Alkalinity and acidity cycling and fluxes in an intermediate fen peatland in northern Ontario

Peatlands are important to global carbon (C) sequestration and surface water acid–base status, both of which are affected by peatland alkalinity and acidity cycling. Relationships among sulfate (SO₄ ^2?), nitrate (NO₃ ^?), organic acids (OA^?), base cations (i.e., Ca²⁺, Mg²⁺, K⁺, and Na⁺), proton (H⁺) acidity, and bicarbonate (HCO₃ ^?) alkalinity were investigated in an intermediate fen peatland in northern Ontario during 2004 (an average precipitation year) and 2005 (a dry summer). Potential evapotranspiration was higher and the water table, groundwater input from the uplands, and runoff were lower during 2005. Net inputs of base cations, HCO₃ ^?, SO₄ ^2?, and OA^?, and to a lesser degree NO₃ ^?, were lower during the drier year, mainly due to lower groundwater transfer to the fen. Fen porewater HCO₃ ^? concentration and net output were also lower in the drier year, whereas Ca²⁺, Mg²⁺, and SO₄ ^2? concentrations and net output were higher. During the climatically average year, N immobilization, carbonic acid (H₂CO₃) dissociation, and OA dissociation were equally important H⁺-producing reactions. Peat cation exchange accounted for 50% of the H⁺ sink, while SO₄ ^2? reduction and denitrification accounted for an additional 20 and 25% of the H⁺ sink, respectively. During the dry year, S oxidation accounted for 55% of the H⁺ net production, while that for H₂CO₃ dissociation was 70% lower than that during the climatically average year. Peat cation exchange consumed three times the acidity, and accounted for 92% of the H⁺ consumption during the dry year compared to the climatically average year. This was consistent with a three-fold higher net base cation export from the fen during the dry year. Based on the study results, a conceptual model was developed that describes the role of acidity formation and its implications to intermediate fen acidification.     

Local dynamics and structure of the solvated hydroxide ion in water

Details of the structural diffusion mechanism in proton transfer (PT) reactions involving a hydroxide ion in water and the structure of the solvated ions and transfer intermediates are in dispute. Here, we elucidate the mechanism of PT involving a hydroxide ion in water by molecular dynamics simulations using a dissociating water model based on ab initio calculations. We find that the hydroxide ion in bulk water is present as the four-coordinated OH^? (H₂O)₄ complex, which loses a water molecule before a PT occurs through the formation of proton sharing intermediates in general agreement with the previously disputed first principles studies of small systems.     

Effect of root environment on proton efflux in wheat roots

Proton net efflux of wheat (Triticum aestivum L.) roots growing in sand culture or hydroponics was determined by measuring the pH values of the solution surrounding the roots by pH microelectrodes, by base titration and by color changes of a pH indicator in solid nutrient media. The proton net efflux was dependent on light, aeration, and source of nitrogen (NH ₄ ⁺ , NO ₃ ^? ). Ammonium ions caused the highest proton efflux, whereas nitrate ions decreased the proton efflux. Iron deficiency had no significant effect on proton efflux. Replacement of ammonium by nitrate inhibited proton efflux, whereas the reverse enhanced proton extrusion. A lag period between changes in plant environment and proton efflux was observed. The proton net efflux occurred at the basal portion of the roots but not in the root tips or at the elongation zone. Under optimal conditions, proton efflux capacity reached a maximum value of 5.7 μmole H⁺ g^?1 fresh weight h^?1 with an average (between different measurements) of 3.4 μmole H⁺ g^?1 fresh wth^?1 whereas the pH value decreased to 3.2–3.7 and reached a minimal value of 2.9. Inhibition of ATPase activity by orthovanadate inhibited proton efflux. The results indicate that proton efflux in wheat roots is ammonium ion and light dependent and probably governed by ATPase activity.     

