Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus: Growth yields and morphological characterization

The growth stoichiometry of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus was determined in glucose-limited chemostat cultivations using a chemically defined medium. This strain produces adipoyl-7-aminodeacetoxycephalosporanic acid (ad-7-ADCA) when it is fed with adipic acid. The biomass yield and maintenance coefficients for the strain were similar to those found for penicillin-producing strains of Penicillium chrysogenum. The maximum specific growth rate in the chemostat was found to be 0.11 h(-1). Metabolic degradation of adipate was found to take place in significant amounts only at dilution rates below 0.03 h(-1). After three to five residence times, adipate degradation and ad-7-ADCA production disappeared, and this allowed determination of the biomass yield coefficient on adipate. The morphology was measured at different dilution rates and the mean total hyphal length and mean number of tips both increased with an increase in dilution rate from 0.015 to 0.065 h(-1). Both variables decreased when the dilution rate was increased above 0.065 h(-1). A correlation between mean total hyphal length and productivity of ad-7-ADCA was found.     

Physiological characterisation of Penicillium chrysogenum strains expressing the expandase gene from Streptomyces clavuligerus during batch cultivations. Growth and adipoyl-7-aminodeacetoxycephalosporanic acid production.

The production of adipoyl-7-aminodeacetoxy-cephalosporanic acid (ad-7-ADCA) was studied, using two recombinant strains of Penicillium chrysogenum carrying the expandase gene from Streptomyces clavuligerus. The adipoyl-side chain of this compound may easily be removed using an amidase; and this process therefore represents a new route for the production of 7-ADCA, which serves as a precursor for the production of many semi-synthetic cephalosporins. In this study, one low- and one high-yielding strains were characterised and the specific productivities of ad-7-ADCA and byproducts of the biosynthetic pathway were compared. The fluxes through the biosynthetic pathway were quantified and it was found that there was a 30% higher flux through the expandase in the high-yielding strain. In both strains, there was a significant degradation of adipate. Furthermore, the initial adipate concentration in batch cultures was shown to have a positive effect on the formation of ad-7-ADCA.     

Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus: Kinetics of adipoyl-7-aminodeacetoxycephalosporanic acid and byproduct formations

The production kinetics of a transformed strain of Penicillium chrysogenum expressing the expandase gene from Streptomyces clavuligerus was investigated in chemostat cultivations. The recombinant strain produces adipoyl-7-aminodeacetoxycephalosporanic acid (ad-7-ADCA) as the major product; however, during the cultivations, the appearance of a major unknown and poorly secreted product was observed. Investigations using high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS) showed that this byproduct has a six-membered dihydrothiazine ring, which is characteristic for cephalosporins. The byproduct may be formed via isopenicillin N by as-yet unknown mechanisms, but involving expandase. It is likely that the unknown compound (UC) is deacetoxycephalosporin C (DAOC). Investigation of the instability of the various beta-lactams produced showed higher instability for compounds with a five-membered thiazolidine ring than those with a six-membered dihydrothiazine ring. Furthermore, secretion of products and byproducts was shown to be quite different. The productivity was studied as a function of the dilution rate in the range 0.015 to 0.090 h(-1). The specific productivity of total beta-lactams was compared with that of the penicillin-G-producing host strain, and it was found to be lower at dilution rates of <0.06 h(-1). Quantification of the fluxes through the pathway leading to ad-7-ADCA showed a decrease in flux toward ad-7-ADCA, and an increase in flux toward UC as the dilution rate increased. Northern analysis of the biosynthetic genes showed that expression of the enzymes involved in the ad-7-ADCA pathway decreased as the dilution rate increased.     

