Interspecific variability and environmental influence on particulate organic carbon {delta}13C in cultured marine phytoplankton

The stable carbon isotope composition of particulate organicmatter expressed as ¹³C was measured in cultures of 13 speciesof marine microalgae in different phylogenetic groups. The effectsof salinity variations and changes in photoperiod were alsoassayed for three of them (i.e. Skeletonema costatum, Amphidiniumopercularum and Isochrysis galbana); the effect of nature ofnitrogen supply (nitrate. ammonium) was studied for one (S.costatum).These environmental parameters were chosen because of theirvariability in the ocean and their possible effects on ¹³C valuesof phytoplankton organic carbon. Batch culture conditions andsampling time after inoculum were strongly controlled in orderto provide cells in good physiological state which were comparablefrom one culture to the other. In the same way, sampling waslimited to the first 2 days of exponential growth, in orderto avoid a possible dissolved inorganic carbon (DIC) limitation.Carboxylase activities [of the enzyme ribulose 1,5-bisphosphatecarboxylase oxygenase (Rubisco), and the three ß carboxylases:phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvatecarboxykinase (PEPCK) and pyruvate carboxylase (PC)] and totalchlorophyll a concentrations were assayed simultaneously. The¹³C values observed were between –30.2 and –12.7i.e. comparable to those observed in the world's oceans. Theisotopic composition of phytoplankton organic carbon was shownto be under the influence of the parameters tested but ¹³C variationsare specific to the species considered. The nature of ßcarboxylase found in each species, or systematic position, couldnot be linked to the isotopic composition of organic carbon.No linear or single correlation between ¹³C variations and environmentalmodifications were observed and there is no evidence for a simpleand universal relation between ¹³C of phytoplankters and theirenvironment. In monospecific cultures as in the field, ¹³C fractionationby Rubisco (and eventually by PEPCK) may be counterbalancedby other mechanisms.     

水体无机碳条件对常见沉水植物生长和生理的影响

为了解水华引起的水体无机碳变化对沉水植物生长的影响,对8种沉水植物:金鱼藻、穗花狐尾藻、篦齿眼子菜、光叶眼子菜、微齿眼子菜、伊乐藻、菹草和黑藻在不同无机碳浓度下的生物量、株高、叶绿素以及光合和呼吸速率进行了比较研究.结果表明8种沉水植物均能利用HCO3-作为光合无机碳源,在1.5 mmoL/L外源HCO3-浓度下能促进金鱼藻、菹草和伊乐藻的生长,提高其光合速率;在2.5 mmol/L外源HCO3-浓度下能促进狐尾藻、光叶眼子菜、黑藻、微齿眼子菜和蓖齿眼子菜的生长,提高其光合速率.在CO32-为优势碳源时,8种沉水植物表现出不同的适应性,发现微齿眼子菜、篦齿眼子菜和黑藻在整个实验范围内生长未受抑制,且在不同浓度下表现生长和光合速率的促进,说明这三种沉水植物对[HCO3-]/[CO32-]比值和pH具有较广适应范围.而当CO32-成为优势碳源时,金鱼藻和伊乐藻的生长受到抑制,狐尾藻、菹草和光叶眼子菜均死亡,表明[HCO3-]/[CO32-]比值和pH是这5种沉水植物生长的重要限制因子.     

The effect of hydrogen sulfide on the metabolism of Solemya velum and enzymes of sulfide oxidation in gill tissue

• 1.1. The addition of sulfide to sea-water in respirometer flasks stimulated oxygen uptake by intact Solemya velum; at concentrations of 0.5 and 0.8 mM, the experimental rates were 1.8 and 2.5 times control rates.
• 2.2. Extracts of gill tissue catalyzed the conversion of thiosulfate to sulfite, the production of adenosine phosphosulfate (APS) from AMP and sulfite and the formation of ATP from APS. The enzymes, thiosulfate sulfurtransferase (EC 2.8.1.1), adenylsulfate reductase (EC 1.8.99.2) and sulfate adenylyl transferase (EC 2.7.7.4) have K_m and V_max in the same range as similar enzymes in other species.
• 3.3. Calculations based on these experiments suggest that adenylylsulfate reduction is ordinarily catalyzed at no more than 8% of maximum velocity.

