Optimum growth requirements of nitrifying consortia developed from treated sewage

The optimum growth requirements of two nitrifying consortia developed from treated sewage by enrichment technique were determined by a series of experiments. There was total inhibition of nitrification at above 2.75 g l(-1) NH4(+)- N and 2.5 g l(-1) NO2(-)-N and the ammonia oxidizing consortium preferred a pH at 8.5 and the nitrite oxidizing consortium a pH of 7.5 as the optima for nitrification. Optimum temperatures were between 20 degrees and 30 degrees C for both the groups. As the rate of airflow was increased from 1 to 7 l/min, the build-up of NO2(-)-N increased 10-fold and the consumption of NO2(-)-N increased by a factor of 28.8 implying that the ammonia oxidizing consortium in a bioreactor required three times more aeration than that for nitrite oxidizers for expressing their full nitrifying potential. These data directly contribute for developing a fermentation process for the mass production of nitrifiers as well as for designing bioreactors for nitrifying sewage.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using semicontinuous fermentation in bioreactor

Semicontinuous fermentation using pellets of Rhizopus oryzae has been recognized as a promising technology for l-lactic acid production. In this work, semicontinuous fermentation of R. oryzae AS 3.819 for l-lactic acid production has been developed with high l-lactic acid yield and volumetric productivity. The effects of factors such as inoculations, CaCO₃ addition time, and temperature on l-lactic acid yield and R. oryzae morphology were researched in detail. The results showed that optimal fermentation conditions for the first cycle were: inoculation with 4% spore suspension, CaCO₃ added to the culture medium at the beginning of culture, and culture temperature of 32–34°C. In orthogonal experiments, high l-lactic acid yield was achieved when the feeding medium was (g/l): glucose, 100; (NH₄)₂SO₄, 2; KH₂PO₄, 0.1; ZnSO₄·7H₂O, 0.33; MgSO₄·7H₂O, 0.15; CaCO₃, 50. Twenty cycles of semicontinuous fermentation were carried out in flask culture. l-lactic acid yield was 78.75% for the first cycle and 80–90% for the repeated cycles; the activities of lactate dehydrogenases (LDH) were 7.2–9.2 U/mg; fermentation was completed in 24 h for each repeated cycle. In a 7-l magnetically stirred fermentor, semicontinuous fermentation lasted for 25 cycles using pellets of R. oryzae AS 3.819 under the optimal conditions determined from flask cultures. The final l-lactic acid concentration (LLAC) reached 103.7 g/l, and the volumetric productivity was 2.16 g/(l·h) for the first cycle; in the following 19 repeated cycles, the final LLAC reached 81–95 g/l, and the volumetric productivities were 3.40–3.85 g/(l·h).     

Efficient conversion of lactic acid to butanol with pH-stat continuous lactic acid and glucose feeding method by Clostridium saccharoperbutylacetonicum

In order to achieve high butanol production by Clostridium saccharoperbutylacetonicum N1-4, the effect of lactic acid on acetone–butanol–ethanol fermentation and several fed-batch cultures in which lactic acid is fed have been investigated. When a medium containing 20 g/l glucose was supplemented with 5 g/l of closely racemic lactic acid, both the concentration and yield of butanol increased; however, supplementation with more than 10 g/l lactic acid did not increase the butanol concentration. It was found that when fed a mixture of lactic acid and glucose, the final concentration of butanol produced by a fed-batch culture was greater than that produced by a batch culture. In addition, a pH-controlled fed-batch culture resulted in not only acceleration of lactic acid consumption but also a further increase in butanol production. Finally, we obtained 15.5 g/l butanol at a production rate of 1.76 g/l/h using a fed-batch culture with a pH-stat continuous lactic acid and glucose feeding method. To confirm whether lactic acid was converted to butanol by the N1-4 strain, we performed gas chromatography–mass spectroscopy (GC-MS) analysis of butanol produced by a batch culture during fermentation in a medium containing [1,2,3-¹³C₃] lactic acid as the initial substrate. The results of the GC-MS analysis confirmed the bioconversion of lactic acid to butanol.     

硝化细菌群的富集及其去除城镇寡营养河道中氨氮污染的应用

[背景]随着工农业的发展,污水排放导致的氨氮超标逐渐成为水体污染的重要因素,脱氮已成为人们研究的重点.目前脱氮方法主要集中于硝化细菌的硝化作用,其将氨氮转化为硝酸盐氮,从而减少水体中氨氮的污染.由于工业废水和农业污水中的有机物含量较高,而且异养硝化细菌具有生长较快等优势,因此对异养菌的研究多于自养菌.然而现有的异养硝化...     