Exploring Bacterioplankton Growth and Protein Synthesis to Determine Conversion Factors Across a Gradient of Dissolved Organic Matter

The effect of bacterial specific growth rates of abundance (micro) and protein synthesis (b) on conversion factor (CF) variability was explored in order to provide an alternative approach to the controversial application of just one universal CF to field data. Nine regrowth cultures (RCs) were set up from very diverse aquatic ecosystems, controlling temperature and adding N and P to avoid mineral limitation and force organic carbon limitation. The values of micro varied one order of magnitude from 0.26 to 3.34 d(-1), whereas b values varied two orders of magnitude from 0.28 to 34.87 d(-1). We found no relationships between micro or b values and the dissolved organic carbon (DOC) concentration or the dissolved organic matter (DOM) quality indexes assayed. Abundance and protein synthesis increased exponentially and synchronously in four RCs, leading to balanced growth (micro = b). In contrast, abundance and protein synthesis increased logistically in the other five RCs and b values were significantly higher than g values, leading to unbalanced growth (micro not equal b). CFs ranged from 0.0062 to 0.0576 x 10(18) cells mol leucine(-1) with an average of 0.0305 x 10(18) cells mol leucine(-1). CFs obtained in RCs with balanced growth were generally higher than CFs obtained in RCs with unbalanced growth and were not alike, impeding the establishment of an upper limit for CFs. A positive and significant relationship (n = 8, p < 0.0 1, r2 = 0.71) was found between CFs and DOC concentration (CF (x10(18) cells mol leucine(-1)) = 0.0104 + 0.0094 DOC (mM)) when the value for the most productive system was excluded. This function permits the estimation of site-specific CFs based on DOC concentration instead of the controversial use of a single CF for different systems.     

Influence of medium composition on the cephalosporin C production with a highly productive strain Cephalosporium acremonium.

Cephalosporin production by a highly productive Cephalosporium acremonium strain was carried out and optimized by fed-batch operation in a 40 l stirred tank reactor using a complex medium containing 30-120 g l-1 peanut flour. The concentrations of cephalosporin C (CPC) and its precursors: penicillin N (PEN N), deacetoxy cephalosporin C (DAOC), and deacetyl cephalosporin C (DAC) were monitored with an on-line HPLC. The concentrations of amino acids valine (VAL), cysteine (CYS), alpha-amino adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV), were determined off-line by HPLC. The RNA content and dry weight of the sediment as well as the oxygen transfer rate (OTR) and the CO2 production rate (CPR) were used to calculate the cell mass concentration (X). The influences of peanut flour (PF) and the on-line monitored and controlled medium components: glucose (GLU), phosphate, methionine (MET) as well as the dissolved oxygen (DOC) on the cell growth, the product formation, and the pathway of cephalosporin C biosynthesis were investigated and evaluated. When the glucose fed-batch cycle was optimized and oxygen transfer limitation was avoided (DOC greater than 20% of the saturation value), high process performance (103.5 g l-1 X, 11.84 g l-1 CPC, a maximum CPC productivity of 118 mg l-1 h-1, and the whole concentration of the beta-lactam antibiotics CPC, DAC, DAOC, PEN N 17.34 g l-1) was achieved by using 100 g l-1 PF in the medium with the optimum concentration of phosphate (260-270 mg l-1) and a low glucose concentration (less than 0.5 g l-1). The cultivations with different medium concentrations demonstrated that the product formation was directly proportional to the cell mass concentration. On the average, the cell mass-based yield coefficient of CPC: YCPC/X amounted to 0.115 g CPC per g cell mass.     

Metabolic network analysis of an adipoyl-7-ADCA-producing strain of Penicillium chrysogenum: elucidation of adipate degradation

An adipoyl-7-ADCA-producing, recombinant strain of Penicillium chrysogenum was characterized by metabolic network analysis, with special focus on the degradation of adipate and determination of the metabolic fluxes. Degradation of the side-chain precursor, adipate, causes an undesired consumption of adipate in the production of 7-ADCA. Using (13)C-labeled glucose and measurement of metabolite labeling patterns, it was shown that adipate was degraded by beta-oxidation to succinyl-CoA and acetyl-CoA. The labeling analysis indicated that degradation of adipate was taking place in the microbodies and the formed acetyl-CoA was metabolized in the glyoxylate shunt. This hypothesis was further substantiated by an enzyme assay, which showed activity of the key enzyme in the glyoxylate shunt. Flux estimations in two chemostat cultures, one with and one without adipate in the feed, revealed that degradation of adipate replaces the net anaplerotic reaction from pyruvate to oxaloacetate. Thus, with a combination of labeling experiments and enzyme assays, the pathway of adipate degradation was elucidated, and the effect of adipate degradation on the primary metabolism was quantified.     

