostocalean Nodularia spumigena and the oscillatorialeans Leptolyngbya sp. and Planktothrix rubescens, exhibit higher tolerances to bacillamides, similar to that shown by different eukaryotic microalgae. Diatoms, on the other hand, proved to be quite as sensitive to most bacillamides as the most affected cyanobacteria. The properties of 5-iodo-Bacillamide (algicide or algistatic) were further investigated. This compound acted as an algistactic agent against eukaryotic algae and, depending on its concentration, acted as either an algicide or algistactic agent against most of the cyanobacteria tested. Although bacillamides cannot be considered as broad spectrum cyanobacterial algicides, different bacillamides might be of use in selectively controlling the growth of particular species of cyanobacteria.     

DISTRIBUTION AND PHYSIOLOGICAL DETERMINANTS OF BLUE-GREEN ALGAL NITROGEN FIXATION ALONG A THERMOGRADIENT1

The capacity of thermal algal-bacterial mats to fix nitrogen (N₂) was examined in an alkaline thermal stream, Rabbit Creek, of Yellowstone National Park. Nitrogenase activity and nitrogen-fixation rates of mat cores placed in serum bottles and incubated in situ were estimated by the acetylene-reduction technique. Active nitrogenase was not detected at 60 or 65 C in either the blue-green algal or bacterial undermat components of the mats. Acetylene was reduced by all mats ≤55 C along the thermogradient; mean fixation estimates for the mats ranged from 7 to 5,028 nmoles N₂ fixed · mg Chl a^?1· hr^?1. Maximum fixation occurred at 35 C in the stream; statistical comparison of mean rates ordered the thermogradient mats according to estimated activities: 35 > 40 > 30 > 50 ≥ 55 ≥ 45 C. Mats (≤40 C) dominated by species of Calothrix accounted for ca. 97% of the total nitrogen fixation observed in the stream; the remaining activity was associated with mats containing Mastigocladus laminosus Cohn. Light intensity significantly affected fixation rates of the Calothrix mats which responded in a linear fashion from 9–100% full sunlight (ca. 1,900 μEin · m^?2· sec^?1). Calothrix mats from 30 and 40 C had maximum nitrogenase activity at their growth temperature suggesting that nitrogen fixation along the thermogradient was optimally adapted to in situ temperatures.     

Estimation of periphytic microalgae gross primary production with DCMU-fluorescence method in Yenisei River (Siberia, Russia)

Periphyton (epilithon) gross primary production (GPP) was estimated using the DCMU-fluorescence method in the Yenisei River. In the unshaded littoral zone, chlorophyll a concentration (Chl a) and GPP value varied from 0.83 to 973.74 mg m⁻²and 2–304,425 O₂ m⁻² day⁻¹ (0.64–95 133 mg C m⁻² day⁻¹), respectively. Positive significant correlation (r = 0.8) between daily GPP and periphyton Chl a was found. Average ratio GPP:Chl a for periphyton was 36.36 mg C mg Chl a m⁻² day⁻¹. The obtained GPP values for the Yenisei River have a high significant correlation with values predicted by a conventional empirical model for stream periphyton. We concluded that the DCMU-fluorescence method can be successfully used for measuring of gross primary production of stream phytoperiphyton at least as another useful tool for such studies.     

Optical properties and optical constituents of the Faroe Islands shelf and off-shelf waters,North East Atlantic

The study comprises a data set of CTD, optical properties—K ₀(PAR), c _p, a(PAR), b(PAR)—and optical constituents—Chl a, SPM, CDOM—from 72 shelf and off-shelf stations in the Faroe Islands (62°N, 7°W) North East Atlantic, in early spring 2005. Results showed that shelf waters surrounding the islands were cold and low saline, whereas off-shelf waters were warmer (~1°C) and more saline (~0.05) PSU. A pronounced oceanographic front separated the two waters, and diffuse light attenuation K ₀(PAR), beam attenuation c _p, Chl a, absorption a(PAR), and scattering coefficient b(PAR) were all significantly higher on the shelf. Analyses showed that off-shelf light attenuation K ₀(PAR) was governed by Chl a, shown by a high (r ² = 0.64) Chl a–K ₀(PAR) correlation, whereas light attenuation on the shelf was governed by both Chl a, SPM, and CDOM in combination. A Chl a specific diffuse attenuation coefficient K₀^* ( \textPAR ) K_{0}^{*} \left( {\text{PAR}} \right) of 0.056 (m² mg⁻¹ Chl a) and a Chl a specific beam attenuation ( c_\textp^* c_{\text{p}}^{*} ) of 0.27 (m² mg⁻¹ Chl a) coefficients were derived for the off-shelf. It is pointed out that Chl a is the single variable that changes over time as no rivers with high SPM and CDOM enter the shelf area. Data were obtained in early spring, and Chl a concentrations were low ~0.5 mg Chl a m⁻³. Spring bloom Chl a are about 10 mg Chl a m⁻³ and estimations showed that shelf K ₀(PAR) will increase about 5 times and beam attenuation about 10 times. The Faroe Islands shelf–off-shelf waters is a clear example where physical conditions maintain some clear differences in optical properties and optical constituents. The complete data set is enclosed.     