Optimization of glucose feeding approaches for enhanced glucosamine and N-acetylglucosamine production by an engineered Escherichia coli

In this work, a recombinant Escherichia coli was constructed by overexpressing glucosamine (GlcN) synthase and GlcN-6-P N-acetyltransferase for highly efficient production of GlcN and N-acetylglucosamine (GlcNAc). For further enhancement of GlcN and GlcNAc production, the effects of different glucose feeding strategies including constant-rate feeding, interval feeding, and exponential feeding on GlcN and GlcNAc production were investigated. The results indicated that exponential feeding resulted in relatively high cell growth rate and low acetate formation rate, while constant feeding contributed to the highest specific GlcN and GlcNAc production rate. Based on this, a multistage glucose supply approach was proposed to enhance GlcN and GlcNAc production. In the first stage (0–2 h), batch culture with initial glucose concentration of 27 g/l was conducted, whereas the second culture stage (2–10 h) was performed with exponential feeding at μ _set = 0.20 h⁻¹, followed by feeding concentrated glucose (300 g/l) at constant rate of 32 ml/h in the third stage (10–16 h). With this time-variant glucose feeding strategy, the total GlcN and GlcNAc yield reached 69.66 g/l, which was enhanced by 1.59-fold in comparison with that of batch culture with the same total glucose concentration. The time-dependent glucose feeding approach developed here may be useful for production of other fine chemicals by recombinant E. coli.     

Measurement of potential activity of fixed nitrifying bacteria in biological filters used in drinking water production

Nitrification during biological filtration is being used more and more in drinking water production to remove ammonia, which can be the source of several water quality problems during distribution. In this process, ammonia is converted into nitrite and then into nitrate by fixed autotrophic nitrifying bacteria. The purpose of this work was to develop a technique to estimate fixed nitrifying biomass (sum of ammonia- and nitrite-oxidizing populations). The quantification of autotrophic nitrifying biomass was determined by potential nitrifying activity measurement. The production of oxidized forms of inorganic nitrogen (nitrates and nitrites) was measured after an incubation of 2 cm³ of colonized solid support in the presence of a 5-ml nitrifier medium containing 10 mg N-NH₄ L⁻¹ for 30 min at 32°C. The production rate of oxidized nitrogen in optimal conditions was measured and converted into nitrifying biomass by using the maximum specific oxidizing activity. This technique was shown to be appropriate for conditions encountered in the biological filters used in drinking water production and sufficiently simple to be used for routine measurements. Journal of Industrial Microbiology & Biotechnology (2000) 24, 161–166. Received 28 July 1999/ Accepted in revised form 11 November 1999     

Activated packed bed bioreactor for rapid nitrification in brackish water hatchery systems

A packed bed bioreactor (PBBR) was developed for rapid establishment of nitrification in brackish water hatchery systems in the tropics. The reactors were activated by immobilizing ammonia-oxidizing (AMONPCU-1) and nitrite-oxidizing (NIONPCU-1) bacterial consortia on polystyrene and low-density polyethylene beads, respectively. Fluorescence in situ hybridization demonstrated the presence of autotrophic nitrifiers belong to Nitrosococcus mobilis, lineage of β ammonia oxidizers and nitrite oxidizer Nitrobacter sp. in the consortia. The activated reactors upon integration to the hatchery system resulted in significant ammonia removal (P < 0.01) culminating to its undetectable levels. Consequently, a significantly higher percent survival of larvae was observed in the larval production systems. With spent water the reactors could establish nitrification with high percentage removal of ammonia (78%), nitrite (79%) and BOD (56%) within 7 days of initiation of the process. PBBR is configured in such a way to minimize the energy requirements for continuous operation by limiting the energy inputs to a single stage pumping of water and aeration to the aeration cells. The PBBR shall enable hatchery systems to operate under closed recirculating mode and pave the way for better water management in the aquaculture industry.     