Effects of dissolved oxygen on the morphology of an arachidonic acid production by Mortierella alpina 1S-4

Arachidonic acid (AA) production by Mortierella alpina 1S-4 was investigated using a 50-L fermentor. In order to optimize the dissolved oxygen (DO) concentration and to investigate the effect of DO on morphology, cultivation was carried out under constant DO at various levels in the range of 3-50 ppm. To maintain a DO concentration above 7 ppm, two methods, i.e., the oxygen-enrichment (OE) method (experimental range, 25-90% oxygen gas supplied) and the pressurization (PR) method (experimental range, 180-380 kPa headspace pressure), were used. As a result, the optimum DO concentration range was found to be 10-15 ppm. In this optimum DO concentration range, the AA yield was enhanced about 1.6-fold compared to that obtained at 7 ppm DO, and there was no difference in the AA productivity between the OE and PR methods. When the DO concentration was maintained at 20-50 ppm using the OE method, the morphology changed from filaments to pellets, and the AA yield decreased drastically because of stress due to the limited mass transfer through the pellet wall. When the DO concentration was maintained at 15-20 ppm using the PR method, the morphology did not change, and the AA yield decreased gradually.     

The dual influences of dissolved organic carbon on hypolimnetic metabolism: organic substrate and photosynthetic reduction

We investigated the effect of dissolved organic carbon (DOC) on hypolimnetic metabolism (accumulation of dissolved inorganic carbon (DIC) and methane (CH₄)) in 21 lakes across a gradient of DOC concentrations (308 to 1540 mol C L^–1). The highly colored nature of the DOC in these lakes suggests it is mostly of terrestrial origin. Hypolimnetic methane accumulation was positively correlated with epilimnetic DOC concentration (Spearman rank correlation = 0.67; p < 0.01), an indicator of allochthonous DOC inputs, but not with photic zone chlorophyll a concentration (Spearman rank correlation = 0.30; p = 0.22). Hypolimnetic DOC concentrations declined in 19 of 21 lakes during the stratified period at rates that ranged from 0.06 to 53.9 mmol m^–2 d^–1. The hypolimnetic accumulation of DIC + CH₄ was positively correlated with, and, in most cases of comparable magnitude to, this DOC decline suggesting that DOC was an important substrate for hypolimnetic metabolism. The percentage of surface irradiance reaching the thermocline was lower in high DOC lakes (0.3%) than in low DOC lakes (6%), reducing hypolimnetic photosynthesis (as measured by the depth and magnitude of the deep dissolved oxygen maxima) in the high DOC lakes. In June, the hypolimnia of lakes with < 400 mol L^–1 DOC had high concentrations of dissolved oxygen and no CH₄, while the hypolimnia of lakes with DOC > 800 mol L^–1 were completely anoxic and often had high CH₄ concentrations. Thus, DOC affects hypolimnetic metabolism via multiple pathways: DOC was significant in supporting hypolimnetic metabolism; and at high concentrations depressed photosynthesis (and therefore oxygen production and DIC consumption) in the hypolimnion.     

The Siak, a tropical black water river in central Sumatra on the verge of anoxia

The Siak is a black water river in central Sumatra, Indonesia, which owes its brown color to dissolved organic matter (DOM) leached from surrounding, heavily disturbed peat soils. The dissolved organic carbon (DOC) concentrations measured during five expeditions in the Siak between 2004 and 2006 are among the highest reported world wide. The DOM decomposition appeared to be a main factor influencing the oxygen concentration in the Siak which showed values down to 12 μmol l⁻¹. Results derived from a box-diffusion model indicated that in addition to the DOC concentration and the associated DOM decomposition the water-depth also plays a crucial role in regulating the oxygen levels in the river because of its impact on the turbulence in the aquatic boundary layer and the surface/volume ratio of water in the river. Model results imply furthermore that a reduced water-depth could counteract an increased oxygen consumption caused by an enhanced DOM leaching during the transition from dry to wet periods. This buffer mechanism seems to be close to its limits as indicated by sensitivity studies which showed in line with measured data that an increase of the DOC concentrations by ~15% could already lead to anoxic conditions in the Siak. This emphasizes the sensitivity of the Siak against further peat soil degradation, which is assumed to increase DOC concentrations in the rivers.     