Hydrogen and oxygen trapping at the H-cluster of [FeFe]-hydrogenase revealed by site-selective spectroscopy and QM/MM calculations

[FeFe]-hydrogenases are superior hydrogen conversion catalysts. They bind a cofactor (H-cluster) comprising a four-iron and a diiron unit with three carbon monoxide (CO) and two cyanide (CN^?) ligands. Hydrogen (H₂) and oxygen (O₂) binding at the H-cluster was studied in the C169A variant of [FeFe]-hydrogenase HYDA1, in comparison to the active oxidized (Hox) and CO-inhibited (Hox-CO) species in wildtype enzyme. ⁵⁷Fe labeling of the diiron site was achieved by in vitro maturation with a synthetic cofactor analogue. Site-selective X-ray absorption, emission, and nuclear inelastic/forward scattering methods and infrared spectroscopy were combined with quantum chemical calculations to determine the molecular and electronic structure and vibrational dynamics of detected cofactor species. Hox reveals an apical vacancy at Fe_d in a [4Fe4S-2Fe]^3 ? complex with the net spin on Fe_d whereas Hox-CO shows an apical CN^? at Fe_d in a [4Fe4S-2Fe(CO)]^3 ? complex with net spin sharing among Fe_p and Fe_d (proximal or distal iron ions in [2Fe]). At ambient O₂ pressure, a novel H-cluster species (Hox-O₂) accumulated in C169A, assigned to a [4Fe4S-2Fe(O₂)]^3 ? complex with an apical superoxide (O₂^?) carrying the net spin bound at Fe_d. H₂ exposure populated the two-electron reduced Hhyd species in C169A, assigned as a [(H)4Fe4S-2Fe(H)]^3 ? complex with the net spin on the reduced cubane, an apical hydride at Fe_d, and a proton at a cysteine ligand. Hox-O₂ and Hhyd are stabilized by impaired O₂^– protonation or proton release after H₂ cleavage due to interruption of the proton path towards and out of the active site.     

In situ measurements confirm the seasonal dominance of benthic algae over phytoplankton in nearshore primary production of a large lake

1. Despite the recognition of its importance, benthic primary production is seldom reported, especially for large lakes. We measured in situ benthic net primary production by monitoring flux in dissolved inorganic carbon (DIC) concentration in benthic incubation chambers, based on continuous measurements of CO_2(aq) flux, alkalinity, and the temperature‐dependent dissociation constants of carbonic acid (K₁ and K₂). This methodology has the advantages of monitoring net primary production directly as change in carbon, maintaining continuous water recirculation, and having sufficient precision to detect change in DIC over short (i.e. 15 min) incubations, even in alkaline waters. 2. Benthic primary production on Cladophora‐dominated rocky substrata in western Lake Ontario was measured biweekly. Maximum biomass‐specific net photosynthetic rates were highest in the spring (2.39 mgC g Dry Mass^?1 h^?1), decreased to negative rates by early summer (?0.76 mgC g DM^?1 h^?1), and exhibited a regrowth in late summer (1.98 mgC g DM^?1 h^?1). 3. A Cladophora growth model (CGM), previously validated to predict Cladophora biomass accrual in Lake Ontario, successfully simulated the seasonality and magnitude of biomass‐specific primary production during the first cohort of Cladophora growth. Averaged over this growing season (May–Aug), mean areal net benthic production at the estimated depth of peak biomass (2 m) was 405 mg C m^?2 d^?1. 4. We measured planktonic primary production in proximity to the benthic study and constructed a depth‐resolved model of planktonic production. Using the CGM, benthic primary production was compared with planktonic primary production for the period May–Aug. Net benthic production from the shoreline to the 12 m contour (1–2 km offshore) equalled planktonic production. Closer to shore, benthic primary production exceeded planktonic primary production. Failure to account for benthic primary production, at least during abundant Cladophora growth, will lead to large underestimates in carbon and nutrient flows in the nearshore zone of this Great Lake.     