N-removal performance and underlying bacterial taxa of upflow filter bioreactor system under different dissolved oxygen and internal recycle conditions

Biological N-removal treatment of piggery wastewater in the upflow anaerobic–anoxic–aerobic floating filter (UA³FF) bioreactor based on the concept of nitritation–denitritation was studied along with the changes in internal recycle ratio and dissolved oxygen concentration (DO). Consecutive changes in the recirculation ratio between the anoxic and aerobic reactors has resulted in abundance and composition shifts of N-cycling bacteria as well as other bacterial groups, reflecting different survival strategies across (bio/physico)chemical milieu. The DO concentration was optimized to achieve nitritation in the aerobic reactor and denitritation in the anoxic reactor. Optimal nitritation–denitritation (270 and 130 g NO₂ ⁻–N produced or reduced/m³ filter media/day) was obtained at DO of 1.0–1.5 mg/l, inter-reactor recirculation ratio of 1:1–2:1, HRT of 24 h, pH of 7.6 ± 0.3, and temperature of 28 ± 4 °C. Since only well known nitrifying and denitrifying taxa were found, nitritation–denitritation was likely carried out by these bacteria rather than the yet unidentified novel taxa. Archaeal nitrifiers recently discovered to be important in the global N-cycle were not detected.     

Microbial population dynamics, especially stress tolerant Bacillus thuringiensis, in partially anaerobic rice field soils during post-harvest period of the Himalayan, island, brackish water and coastal habitats of India

Population ( × 10⁷ c.f.u./g dr. soil) of the aerobic (30.5–154.1) and anaerobic (5.9–91.4) heterotrophic, aerobic (24.0–56.0) and anaerobic (2.4–4.2) spore forming, Gram (-)ve (1.6–2.9), phosphate solubilizing (10–20), asymbiotic N₂-fixing (0.5–0.9), sulfur oxidizing (1.1–2.0), nitrifying (1.0–5.8) and denitrifying (12.1–18.7) bacteria; as well as, the actinomycetes (about 10⁴ c.f.u./g dr. soil) and fungi (about 10⁵ c.f.u./g dr. soil) were variable in the partially anaerobic, saline and drain out flooded rice soils during the post harvest period of the Himalayan, brackish water flooded, island and coastal habitats of India. The aerobic heterotrophic and spore forming bacteria were more than the anaerobic counterparts in the soils. Population (0.51–3.51 × 10⁶ c.f.u./g dr. soil) and crystal morphotype (spherical, bipyramidal and polymorphic) of the Bacillus thuringiensis (Bt) isolates of different soils were variable. Bt index was 0.002 at Mahe but 0.006 in other soils. The Bt isolates tolerated 5–12% NaCl. The osmolytes (mg/g dr. wt.) like the amino acids (0.38–99.45) and proline (0.38–0.80); and the antioxidative enzymes (units (U)/mg protein/min) viz. the catalase (0.17–5.59) and superoxide dismutase (0.35–74.46) were related with intrinsic osmotic stress tolerance of the Bt but they formed spores to overcome anoxic stress. Two Bt isolates were potent tolerant to both osmotic and anoxic stresses.     

Nitrite effect on ammonium and nitrite oxidizing processes in a nitrifying sludge

Nitrite accumulation can be undesirable in nitrifying reactors used for the biological elimination of nitrogen from wastewaters because the ammonium oxidation process was seen to be inhibited. There is a need to better understand the effects of nitrite on both ammonium and nitrite oxidizing processes. In this paper, the effect of nitrite on the nitrifying activity of a sludge produced in steady-state nitrification was evaluated in batch cultures. At 25 mg N/l of added nitrite, nitrification was successfully carried out. Addition of higher nitrite concentrations to nitrifying cultures (100 and 200 mg N/l) provoked inhibitory effects on the nitrification respiratory process. Nitrite at 100 and 200 mg N/l induced a significant decrease in the values for nitrate yield (−20% and −34%, respectively) and specific rate of nitrate formation (−26% and −67%, respectively), while the ammonium consumption efficiency kept high and the specific rate of ammonium oxidation did not significantly change. This showed that the nitrite oxidizing process was more sensitive to the presence of nitrite than the ammonium oxidizing process. These results showed that as a consequence of nitrite accumulation in nitrification systems, the activity of the nitrite oxidizing bacteria could be more inhibited than that of the ammonium oxidizing bacteria, provoking a higher accumulation of nitrite in the medium.     