Concentrations and fluxes of dissolved organic carbon in an age-sequence of white pine forests in Southern Ontario,Canada

We determined concentrations and fluxes of dissolved organic carbon (DOC) in precipitation, throughfall, forest floor and mineral soil leachates from June 2004 to May 2006 across an age-sequence (2-, 15-, 30-, and 65-year-old) of white pine (Pinus strobus L.) forests in southern Ontario, Canada. Mean DOC concentration in precipitation, throughfall, leachates of forest floor, Ah-horizon, and of mineral soil at 1 m depth ranged from ∼2 to 7, 9 to 18, 32 to 88, 20 to 66, and 2 to 3 mg DOC L⁻¹, respectively, for all four stands from April (after snowmelt) through December. DOC concentration in forest floor leachates was highest in early summer and positively correlated to stand age, aboveground biomass and forest floor carbon pools. DOC fluxes via precipitation, throughfall, and leaching through forest floor and Ah-horizon between were in the range of ∼1 to 2, 2 to 4, 0.5 to 3.5, and 0.1 to 2 g DOC m⁻², respectively. DOC export from the forest ecosystem during that period through infiltration and groundwater discharge was estimated as ∼7, 4, 3, and 2 g DOC m⁻² for the 2-, 15-, 30-, and 65-year-old sites, respectively, indicating a decrease with increasing stand age. Laboratory DOC sorption studies showed that the null-point DOC concentration fell from values of 15 to 60 mg DOC L⁻¹ at 0 to 5 cm to <15 mg DOC L⁻¹ at 50 cm. Specific ultraviolet light absorption at 254 nm (SUVA₂₅₄) increased from precipitation and throughfall to a maximum in forest floor and decreased with mineral soil depth. No age-related pattern was observed for SUVA₂₅₄ values. DOC concentration in forest floor soil solutions showed a positive exponential relationship with soil temperature, and a negative exponential relationship with soil moisture at all four sites. Understanding the changes and controls of DOC concentrations, chemistry, and fluxes at various stages of forest stand development is necessary to estimate and predict DOC dynamics on a regional landscape level and to evaluate the effect of land-use change.     

Effect of Dissolved Oxygen, Temperature, Initial Cell Count, and Sugar Concentration on the Viability of Saccharomyces cerevisiae in Rapid Fermentations

By using 7 x 10(8) cells of Saccharomyces cerevisiae per ml with which 25 degrees Brix honey solutions were fermented to 9.5% (wt/vol; 12% vol/vol) ethanol in 2.5 to 3 h at 30 C, i.e., rapid fermentation, the death rate was found to be high, with only 2.1% of the yeast cells surviving at the end of 3 h under anaerobic conditions. As the dissolved oxygen in the medium was increased from 0 to 13 to 20 to 100% in rapid fermentations at 30 C, there was a progressive increase in the percentage of cells surviving. The ethanol production rate and total were not seriously affected by a dissolved oxygen concentration of 13%, but fermentation was retarded by 20% dissolved oxygen and still further decreased as the dissolved oxygen content reached 100%. When the fermentation temperature was decreased to 15 C (at 13% dissolved oxygen), the rate of fermentation decreased, and the fermentation time to 9.5% ethanol (wt/vol) increased to 6 h. It was found that the higher the temperature between 15 and 30 C, the greater the rate of death as initial cell counts were increased from 1.1 x 10(7) to 7.8 x 10(8) cells per ml. At the lowest level of inoculum, 1.1 x 10(7) cells per ml, there was actual multiplication, even at 30 C; however, the fermentation was no longer rapid. The addition of 15% sugar, initially followed after an hour by the remaining 10%, or addition of the sugar in increments of 2.5 or 5% yielded a better survival rate of yeast cells than when the fermentation was initiated with 25% sugar.     

Influence of dissolved oxygen concentration on the biosynthesis of cephalosporin C.