     

集约化生产对农田土壤碳氮含量及δ13C和δ15N同位素丰度的影响

集约化生产下农田土壤碳、氮含量变化是衡量土壤肥力持久性的重要指标.对常规水稻-蚕豆轮作地、露地蔬菜地、3年塑料大棚地和10年以上塑料大棚地的土壤pH、电导率(EC)、土壤有机碳(SOC)和总氮(TN)含量及δ13C和δ15N同位素丰度进行测定,研究了集约化生产程度对土壤特性的影响.结果表明:与水稻-蚕豆轮作地相比,露地蔬菜地、3年塑料大棚地和10年以上塑料大棚地0 ～20 cm耕层土壤pH分别降低1.1、0.8和0.7,而土壤EC分别是水稻-蚕豆轮作地的4.2、4.9和5.2倍;土壤碳、氮含量随塑料大棚地生产年限的增加总体上呈先增大后减小的趋势.与水稻-蚕豆轮作地相比,10年以上塑料大棚地0～20、20～40、40 ～60、60 ～ 80、80 ～ 100 cm土层的土壤SOC含量分别下降了54％、46％、60％、63％和59％,土壤TN含量分别下降了53％、53％、71％、82％和85％.农田集约化生产程度显著影响土壤SOC、TN含量和δ13C、δ15N丰度,土壤δ13C丰度与SOC含量呈显著负相关.土壤δ13C丰度可作为评价农田土壤碳循环受人为干扰强度的指标.     

温度和无机碳浓度对龙须眼子菜（Potamogeton pectinatus）碳同位素分馏的影响

温度和无机碳浓度是沉水植物碳同位素分馏的重要影响因素.通过在16、19、22、25、28、31℃ 6个温度条件和溶解无机碳浓度(DIC)为0.0001mol·L-1和0.0001mol·L-1两种条件下培养沉水植物--龙须眼子菜(Potamogeton pectinatus),获得了不同条件下生长的龙须眼子菜植物样品,随后进行了定量和碳同位素组成(δ13CP)分析.分析结果表明,随生长温度的升高,龙须眼子菜碳δ13CP均升高,具有明显的温度相关性(R2＞0.90).在DIC浓度为0.0001mol·L-1时,随着生长温度的升高, δ13CP从-14.83‰增加至-13.47‰;而在DIC浓度为0.001mol·L-1时, δ13CP从-18.56‰增加至-15.15‰.同一生长温度条件下, DIC浓度对δ13CP也有明显影响, δ13CP随DIC浓度增高而降低.在不同生长温度条件下, DIC浓度对δ13CP的影响大小随生长温度的增高而降低,在温度为16℃时,两个DIC浓度下的δ13CP相差3.73‰;而在温度为31℃时,这一差值减少为1.68‰.同时,通过建模进行计算,获得了不同温度下龙须眼子菜(P. pectinatus)碳同位素分馏与初始DIC浓度的关系,并对有关的参数的意义进行了探讨.     

大亚湾春季溶解无机碳的分布特征

于2010年5月对大亚湾溶解无机碳(DIC)进行采样调查,分析了大亚湾溶解无机碳空间分布特征,并讨论了大亚湾溶解无机碳与各环境要素(pH、水温、溶解氧和叶绿素a等)的相关性。大亚湾表层水域的DIC含量在24.26～26.13mg.L-1之间;底层水域的DIC含量在24.06～26.89mg.L-1之间。大亚湾表层水体中DIC含量的高值区集中在大辣甲-桑洲连线以北水域,然后呈现出向四周逐渐递减的分布趋势;底层水体中DIC含量高值区集中于西南部水域(大辣甲南部),总体呈现出由南部向北部逐渐递减的分布趋势。大亚湾DIC与水温和叶绿素a呈现负相关系,与pH、DO和盐度呈现正相关关系,但从相关的显著性来看,DIC与以上各环境因子的相关系数均不高。     