Degradation of cyanide in agroindustrial or industrial wastewater in an acidification reactor or in a single-step methane reactor by bacteria enriched from soil and peels of cassava

During cassava starch production, large amounts of cyanoglycosides were released and hydrolysed by plant-borne enzymes, leading to cyanide concentrations in the wastewater as high as 200 mg/l. For anaerobic degradation of the cyanide during pre-acidification or single-step methane fermentation, anaerobic cultures were enriched from soil residues of cassava roots and sewage sludge. In a pre-acidification reactor this culture was able to remove up to 4 g potassium cyanide/l of wastewater at a hydraulic retention time (t _HR) of 4 days, equivalent to a maximal cyanide space loading of 400 mg CN⁻ l⁻¹ day⁻¹. The residual cyanide concentration was 0.2–0.5 mg/l. Concentrated cell suspensions of the mixed culture formed ammonia and formate in almost equimolar amounts from cyanide. Little formamide was generated by chemical decay. A concentration of up to 100 mmol ammonia/l had no inhibitory effect on cyanide degradation. The optimal pH for cyanide degradation was 6–7.5, the optimal temperature 25–37 °C. At a pH of 5 or lower, cyanide accumulated in the reactor and pre-acidification failed. The minimal t _HR for continuous cyanide removal was 1.5 days. The enriched mixed culture was also able to degrade cyanide in purely mineralic wastewater from metal deburring, either in a pre-acidification reactor with a two-step process or in a one-step methanogenic reactor. It was necessary to supplement the wastewater with a carbon source (e.g. starch) to keep the population active enough to cope with any possible inhibiting effect of cyanide. Received: 29 April 1998 / Received revision: 8 June 1998 / Accepted: 14 June 1998     

Kinetics of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var. <Emphasis Type="Italic">israelensis</Emphasis> growth on high glucose concentrations

The kinetic and general growth features of Bacillus thuringiensis var. israelensis were evaluated. Initial glucose concentration (S ₀) in fermentation media varied from 10 to 152 g/l. The results afforded to characterize four morphologically and physiologically well-defined culture phases, independent of S ₀ values: Phase I, vegetative growth; Phase II, transition to sporulation; Phase III, sporulation; and Phase IV, spores maturation and cell lysis. Important process parameters were also determined. The maximum specific growth rates (μ _X,m) were not affected with S ₀ up to 75 g/l (1.0–1.1 per hour), but higher glucose concentrations resulted in growth inhibition by substrate, revealed by a reduction in μ _X,m values. These higher S ₀ values led to longer Phases III and IV and delayed sporulation. Similar biomass concentrations (X _m = 15.2–15.9 g/l) were achieved with S ₀ over 30.8 g/l, with increasing residual substrate, suggesting a limitation in some other nutrients and the use of glucose to form other metabolites. In this case, with S ₀ from 30.8 to 152 g/l, cell yield (Y _X/S ) decreased from 0.58 to 0.41 g/g. On the other hand, with S ₀ = 10 g/l growth was limited by substrate, and Y _X/S has shown its maximum value (0.83 g/g).     

Efficient isolation of high quality nucleic acids from different tissues of <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew (Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform extraction steps from the isolation procedure. Both spectrophotometric (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the average yield of 100–300 μg/g leaf and stem tissue and total RNA with an average yield of 20–30 μg/g cell culture and 80–100 μg/g leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively.     

Simultaneous flue gas bioremediation and reduction of microalgal biomass production costs

A flue gas originating from a municipal waste incinerator was used as a source of CO₂ for the cultivation of the microalga Chlorella vulgaris, in order to decrease the biomass production costs and to bioremediate CO₂ simultaneously. The utilization of the flue gas containing 10–13% (v/v) CO₂ and 8–10% (v/v) O₂ for the photobioreactor agitation and CO₂ supply was proven to be convenient. The growth rate of algal cultures on the flue gas was even higher when compared with the control culture supplied by a mixture of pure CO₂ and air (11% (v/v) CO₂). Correspondingly, the CO₂ fixation rate was also higher when using the flue gas (4.4 g CO₂ l⁻¹ 24 h⁻¹) than using the control gas (3.0 g CO₂ l⁻¹ 24 h⁻¹). The toxicological analysis of the biomass produced using untreated flue gas showed only a slight excess of mercury while all the other compounds (other heavy metals, polycyclic aromatic hydrocarbons, polychlorinated dibenzodioxins and dibenzofurans, and polychlorinated biphenyls) were below the limits required by the European Union foodstuff legislation. Fortunately, extending the flue gas treatment prior to the cultivation unit by a simple granulated activated carbon column led to an efficient absorption of gaseous mercury and to the algal biomass composition compliant with all the foodstuff legislation requirements.     