Cephalosporin C was produced by a highly productive strain of Cephalosporium acremonium under industrial production conditions by fed-batch cultivation in a 40-l stirred-tank reactor using a complex medium containing 50 g l-1 peanut flour. The influence of dissolved oxygen concentration (pO2, DOC), which was maintained at different constant levels between 5 and 40% of its saturation value, during the production phase by means of a parameter-adaptive pO2-controller, on the cephalosporin C biosynthesis, was investigated. The concentrations of cephalosporin C (CPC) and its precursors penicillin N (PEN N), deacetoxycephalosporin C (DAOC), and deacetylcephalosporin C (DAC) were monitored by on-line HPLC. The concentrations of amino acids, valine (VAL), cysteine (CYS), alpha-amino-adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV) were determined by off-line HPLC. By reducing the pO2 in the production phase from 40 to 5% of its saturation value, the CPC concentration diminished from 7.2 to 1.1 g l-1 and the PEN N concentration increased from 2.57 to 7.65 g l-1. The DAC concentration also dropped from 3.13 to 0.42 g l-1; however, the DAOC concentration was less influenced. The concentrations of AC and ACV were also less affected. The small DOC did not lead to an accumulation of the intermediate AC and ACV during the production phase. With increasing DOC in the range of 5-20%, the maximal specific production rate, the cell mass concentration-based and the substrate-based yield coefficients for CPC increased almost linearly, and fell back for PEN N.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the emergent macrophyte Juncus effusus L. on the chemical composition of interstitial water and bacterial productivity

Release of oxygen from the roots ofaquatic macrophytes into anaerobic sediments canaffect the quantity of interstitial dissolved organicmatter and nutrients that are available to bacteria. Nutrient and dissolved organic carbon (DOC)concentrations were compared between subsurface(interstitial) waters of unvegetated sediments andsediments among stands of the emergent herbaceousmacrophyte Juncus effusus L. in a lotic wetlandecosystem. Concentrations of inorganic nitrogen(NH₄ ⁺, NO₃ ^-, and NO₂ ^-)were greater from sediments of the unvegetatedcompared to the vegetated zone. DOC concentrations ofinterstitial waters were greater in sediments of theunvegetated zone both in the winter and springcompared to those from the vegetated zone. AlthoughDOC concentrations in hydrosoils collected from bothzones increased from winter to spring, bacterialproductivity per mg DOC in spring decreased comparedto winter. Greater initial bacterial productivityoccurred on DOM collected from the vegetated comparedto the unvegetated zone in winter samples (days 1 and4), with increased bacterial productivity on samplescollected from the unvegetated zone at the end of thestudy (day 20). Bacterial productivity wassignificantly greater on all sampling days on DOM fromvegetated samples compared to unvegetated samples. In nutrient enrichment experiments, bacterialproductivity was significantly increased (p < 0.05)with phosphorus but not nitrogen only amendments.     

大口黑鲈池塘工程化循环水养殖系统的溶解氧时空变化及菌群响应特征

采用基于Illumina Miseq测序平台的高通量测序技术, 从不同角度(密度、区域、溶氧、季节)分析大口黑鲈(Micropterus salmoides)池塘工程化循环水养殖系统中溶解氧和水温对细菌群落结构和丰富度的影响。结果表明: 菌群丰富度在9月份最高, 在10月份最低。在昼夜变化中, 溶解氧最低时的菌群丰富度整体上高于溶解氧最高时。在不同区域中, 粪便收集区的菌群丰富度高于养殖区。在7—11月份的季节变化中, 变形菌、放线菌、拟杆菌和蓝细菌的相对丰度占据前四位; 在属水平上, 假单胞菌、黄杆菌和聚球菌为优势物种; 几乎每种细菌都具有显著或极显著的月份差异。假单胞菌的相对丰度与溶解氧和水温皆具有极显著的相关性。聚球菌、蓝细菌、CL-500_marine_group和Alpinimonas皆与水温具有极显著相关性。分枝杆菌、MNG7与溶解氧极显著相关。此外, 不同菌群之间也具有显著或极显著的相关性。实验结果表明放养密度、养殖区域、溶氧浓度和季节变化都会影响水体菌群丰富度。     

Transport and fate of dissolved organic carbon in the Lake Pontchartrain esutary,Louisiana, U.S.A.