Kinetics of urea uptake by Melosira italica (Ehr.) Kütz at different luminosity conditions

The kinetic parameters K_m and V_max for urea uptake by Melosira italica were determined at 160 μeinsteins m⁻² s⁻¹ and in the dark. The transport systems showed an affinity for the substrate and a storing capacity in the dark (K_m = 65.07 μM; V_max = 2.18 nmoles 10⁵ cells ⁻¹ h⁻¹) greater than under 160 μE m⁻² s ⁻¹ (K_m = 111.2 μM; V_max = 1.11 nmoles 105 cells⁻¹ h⁻¹). Similarly, a reduction in consumption rate of urea under increasing photon flux densities was observed. The use of an inhibitor (potassium cyanide) indicated that the uptake process requires metabolic energy. That urea transport is more important in darkness, may constitute a survival strategy in which this compound is utilized by cells mainly during heterotrophic growth.     

Light‐dependent oxygen consumption in nitrogen‐fixing cyanobacteria plays a key role in nitrogenase protection1

All colonial diazotrophic cyanobacteria are capable of simultaneously evolving O₂ through oxygenic photosynthesis and fixing nitrogen via nitrogenase. Since nitrogenase is irreversibly inactivated by O₂, accommodation of the two metabolic pathways has led to biochemical and/or structural adaptations that protect the enzyme from O₂. In some species, differentiated cells (heterocysts) are produced within the filaments. PSII is absent in the heterocysts, while PSI activity is maintained. In other, nonheterocystous species, however, a “division of labor” occurs whereby individual cells within a colony appear to ephemerally fix nitrogen while others evolve oxygen. Using membrane inlet mass spectrometry (MIMS) in conjunction with tracer ¹⁸O₂ and inhibitors of photosynthetic and respiratory electron transport, we examined the light dependence of O₂ consumption in Trichodesmium sp. IMS 101, a nonheterocystous, colonial cyanobacterium, and Anabaena flos‐aquae (Lyngb.) Bréb. ex Bornet et Flahault, a heterocystous species. Our results indicate that in both species, intracellular O₂ concentrations are maintained at low levels by the light‐dependent reduction of oxygen via the Mehler reaction. In N₂‐fixing Trichodesmium colonies, Mehler activity can consume ～75% of gross O₂ production, while in Trichodesmium utilizing nitrate, Mehler activity declines and consumes ～10% of gross O₂ production. Moreover, evidence for the coupling between N₂ fixation and Mehler activity was observed in purified heterocysts of Anabaena, where light accelerated O₂ consumption by 3‐fold. Our results suggest that a major role for PSI in N₂‐fixing cyanobacteria is to effectively act as a photon‐catalyzed oxidase, consuming O₂ through pseudocyclic electron transport while simultaneously supplying ATP in both heterocystous and nonheterocystous taxa.     

Sensitivity of Metasequoia glyptostroboides to ozone stress

2-year-old seedlings of Metasequoia glyptostroboides were grown in open top chambers and exposed to four ozone concentrations [O₃] (charcoal-filtered air, CF; 50, 100, and 200 mm³ m⁻³) for 25 d. Measurements of growth, leaf chlorophyll (Chl) content, and gas exchange parameters were made before and/or after O₃ exposure. Leaf length, crown width, Chl a/b, net photosynthetic rate, stomatal conductance, and transpiration rate were significantly reduced at 100 and 200 mm³(O₃) m⁻³. A remarkable decrease in stomatal conductance also occurred at 50 mm³(O₃) m⁻³.     

Excess irradiance causes early symptoms of senescence during leaf expansion in photoautotrophically <Emphasis Type="Italic">in vitro</Emphasis> grown tobacco plants

Photosynthetic parameters, growth, and pigment contents were determined during expansion of the fourth leaf of in vitro photoautotrophically cultured Nicotiana tabacum L. plants at three irradiances [photosynthetically active radiation (400–700 nm): low, LI 60 μmol m⁻² s⁻¹; middle, MI 180 μmol m⁻² s⁻¹; and high, HI 270 μmol m⁻² s⁻¹]. During leaf expansion, several symptoms usually accompanying leaf senescence appeared very early in HI and then in MI plants. Symptoms of senescence in developing leaves were: decreasing chlorophyll (Chl) a+b content and Chl a/b ratio, decreasing both maximum (F_V/F_M) and actual (Φ_PS2) photochemical efficiency of photosystem 2, and increasing non-photochemical quenching. Nevertheless, net photosynthetic oxygen evolution rate (P _N) did not decrease consistently with decrease in Chl content, but exhibited a typical ontogenetic course with gradual increase. P _N reached its maximum before full leaf expansion and then tended to decline. Thus excess irradiance during in vitro cultivation did not cause early start of leaf senescence, but impaired photosynthetic performance and Chl content in leaves and changed their typical ontogenetic course.     