Proton stoichiometric imbalance during algae photosynthetic growth on various nitrogen sources: toward metabolic pH control

The use of algae as a potential platform for fuels or biochemical production requires process design and control that can be implemented at agronomic scales. Toward achieving pH control in large unmixed systems, we present a rigorous set of direct measurements of non-buffered proton uptake and efflux during growth on ammonium and nitrate, observing nearly unit molar proton imbalance H⁺/OH^? respectively for these nitrogen sources. This proton imbalance can be shown to be consistent with the initial assimilation steps of nitrogen from glutamate to peptide bonds which indicates that the remainder of metabolism is largely net proton balanced. These results are refined by demonstrating pH balance for growth with incrementally fed nitric acid and ammonium hydroxide. In contrast to the typical assumption of simple charge balance, each displays a slight proton uptake (around 10 % excess) that is considerably lower than urea, which displayed a molar H⁺ uptake per N assimilated of up to 33 %. This work illustrates details of proton imbalance that have been largely obscured in laboratory work due to use of elevated CO₂ and its associated carbonate equilibrium. Combined with the recent demonstration of preferential, mutually exclusive assimilation of ammonium over nitrate in Chlorella and Chlamydomonas, these results provide the stoichiometry and dynamics of photosynthetic algae growth needed to implement large-scale pH control in the absence of buffering.     

Chemical rescue of H+ delivery in proton transfer mutants of reaction center of photosynthetic bacteria

In the native and most mutant reaction centers of bacterial photosynthesis, the electron transfer is coupled to proton transfer and is rate limiting for the second reduction of Q_B^??→?Q_BH₂. In the presence of divalent metal ions (e.g. Cd²⁺) or in some (“proton transfer”) mutants (L210DN/M17DN or L213DN), the proton delivery to Q_B^? is made rate limiting and the properties of the proton pathway can be directly examined. We found that small weak acids and buffers in large concentrations (up to 1?M) were able to rescue the severely impaired proton transfer capability differently depending on the location of the defects: lesions at the protein surface (proton gate H126H/H128H?+?Cd²⁺), beneath the surface (M17DN?+?Cd²⁺, L210DN/M17DN) or deep inside the protein (L213DN) could be completely, partially or to very small extent recovered, respectively. Small zwitterionic acids (azide/hydrazoic acid) and buffers (tricine) proved to be highly effective rescuers consistent with their enhanced binding affinity and access to any of the proton acceptors (including Q_B^? itself) in the pathway. As a consequence, back titration of the protons at L212Glu could be observed as a pH-dependence of the rate constant of the charge recombination in the presence of azide or formate. Model calculations support the collective influence of the acid cluster on the change of the protonation states upon extension of the cluster with the bound small acid. In proton transfer mutants, the rescuing agents decreased the free energy of activation together with their enthalpic and entropic components. This is in agreement with the hypothesis that they function as protein-penetrating protonophores delivering protons into the chain and select dominating paths out of many alternate routes. We estimate that the proton delivery will be accelerated in one pathway out of 100–200 alternate pathways. The implications for design of the chemical recovery of impaired intra-protein proton transfer pathways in proton transfer mutants are discussed.     

Dynamic Structure of Proteins in Solid State. 1H and 13C NMR Relaxation Study

Abstract

Temperature dependencies of ¹H non-selective NMR T₁ and T₂ relaxation times measured at two resonance frequencies and natural abundance ^l3C NMR relaxation times T_l and T_lr measured at room temperature have been studied in a set of dry and wet solid proteins—;Bacterial RNase, lysozyme and Bovine serum albumin (BSA). The proton and carbon data were interpreted in terms of a model supposing three kinds of internal motions in a protein. These are rotation of the methyl protons around the axis of symmetry of the methyl group, and fast and slow oscillations of all atoms. The correlation times of these motions in solid state are found around 10^?11, 10^?9 and 10^?6 s, respectively. All kinds of motion are characterized by the inhomogeneous distribution of the correlation times. The protein dehydration affects only the slow internal motion. The amplitude of the slow motion obtained from the carbon data is substantially less than that obtained from the proton data. This difference can be explained by taking into account different relative inter- and intra- chemical group contributions to the proton and carbon second moments. The comparison of the solid state and solution proton relaxation data showed that the internal protein dynamics in these states is different: the slow motion seems to be few orders of magnitude faster in solution.     