Cycling dynamics of dissolved organic carbon (DOC) were examinedin Lake Pontchartrain estuary, Louisiana, in relation to changesin freshwater inputs. DOC concentrations ranged from 5.3 to 8.5mg C L-1 reaching their highest during high river inflow.The percentage of DOC represented by HMW DOC (or colloidal material)was greatest (ca. 11%) at stations where freshwaterdischarge from rivers and surrounding wetlands was most significant.Moreover, the lignin-phenol content of this material (ranged from 0.09 to 0.33 and from 0.11 to 0.39)confirmed that a significant fraction of colloidal organic carbon wasderived from terrestrial sources. Riverine and benthic fluxes representedthe dominant sources of DOC to the estuary. On an annual basis, riverineand benthic DOC concentrations were estimated to be 2.8 ×10 10 g C yr-1 and 8.8 × 10 10 g C yr-1, respectively, while the totalDOC pool in the estuary was 3.8 × 10 10 gC. Annual average concentrations of dissolved inorganic carbon (DIC)(1298 M) and pCO2 (5774 atm)were comparable to those found in other freshwater systems that reachedCO2 saturation levels. Net losses of DOC in the LakePontchartrain estuary appeared to be primarily controlled by heterotrophicconsumption (conversion of CO2) – whichmay have been amplified by the long residence time (approximately 120days) of DOC in this system.     

以纸为碳源去除地下水硝酸盐的研究

研究了以纸为碳源和反应介质的生物反应器对水中硝酸盐的去除。结果表明,以纸为碳源的反应器启动快．反硝化反应受温度及水力停留时间影响大。25℃的反硝化速率是14℃的1．7倍。在室温25±1℃,进水硝酸盐氮浓度为45．2mg·L^-1、水力停留时间8．6h时,反应器对硝酸盐氮的去除率在99．6％以上,当水力停留时间为7．2h,氮去除率只有50％。反硝化反应受pH值和溶解氧的影响小,反应进行过程中,纸表面形成了生物膜,纸也被消耗了．采用反应器出水再经活性炭吸附的工艺流程处理高硝酸盐氮地下水,<33．9mg·L^-1的硝酸盐氮完全去除,没有出现NC2-N,最终出水水质DOC<11mg·L^-1。     

Potential capacity of coprecipitation of dissolved organic carbon (DOC) with iron(III) precipitates

Hypolimnetic anoxic water of Lake Onogawa was subjected to aeration experiments. When the samples were agitated by magnetic stirrers for 24 h, dissolved oxygen increased from 0 to more than 7.6 mg l⁻¹, dissolved iron decreased from 98% to about 5% of the initial total iron, and from 32% to 48% of the dissolved organic carbon (DOC) disappeared. On the other hand, when the anoxic waters were left unstirred, dissolved oxygen increased from 0 to 2.2 mg l⁻¹, dissolved iron decreased from 98% to 31%, and 20% of the DOC disappeared within 48 h. Further 24-h incubation had little effect on the DOC loss, although dissolved oxygen increased to 3.9 mg l⁻¹ and dissolved iron decreased to 5%. These rates of DOC disappearance are too large to be explained by bacterial decomposition. It is quite conceivable that a part of the DOC is coprecipitated with iron(III) precipitates. When Fe(II) in the anoxic hypolimnion is oxidized by autumnal water mixing, probably anoxic water is mixed with aerobic water. The anoxic water must receive oxygen from the aerobic water during this mixing and be simultaneously diluted with the aerobic water. Because the present experimental conditions, especially the stirred one, significantly differ from in situ conditions, the present results are thought to be a potential capacity of DOC coprecipitation.     

A mixing model analysis of stream solute dynamics and the contribution of a hyporheic zone to ecosystem function*