Limitation of lowland riverine bacterioplankton by dissolved organic carbon and inorganic nutrients

Flow regulation in lowland rivers has reduced the amount of allochthonous dissolved organic carbon (DOC) entering main channels through less frequent wetting of benches, flood runners and floodplains. The hypothesis tested was that lowland riverine bacterioplankton are DOC limited when flow events are absent and simulating an increase in assimilable DOC similar to that expected during an environmental flow will lead to heterotrophic dominance. Experiments took place in the Namoi River, a highly regulated lowland river in Australia. Specifically, in situ microcosms were used to examine the responses of bacterioplankton and phytoplankton to various additions of DOC as glucose or leaf leachate, with and without additions of inorganic nutrients. The results indicated that ambient DOC availability limited the bacterioplankton for the three seasons over which we conducted the experiments. When DOC was added alone, dissolved oxygen concentrations decreased primarily because of increased bacterial respiration and bacterioplankton growth generally increased relative to controls. Additions of DOC alone led to a pattern of decreased chlorophyll a concentration relative to controls, except for willow leachate. Additions of inorganic nutrients alone increased chlorophyll a concentrations above controls, indicating limitation of phytoplankton. These findings support our hypothesis. Based on the present results, environmental flows should increase the duration of allochthonously driven heterotrophic dominance, thus shifting regulated lowland rivers to more natural (pre-regulation) conditions for greater periods.     

Controls on the origin and cycling of riverine dissolved inorganic carbon in the Brazos River, Texas

Rivers draining watersheds that include carbonate bedrock or organic matter (OM)-rich sedimentary rocks frequently have ¹⁴C-depleted dissolved inorganic carbon (DIC) relative to rivers draining carbonate- and OM-free watersheds, due to dissolution of carbonate and/or decomposition of ancient OM. However, our results from a subtropical river, the Brazos River in Texas, USA, show that in this watershed human activities appear to dominate basin lithology in controlling the origin and metabolism of DIC. The middle Brazos flows through limestone and coal-bearing bedrock, but DIC isotope data suggest no limestone dissolution or respiration of ancient OM, and instead reflect efficient air?Cwater CO₂ exchange, degradation of relatively young OM and photosynthesis in the river as a result of river damming and urban treated wastewater input. The lower Brazos drains only small areas of carbonate and coal-bearing bedrock, but DIC isotope data suggest the strong influence of carbonate dissolution, with a potentially minor contribution from decomposition of old soil organic matter (SOM). Oyster shells and crushed carbonate minerals used in road construction are likely sources of carbonate in the lower Brazos, in addition to natural marl and pedogenic carbonate. Additionally, the generally low pCO₂ and high DIC concentration in the Brazos River lead to a low CO₂ outgassing:DIC export ratio, distinguishing the Brazos River from other rivers.     

Effect of dissolved inorganic carbon on the expression of carboxysomes,localization of Rubisco and the mode of inorganic carbon transport in cells of the cyanobacterium Synechococcus UTEX 625

In the cyanobacterium Synechococcus UTEX 625, the extent of expression of carboxysomes appeared dependent on the level of inorganic carbon (CO₂+HCO _inf3 ^sup- ) in the growth medium. In cells grown under 5% CO₂ and in those bubbled with air, carboxysomes were present in low numbers (<2 · longitudinal section^-1) and were distributed in an apparently random manner throughout the centroplasm. In contrast, cells grown in standing culture and those bubbled with 30 l CO₂ · 1^-1 possessed many carboxysomes (>8 · longitudinal section^-1). Moreover, carboxysomes in these cells were usually positioned near the cell periphery, aligned along the interface between the centroplasm and the photosynthetic thylakoids. This arrangement of carboxysomes coincided with the full induction of the HCO _inf3 ^sup- transport system that is involved in concentrating inorganic carbon within the cells for subsequent use in photosynthesis. Immunolocalization studies indicate that the Calvin cycle enzyme ribulose bisphosphate carboxylase was predominantly carboxysome-localized, regardless of the inorganic carbon concentration of the growth medium, while phosphoribulokinase was confined to the thylakoid region. It is postulated that the peripheral arrangement of carboxysomes may provide for more efficient photosynthetic utilization of the internal inorganic carbon pool in cells from cultures where carbon resources are limiting.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon (CO₂+HCO _inf3 ^sup- +CO _inf3 ^sup2- ) - PRK phosphoribulokinase - RuBP ribulose 1,5-bisphosphate - Rubisco LS large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase     