Effect of phosphate and oxygen concentrations on alginate production and stoichiometry of metabolism of Azotobacter vinelandii under microaerobic conditions

Alginate production by Azotobacter vinelandii was studied in batch and continuous cultures under microaerobic conditions. In batch culture at a pO₂ of 2–3% (air saturation) alginate production was enhanced by decreasing the PO³⁻ ₄ level in the medium. Alginate yield from biomass (Y _P/X) reached the highest value of 0.66 g/g at the lowest phosphate level (100 mg/l), compared to 0.40 g/g and 0.25 g/g at higher phosphate levels (200 mg/l and 400 mg/l, respectively). In contrast, biomass formation behaved differently and the growth yield (Y _X/S) decreased with decreasing PO₄ ³⁻ concentrations. Moreover, the respiratory quotient (RQ) of the culture was dependent on the initial phosphate concentration, especially in the phosphate-limited phase of growth. As the initial phosphate level decreased from 400 mg/l to 100 mg/l, the average RQ value of the culture declined from 1.46 to 0.89. The low RQ value is very close to the theoretical optimum RQ, calculated to be 0.8 on the basis of the stoichiometry of the metabolic pathways for alginate formation from sucrose. This optimum RQ was also confirmed in continuous culture at different dilution rates. Independent of the dilution rate, a pO₂ value of 2–5% (air saturation) was found to be optimal for alginate production, the corresponding RQ values being 0.80–0.84. In addition, the molecular mass and composition of alginate were also found to be affected by both phosphate and oxygen concentrations. In conclusion, the RQ appears to be a useful parameter for optimum control of alginate production with this microorganism. Received: 31 March 1999 / Received revision: 2 July 1999 / Accepted: 5 July 1999     

Start-up and inhibition analysis of the Anammox process seeded with anaerobic granular sludge

The longer start-up period of the Anammox process is due to the very low cellular yield and growth rates of Anammox bacteria. Nitrite inhibition is considered to be the key factor in the instability of the Anammox process during the operation. However, little attention was paid to the inhibitory effect of pH and free ammonia. This paper presents start-up and inhibition analysis of an Anammox biofilm reactor seeded with anaerobic granular sludge. Results showed that the start-up period could be divided into the sludge lysis phase, lag phase, propagation phase, stationary phase and inhibition phase. Optimization control could be implemented correspondingly to accelerate the start-up of Anammox bioreactors. Effluent pH increased to 8.7–9.1 when the nitrogen removal rate was higher than 1,200 mg l⁻¹ day⁻¹. The free ammonia concentration was accompanied with a higher level of 64–73 mg l⁻¹. Inhibitory effects of high pH and free ammonia on Anammox bacteria contributed to the destabilization of the Anammox bioreactor during the first 125 days with influent KHCO₃ of 0.5 g l⁻¹. Increasing the suffering capacity in the inlet by dosing 1.25 g KHCO₃ l⁻¹ effectively reduced the pH variation, and the nitrogen removal performance of the reactor was further developed.     

<Emphasis Type="Italic">Desulfovibrio legallis</Emphasis> sp. nov.: A Moderately Halophilic,Sulfate-Reducing Bacterium Isolated from a Wastewater Digestor in Tunisia

A new moderately halophilic sulfate-reducing bacterium (strain H₁^T) was enriched and isolated from a wastewater digestor in Tunisia. Cells were curved, motile rods (2–3 x 0.5 μm). Strain H₁^T grew at temperatures between 22 and 43°C (optimum 35°C), and at pH between 5.0 and 9.2 (optimum 7.3–7.5). Strain H₁^T required salt for growth (1–45 g of NaCl/l), with an optimum at 20–30 g/l. Sulfate, sulfite, thiosulfate, and elemental sulfur were used as terminal electron acceptors but not nitrate and nitrite. Strain H₁^T utilized lactate, pyruvate, succinate, fumarate, ethanol, and hydrogen (in the presence of acetate and CO₂) as electron donors in the presence of sulfate as electron acceptor. The main end-products from lactate oxidation were acetate with H₂ and CO₂. The G + C content of the genomic DNA was 55%. The predominant fatty acids of strain H₁^T were C_15:0 iso (38.8%), C_16:0 (19%), and C_14:0 iso 3OH (12.2%), and menaquinone MK-6 was the major respiratory quinone. Phylogenetic analysis of the small-subunit (SSU) ribosomal RNA (rRNA) gene sequence indicated that strain H₁^T was affiliated to the genus Desulfovibrio. On the basis of SSU rRNA gene sequence comparisons and physiological characteristics, strain H₁^T is proposed to be assigned to a novel species of sulfate reducers of the genus Desulfovibrio, Desulfovibrio legallis sp. nov. (= DSM 19129^T = CCUG 54389^T).     