1. We monitored streamwater and streambed sediment porewaters from White Clay Creek (WCC), SE Pennsylvania, for dissolved organic carbon (DOC), dissolved oxygen (DO) and conductivity to investigate organic matter processing within the hyporheic zone. Dissolved organic carbon and DO concentrations were higher in the streamwater than in the porewaters and, in many cases, concentrations continued to diminish with increasing depth into the streambed. 2. Hydrological exchange data demonstrated that the permeability of the stream bed declines with depth and constrains downwelling, effectively isolating porewaters >30 cm from streamwater. 3. End‐member mixing analysis (EMMA) based on conductivity documented a DOC source and DO sink in the hyporheic zone. We calculated hyporheic streambed DOC fluxes and respiration from the EMMA results and estimates of water flux. Based upon our calculations of biodegradable DOC entering the hyporheic zone, we estimate that DOC supports 39% of the hyporheic zone respiration, with the remaining 61% presumably being supported by entrained particulate organic carbon. Hyporheic respiration averaged 0.38 g C m^?2 d^?1, accounted for 41% of whole ecosystem respiration, and increased baseflow ecosystem efficiency from 46 to 59%. 4. Advective transport of labile organic molecules into the streambed concentrates microbial activity in near‐surface regions of the hyporheic zone. Steep gradients in biogeochemical activity could explain how a shallow and hydrologically constrained hyporheic zone can dramatically influence organic matter processing at the ecosystem scale.     

Organic Carbon Composition and Oxygen Metabolism across a Gradient of Seasonally Inundated Limesink and Riparian Wetlands in the Southeast Coastal Plain,USA

A set of three relatively pristine seasonally inundated limesink wetlands and one riparian wetland was studied over a 4–6 month long inundation period in 2001. Patterns in organic matter properties and oxygen consumption in the water column followed a previously documented ecological gradient based on soil composition, vegetation type, and canopy cover. The full canopy, cypress-gum swamp had the highest mean concentrations of dissolved organic carbon (DOC; 26.2 mg/l) and dissolved lignin (sum 6; 299 μg/l) with lower concentrations observed in the partial canopy, cypress savanna (22.0 mg/l DOC; 252 μg/l sum 6) and the open marsh savanna (20.6 mg/l DOC; 135 μg/l sum 6), respectively. During the inundation period, DOC increased in concentration, dissolved lignin decreased, and δ¹³C shifted to more positive values which collectively indicate a large reduction in the percentage of aromatic carbon during the inundation period. All wetlands had very high concentrations of organic matter, yet microbial oxygen consumption was almost always stimulated by the addition of glucose rather than inorganic nutrients. Stimulation by glucose suggests that there were very small pools of highly bioavailable forms of DOC in the wetlands. A larger pool of moderately bioavailable organic matter had the capacity to sustain microbial oxygen consumption rates under dark conditions for at least 15 d. During the inundation period, the cypress-gum swamp had the lowest average rates of whole water oxygen consumption (1.0 μM/h) with increasing rates observed in the cypress savanna (1.3 μM/h), marsh savanna (1.6 μM/h), and riparian wetland (1.9 μM/h), respectively. The lignin compositional fingerprint varied across the gradient of limesink wetlands, and was useful for identifying different sources of vascular plant-derived DOM. Vascular plant production, algal production, microbial respiration, and UV degradation are all important drivers of DOM cycling, and the consistencies observed in this initial assessment of seasonally inundated limesink wetlands suggest they vary in predictable ways across the ecological gradient.     

Experimental whole‐lake increase of dissolved organic carbon concentration produces unexpected increase in crustacean zooplankton density

The observed pattern of lake browning, or increased terrestrial dissolved organic carbon (DOC) concentration, across the northern hemisphere has amplified the importance of understanding how consumer productivity varies with DOC concentration. Results from comparative studies suggest these increased DOC concentrations may reduce crustacean zooplankton productivity due to reductions in resource quality and volume of suitable habitat. Although these spatial comparisons provide an expectation for the response of zooplankton productivity as DOC concentration increases, we still have an incomplete understanding of how zooplankton respond to temporal increases in DOC concentration within a single system. As such, we used a whole‐lake manipulation, in which DOC concentration was increased from 8 to 11 mg L^?1 in one basin of a manipulated lake, to test the hypothesis that crustacean zooplankton production should subsequently decrease. In contrast to the spatially derived expectation of sharp DOC‐mediated decline, we observed a small increase in zooplankton densities in response to our experimental increase in DOC concentration of the treatment basin. This was due to significant increases in gross primary production and resource quality (lower seston carbon‐to‐phosphorus ratio; C:P). These results demonstrate that temporal changes in lake characteristics due to increased DOC may impact zooplankton in ways that differ from those observed in spatial surveys. We also identified significant interannual variability across our study region, which highlights potential difficulty in detecting temporal responses of organism abundances to gradual environmental change (e.g., browning).