Disruption of sulfur cycling and acid neutralization in lakes at low pH

Experimental acidification of a softwater lake to below pH 5 fundamentally changed the sulfur cycle and lowered internal alkalinity generation (IAG). Prior to reaching pH 4.5, the balance of sulfur reduction and oxidation reactions within the lake was in favour of reduction, and the lake was a net sink for sulfate. In the four years at pH 4.5 the balance of reduction and oxidation reactions was in favour of oxidation, and there was a net production of sulfate (SO₄ ^2–) within the lake. Evidence indicating a decrease in net SO₄ ^2– reduction at pH 4.5 was also obtained in an anthropogenically acidified lake that had been acidified for many decades. In both lakes, the decrease in net SO₄ ^2– reduction appeared to be linked not to a simple inhibition of SO₄ ^2– reduction but rather to changes in benthic ecosystem structure, especially the development of metaphytic filamentous green algae, which altered the balance between SO₄ ^2– reduction and sulfur oxidation.At pH's above 4.5, net SO₄ ^2– reduction was the major contributor to IAG in the experimental lake, as it is in many previously studied lakes at pH 5 and above. At pH 4.5, the change in net annual SO₄ ^2– reduction (a decrease of 110%) resulted in a 38% decrease in total IAG. Because of the important role of net SO₄ ^2– reduction in acid neutralization in softwater lakes, models for predicting acidification and recovery of lakes may need to be modified for lakes acidified to pH <5.     

Proton transfer kinetics of 5′-deoxypyridoxal

The kinetics of the isomerizaton between the zwitterion and the unpolar form of 5′-deoxypyridoxal in its monoprotonated state have been studied by means of the T-jump technque; and the equilibrium constant of the isomerization process has been determined.Several parallel reactions contribute to the kinetics of interconvention: a direct intramolecular proton transfer and proton exchange with H₃O⁺, OH^?, the totally protonated and the totally depronated 5′-deoxypyridoxal molecule.The Rate constants for these parallel reactions are evaluated from the measured relaxation times. They are discussed in the light of general aspects of proton transfer reactions.     

Kinetic and thermodynamic study of the substituent effect on the amino-Claisen rearrangement of para-substituted N-allyl-N-arylamine: a Hammett study via DFT

In order to find the susceptibility of the amino-Claisen rearrangement and the next proton shift reaction of N-allyl-N-arylamine to the substituent effects in the para position, the kinetic and thermodynamic parameters were calculated at the B3LYP level using the 6-31G^** basis set. The calculated activation energies for the rearrangements and proton shift reactions are close to 44.4 and 49.5 kcal mol^? 1, respectively. The transition states of the rearrangement with electron-donor substituents are more stable than those with electron-withdrawing substituent groups, but for the proton shift reaction, this situation is reversed (with the exception of fluorine atom for the rearrangement and fluorine and chlorine atoms for the proton shift reaction). Negative values for the activation entropy confirm the concerted mechanism for the amino-Claisen rearrangement and proton shift reaction. The Hammett ρ values of ? 2.4172 and ? 1.7791 are obtained for σ_p and σ^?  (enhanced sigma) in the amino-Claisen rearrangement, respectively. The correlation between log(k _X/k _H) and σ_p is weaker than that with σ^?  (enhanced sigma). A negative Hammett ρ value indicates that the electron-donating groups slightly increase the rate of amino-Claisen rearrangement. A positive Hammett ρ value (2.4921) for the proton shift reaction indicates that electron-withdrawing groups increase the rate of reaction.     

Moss production in a black spruce Picea mariana forest with permafrost near Fairbanks,Alaska, as compared with two permafrost-free stands

During the 1975 and 1976 seasons the net primary production of five common bryophytes in different stands of mature vegetation near Fairbanks, Alaska was investigated. Overall annual moss production at the intensive black spruce site was about 120 g m^?1 yr^?1 or about twice as high as the corresponding annual spruce production. Maximum rates of net photosynthesis varied from 2.7 mg CO₂ g^?1 h^?1 in Polytrichum commune Hedw. to 0.6 mg CO₂ g^?1 h^?1 in Sphagnum nemoreum Scop. The photosynthesis of overwintered leaves early in the season was low and as a result of new growth a steady increase in net photosynthesis occurred throughout the season. Leaf water content was found to be the most important limiting factor for growth under natural conditions. There was a strong increase in growth and photosynthesis of Sphagnum nemoreum after application of N and P, indicating nutrient deficiency.     