Photosynthesis performance in sweet almond [<Emphasis Type="Italic">Prunus dulcis</Emphasis> (Mill) D. Webb] exposed to supplemental UV-B radiation

Due to anthropogenic influences, solar UV-B irradiance at the earth’s surface is increasing. To determine the effects of enhanced UV-B radiation on photosynthetic characteristics of Prunus dulcis, two-year-old seedlings of the species were submitted to four levels of UV-B stress, namely 0 (UV-B_c), 4.42 (UV-B₁), 7.32 (UV-B₂) and 9.36 (UV-B₃) kJ m⁻² d⁻¹. Effects of UV-B stress on a range of chlorophyll (Chl) fluorescence parameters (FPs), Chl contents and photosynthetic gas-exchange parameters were investigated. UV-B stress promoted an increase in minimal fluorescence of dark-adapted state (F₀) and F₀/F_m, and a decrease in variable fluorescence (F_v, F_v/F_m, F_v/F₀ and F₀/F_m) due to its adverse effects on photosystem II (PSII) activity. No significant change was observed for maximal fluorescence of dark-adapted state (F_m). Enhanced UV-B radiation caused a significant inhibition of net photosynthetic rate (P _N) at UV-B₂ and UV-B₃ levels and this was accompanied by a reduction in stomatal conductance (g _s) and transpiration rate (E). The contents of Chl a, b, and total Chl content (a+b) were also significantly reduced at increased UV-B stress. In general, adverse UV-B effects became significant at the highest tested radiation dose 9.36 kJ m⁻² d⁻¹. The most sensitive indicators for UV-B stress were F_v/F₀, Chl a content and P _N. Significant P<0.05 alteration in these parameters was found indicating the drastic effect of UV-B radiation on P. dulcis.     

Irradiance stress responses of gas exchange and antioxidant enzyme contents in pariparoba [<Emphasis Type="Italic">Pothomorphe umbellata</Emphasis> (L.) Miq.] plants

We evaluated the growth and development of the medicinal species Pothomorphe umbellata (L.) Miq. under different shade levels (full sun and 30, 50, and 70 % shade, marked as I₁₀₀, I₇₀, I₅₀, and I₃₀, respectively) and their effects on gas exchange and activities of antioxidant enzymes. Photosynthetically active radiation varied from 1 254 μmol m⁻² s⁻¹ at I₁₀₀ to 285 μmol m⁻² s⁻¹ at I₃₀. Stomatal conductance, net photosynthetic rate, and relative chlorophyll (Chl) content were maximal in I₇₀ plants. Plants grown under I₁₀₀ produced leaves with lower Chl content and signs of chlorosis and necrosis. These symptoms indicated Chl degradation induced by the generation of reactive oxygen species. Stress related antioxidant enzyme activities (Mn-SOD, Fe-SOD, and Cu/Zn-SOD) were highest in I₁₀₀ plants, whereas catalase activity was the lowest. Hence P. umbellata is a shade species (sciophyte), a feature that should be considered in reforestation programs or in field plantings for production of medicinal constituents.     

Hydrogen production with high yield and high evolution rate by self-flocculated cells of Enterobacter aerogenes in a packed-bed reactor

Continuous hydrogen gas evolution by self-flocculated cells of Enterobacter aerogenes, a natural isolate HU-101 and its mutant AY-2, was performed in a packed-bed reactor under glucose-limiting conditions in a minimal medium. The flocs that formed during the continuous culture were retained even when the dilution rate was increased to 0.9 h⁻¹. The H₂ production rate increased linearly with increases in the dilution rate up to 0.67 h⁻¹, giving maximum H₂ production rates of 31 and 58 mmol l⁻¹ h⁻¹ in HU-101 and AY-2 respectively, at a dilution rate of more than 0.67 h⁻¹. The molar H₂ yield from glucose in AY-2 was maintained at about 1.1 at dilution rates between 0.08 h⁻¹ and 0.67 h⁻¹, but it decreased rapidly at dilution rates more than 0.8 h⁻¹. Received: 27 August 1997 / Received revision: 11 November 1997 / Accepted: 14 December 1997