Kinetic analysis of the inhibitory effect of trichloroethylene (TCE) on nitrification in cometabolic degradation

In this study, the inhibitory effect of TCE on nitrification process was investigated with an enriched nitrifier culture. TCE was found to be a competitive inhibitor of ammonia oxidation and the inhibition constant (K _I ) was determined as 666–802 μg/l. The TCE affinity for the AMO enzyme was significantly higher than ammonium. The effect of TCE on ammonium utilization was evaluated with linearized plots of Monod equation (e.g., Lineweaver–Burk, Hanes–Woolf and Eadie–Hofstee plots) and non-linear least square regression (NLSR). No significant differences were found among these data evaluation methods in terms of kinetic parameters obtained.     

Factors influencing regeneration from protoplasts of Asparagus densiflorus cv. Sprengeri

This article describes conditions to optimize the yield of viable protoplasts from callus tissue of Asparagus densiflorus cv. Sprengeri and their subsequent regeneration into plantlets. Callus tissue was initiated by culturing spear sections (5–7 mm) on Murashige and Skoog (MS) medium supplemented with 0.8% (wt/vol) Bacto agar, 3% (wt/vol) sucrose, 0.5 mg/l each of nicotinic acid, pyridoxine-HCl, and thiamine-HCl, 1 mg/l p-chlorophenoxyaceticacid (pCPA) and 1 mg/l 6-benzylaminopurine (BAP). The maximum protoplast yield was obtained in a mixture of 1% (wt/vol) Cellulysin, 0.8% (wt/vol) Rhozyme HP 150 and 0.3% (wt/vol) Macerase, dissolved in cell protoplast wash salt solution with 7 mm CaCl₂ ^.2H₂O, 3 mm MES, 0.6 m glucose, and 0.1 m mannitol. First divisions were observed after 3–4 days of initial culture. The plating efficiency was highest (7.8%) in half-strength MS semisolid medium containing 1 g/l glutamine, 0.6 m glucose, 0.1 m mannitol, 0.5 mg/l folic acid, 0.05 mg/l biotin, 2 mg/l ascorbic acid, 1 mg/l α-naphthaleneacetic acid, 0.5 mg/l zeatin, and 0.1% (wt/vol) Gelrite. Protoplast-derived microcolonies and microcalli were cultured on the same medium on which the primary callus culture was initiated. After 10–12 weeks, calli were transferred to shoot regeneration medium containing MS salts, 1 mg/l BAP, 0.5 mg/l pCPA and 0.2% Gelrite. Shoots (3–4 cm) were then transferred to MS rooting medium with 2 mg/l indole-3-butyric acid, and 0.2% Gelrite. Plantlets were obtained within 4–5 weeks. Received: 9 August 1995 / Revision received: 27 June 1997 / Accepted: 17 July 1997     

Greenhouse gas fluxes from the eutrophic Temmesjoki River and its Estuary in the Liminganlahti Bay (the Baltic Sea)

We studied concentrations of carbon dioxide (CO₂), methane (CH₄) and nitrous oxide (N₂O) in the eutrophic Temmesjoki River and Estuary in the Liminganlahti Bay in 2003–2004 and evaluated the atmospheric fluxes of the gases based on measured concentrations, wind speeds and water current velocities. The Temmesjoki River was a source of CO₂, CH₄ and N₂O to the atmosphere, whereas the Liminganlahti Bay was a minor source of CH₄ and a minor source or a sink of CO₂ and N₂O. The results show that the fluxes of greenhouse gases in river ecosystems are highly related to the land use in its catchment areas. The most upstream river site, surrounded by forests and drained peatlands, released significant amounts of CO₂ and CH₄, with average fluxes of 5,400 mg CO₂–C m⁻² d⁻¹ and 66 mg CH₄–C m⁻² d⁻¹, and concentrations of 210 μM and 345 nM, respectively, but N₂O concentrations, at an average of 17 nM, were close to the atmospheric equilibrium concentration. The downstream river sites surrounded by agricultural soils released significant amounts of N₂O (with an average emission of 650 μg N₂O–N m⁻² d⁻¹ and concentration of 22 nM), whereas the CO₂ and CH₄ concentrations were low compared to the upstream site (55 μM and 350 nM). In boreal regions, rivers are partly ice-covered in wintertime (approximately 5 months). A large part of the gases, i.e. 58% of CO₂, 55% of CH₄ and 36% of N₂O emissions, were found to be released during wintertime from unfrozen parts of the river.