Ecological studies on the timberline of Mt. Fuji

An ecological study of dry matter production was made in a dwarf forest dominated byAlnus maximowiczii at the timberline of Mt. Fuji. Annual gross production was estimated by two methods, namely the summation method using stem analysis and total photosynthesis calculated from leaf area and photosynthetic rate per leaf area. Seasonal changes in relative light intensity and in leaf area were measured in a quadrat. Photosynthesis and respiration rates of samples were measured in temperature-regulated assimilation chambers. The phytomass was 2,989 g d.w.m^?2, and those of stems and branches, leaves, and roots were 1,672 g, 293 g, and 1,024 g respectively. The growing period of this plant was about four months and this plant expanded leaves quickly. The maximum gross photosynthetic rate was 21 mg CO₂dm^?2 h^?1 on September 1. Annual net production estimated by examining the annual rings was 922 g d.w.m^?2 year^?1 and annual respiration was 735 g. Annual gross production estimated from photosynthetic rates was 1,747 g d.w.m^?2 year^?1. The sum of annual net production by stem analysis and respiration agree closely with gross production estimated from photosynthetic rate. Gross production of this dwarf forest is comparable to the beech forest of the upper cool temperate zone owing to the high photosynthetic rate ofAlnus maximowiczii.     

The European carbon balance. Part 4: integration of carbon and other trace‐gas fluxes

Overviewing the European carbon (C), greenhouse gas (GHG), and non‐GHG fluxes, gross primary productivity (GPP) is about 9.3 Pg yr^?1, and fossil fuel imports are 1.6 Pg yr^?1. GPP is about 1.25% of solar radiation, containing about 360 × 10¹⁸ J energy – five times the energy content of annual fossil fuel use. Net primary production (NPP) is 50%, terrestrial net biome productivity, NBP, 3%, and the net GHG balance, NGB, 0.3% of GPP. Human harvest uses 20% of NPP or 10% of GPP, or alternatively 1‰ of solar radiation after accounting for the inherent cost of agriculture and forestry, for production of pesticides and fertilizer, the return of organic fertilizer, and for the C equivalent cost of GHG emissions. C equivalents are defined on a global warming potential with a 100‐year time horizon. The equivalent of about 2.4% of the mineral fertilizer input is emitted as N₂O. Agricultural emissions to the atmosphere are about 40% of total methane, 60% of total NO‐N, 70% of total N₂O‐N, and 95% of total NH₃‐N emissions of Europe. European soils are a net C sink (114 Tg yr^?1), but considering the emissions of GHGs, soils are a source of about 26 Tg CO₂ C‐equivalent yr^?1. Forest, grassland and sediment C sinks are offset by GHG emissions from croplands, peatlands and inland waters. Non‐GHGs (NH₃, NOx) interact significantly with the GHG and the C cycle through ammonium nitrate aerosols and dry deposition. Wet deposition of nitrogen (N) supports about 50% of forest timber growth. Land use change is regionally important. The absolute flux values total about 50 Tg C yr^?1. Nevertheless, for the European trace‐gas balance, land‐use intensity is more important than land‐use change. This study shows that emissions of GHGs and non‐GHGs significantly distort the C cycle and eliminate apparent C sinks.     

The plastoquinone pool as possible hydrogen pump in photosynthesis

The function of the plastoquinone pool as a possible pump for vectorial hydrogen (H⁺ + e^?) transport across the thylakoid membrane has been investigated in isolated spinach chloroplasts. Measurements of three different optical changes reflecting the redox reactions of the plastoquinone, the external H⁺ uptake and the internal H⁺ release led to the following conclusions:(1) A stoichiometric coupling of 1 : 1 : 1 between the external H⁺ uptake, the electron translocation through the plastoquinone pool and the internal H⁺ release (corrected for H⁺ release due to H₂O oxidation) is valid (pH_out = 8, excitation with repetitive flash groups). (2) The rate of electron release from the plastoquinone pool and the rate of proton release into the inner thylakoid space due to far-red illumination are identical over a range of a more than 10-fold variation.These results support the assumption that the protons taken up by the reduced plastoquinone pool are translocated together with the electrons through the pool from the outside to the inside of the membrane. Therefore, the plastoquinone pool might act as a pump for a vectorial hydrogen (H⁺ + e^?) transport. The molecular mechanism is discussed. The differences between this hydrogen pump of chloroplasts and the proton pump of Halobacteria are outlined.     

The participation of a tryptophan residue in the binding of ferric iron to pyrocatechase

The fast reaction technique of pulse radiolysis was used to produce free radicals in aqueous solution from alcohols, deoxyribose, cytosine, uracil, thymine, dihydrothymine and histidine. The electron transfer reactions from these radicals to p-benzoquinone was observed from the formation kinetics of the semiquinone anion ·BQ^? at 430 nm and the efficiency was found to be as high as 90% or more, with k～5×10⁹ M^?1sec^?1. In acid or neutral solutions in the presence of oxygen the peroxy radicals ·O₂RH formed do not essentially transfer an electron to BQ, and the efficiency is <10%. The significance of these results in the fixation of radiation damage in photobiology and radiation biology are indicated. The reactions of the superoxide ·O₂^? radical with BQ are also presented and discussed.     

M234Glu is a component of the proton sponge in the reaction center from photosynthetic bacteria

Bacterial reaction centers use light energy to couple the uptake of protons to the successive semi-reduction of two quinones, namely Q_A and Q_B. These molecules are situated symmetrically in regard to a non-heme iron atom. Four histidines and one glutamic acid, M234Glu, constitute the five ligands of this atom. By flash-induced absorption spectroscopy and delayed fluorescence we have studied in the M234EH and M234EL variants the role played by this acidic residue on the energetic balance between the two quinones as well as in proton uptake. Delayed fluorescence from the P⁺Q_A^? state (P is the primary electron donor) and temperature dependence of the rate of P⁺Q_A^? charge recombination that are in good agreement show that in the two RC variants, both Q_A^? and Q_B^? are destabilized by about the same free energy amount: respectively ～ 100 ± 5 meV and 90 ± 5 meV for the M234EH and M234EL variants, as compared to the WT. Importantly, in the M234EH and M234EL variants we observe a collapse of the high pH band (present in the wild-type reaction center) of the proton uptake amplitudes associated with formation of Q_A^? and Q_B^?. This band has recently been shown to be a signature of a collective behaviour of an extended, multi-entry, proton uptake network. M234Glu seems to play a central role in the proton sponge-like system formed by the RC protein.     

Nitrate fluxes in soybean seedling roots and their response to amino acids: an approach using 15N

This study was undertaken to determine which of the two NO₃^? fluxes (influx or efflux) across plasma membranes of root cells is the target of those amino acids which have been shown to inhibit net NO₃^? uptake (Muller & Touraine 1992, Journal of Experimental Botany 43 , 617–623). Parallel experiments were performed to mea-sure either the time course of ¹⁵NO₃^? release from roots of soybean seedlings previously labelled with this isotope into non-labelled solution, or the time course of ¹⁵N accumulation from labelled ¹⁵NO₃^? solution in non-labelled seedlings. Focusing on the fate of ¹⁵NO₃^? in the cytoplasmic compartment, a model is developed to describe the time courses of the accumulation and release of tracer across the plasma membranes of root cells. Both time courses can be described by the sum of an exponential plus a linear term. In our material, the linear part of the accumulation time course is obscured by the NO₃^? fluxes exiting the cytoplasm, and the curve thus appears to be quasilinear over several minutes. However, we show that the use of the net tracer accumulation rate during this time period as an estimate of NO₃^? influx does not provide accurate estimates of influx and efflux. By contrast, ¹⁵NO₃^? efflux analysis permits calculation of the unidirectional fluxes across plasma membranes of root cells and the kinetic parameters of the cytoplasmic NO₃^? pool. Under our experimental conditions, efflux accounted for 30 to 50% of influx, and the cytoplasmic NO₃^? content was found to be in the 70–400nmol g^?1 fw range. Using this methodology, the effect of amino acid accumulation on unidirectional fluxes of nitrate was then examined. Pretreatments of the seedlings with an amino acid which has been shown to inhibit net NO₃^? uptake led to concomitant decreases in net accumulation rates of ¹⁵NO₃^? and of reduced ¹⁵N in roots and total ¹⁵N in cotyledons. NO₃^? influx was markedly inhibited by these treatments, while NO₃^? efflux remained essentially unaffected, or even decreased. It is concluded that the target of the regulation of NO₃^? uptake by phloemtranslocated amino acids is the influx system.     

Diurnal variations in the auto- and heterotrophic activity of cyanobacterial phycospheres (Gloeotrichia echinulata) and the identity of attached bacteria

1. We assessed the role of cyanobacterial–bacterial consortia (Gloeotrichia echinulata phycospheres) for net changes in inorganic carbon, primary production (PP) and secondary production in Lake Erken (Sweden). 2. At the time of sampling, large colonies of G. echinulata formed a massive bloom with abundances ranging from 102 colonies L^?1 in the pelagic zone to 5000 colonies L^?1 in shallow bays. These colonies and their surrounding phycospheres contributed between 17 and 92% of total PP, and phycosphere‐associated bacteria contributed between 8.5 and 82% of total bacterial secondary production. PP followed a diurnal cycle, whereas bacterial production showed no such pattern. Over a 24 h period, carbon dioxide measurements showed that the phycospheres were net autotrophic in the top layer of the water column, whereas they were net heterotrophic below 2 m depth. 3. Sequencing and phylogenetic analysis of 16S rRNA genes of attached bacteria revealed a diverse bacterial community that included populations affiliated with Proteobacteria, Bacteriodetes, Acidobacteria, Fusobacteria, Firmicutes, Verrucomicrobia, and other Cyanobacteria. 4. Compared with their planktonic counterparts, bacteria associated with cyanobacterial phycospheres had lower affinity for arginine, used as a model compound to assess uptake of organic compounds. 5. Extrapolation of our data to the water column of lake Erken suggests that microorganisms that were not associated with cyanobacteria dominated CO₂ production at the ecosystem scale during our experiments, as CO₂ fixation balanced CO₂ production in the cyanobacterial phycospheres.     

GHG emissions and other environmental impacts of indirect land use change mitigation

The implementation of measures to increase productivity and resource efficiency in food and bioenergy chains as well as to more sustainably manage land use can significantly increase the biofuel production potential while limiting the risk of causing indirect land use change (ILUC). However, the application of these measures may influence the greenhouse gas (GHG) balance and other environmental impacts of agricultural and biofuel production. This study applies a novel, integrated approach to assess the environmental impacts of agricultural and biofuel production for three ILUC mitigation scenarios, representing a low, medium and high miscanthus‐based ethanol production potential, and for three agricultural intensification pathways in terms of sustainability in Lublin province in 2020. Generally, the ILUC mitigation scenarios attain lower net annual emissions compared to a baseline scenario that excludes ILUC mitigation and bioethanol production. However, the reduction potential significantly depends on the intensification pathway considered. For example, in the moderate ILUC mitigation scenario, the net annual GHG emissions in the case study are 2.3 MtCO₂‐eq yr^?1 (1.8 tCO₂‐eq ha^?1 yr^?1) for conventional intensification and ?0.8 MtCO₂‐eq yr^?1 (?0.6 tCO₂‐eq ha^?1 yr^?1) for sustainable intensification, compared to 3.0 MtCO₂‐eq yr^?1 (2.3 tCO₂‐eq ha^?1 yr^?1) in the baseline scenario. In addition, the intensification pathway is found to be more influential for the GHG balance than the ILUC mitigation scenario, indicating the importance of how agricultural intensification is implemented in practice. Furthermore, when the net emissions are included in the assessment of GHG emissions from bioenergy, the ILUC mitigation scenarios often abate GHG emissions compared to gasoline. But sustainable intensification is required to attain GHG abatement potentials of 90% or higher. A qualitative assessment of the impacts on biodiversity, water quantity and quality, soil quality and air quality also emphasizes the importance of sustainable intensification.