The relationship between the dissolved inorganic carbon concentration and growth rate in marine phytoplankton

A range of marine phytoplankton was grown in closed systems in order to investigate the kinetics of dissolved inorganic carbon (DIC) use and the influence of the nitrogen source under conditions of constant pH. The kinetics of DIC use could be described by a rectangular hyperbolic curve, yielding estimations of KG(DIC) (the half saturation constant for carbon-specific growth, i.e. C mu) and mu max (the theoretical maximum C mu). All species attained a KG(DIC) within the range of 30-750 microM DIC. For most species, NH4+ use enabled growth with a lower KG(DIC) and/or, for two species, an increase in mu max. At DIC concentrations of > 1.6 mM, C mu was > 90% saturated for all species relative to the rate at the natural seawater DIC concentration of 2.0 mM. The results suggest that neither the rate nor the extent of primary productivity will be significantly limited by the DIC in the quasi-steady-state conditions associated with oligotrophic oceans. The method needs to be applied in the conditions associated with dynamic coastal (eutrophic) systems for clarification of a potential DIC rate limitation where cells may grow to higher densities and under variable pH and nitrogen supply.     

Effects of altitudinal gradient on Salix atopantha foliar delta13C

In 2010, measurements were conducted on the foliar delta13C, photosynthesis, CO2 diffusive conductivity, nitrogen content, photosynthetic nitrogen use efficiency (PNUE), and special leaf area (SLA) of Salix atopantha at different altitudes (2350 m, 2700 m, 3150 m, and 3530 m) in Wolong Natural Reserve. With the increase of altitude, the foliar nitrogen content (especially the nitrogen content per unit leaf area, N(area)) and the PNUE increased, and the foliar delta13C had a significant increase, with an increment of 1.4 per thousand per 1000 m altitude. The stomatal and mesophyll CO2 diffusion conductance also increased with increasing altitude, which had definite negative effect on the increase of foliar delta13C, but the effect was not strong enough. Comparing with CO2 diffusion conductance, carboxylation capacity was a more important factor limiting the P(c)/P(a), and even, the foliar delta13C. At altitude 2350-2700 m, air temperature was the main factor affecting the allocation of nitrogen in S. atopantha photosynthetic system, whereas at altitude 2700-3530 m, light could be the main affecting factor. No significant difference was observed in the SLA at different altitudes.     

福建兴化湾溶解无机氮、溶解无机磷时空变化及营养状态评价

根据2007年3月至12月福建省兴化湾海域的水质监测结果,重点分析了该海域溶解无机氮(DIN)、溶解无机磷(DIP)的分布特征及其影响因素,并采用有机污染指数和富营养化指数对兴化湾海域的富营化水平进行了评价。结果表明:兴化湾海域富营养化主要污染物是DIN、DIP,其含量主要受径流排放和海洋浮游植物生长等因素的影响。春夏季节浮游植物生长繁殖旺盛,但雨水增多,最终导致了DIP、DIN含量的升高。秋季水温下降,浮游植物生长繁殖逐渐减弱,DIP、DIN的含量也逐渐升高。兴化湾富营养化水平加重,2007年的富营养化指数是2000年的5.7倍,主要体现在DIN、DIP等指标的升高。     

The effects of pH and dissolved inorganic carbon on external carbonic anhydrase activity in Chlorella saccharophila

External carbonic anhydrase (CA) activity in Chlorella saccharophila is suppressed by growth at high dissolved inorganic carbon and at acid pH. External CA activity was shown to be suppressed by growth at pHs below 7.0, with total repression at pH5.0. Growth in the presence of the buffer 3-[N-Morpholino]propane-sulphonic acid (MOPS) between pH 7 and 8 suppressed CA activity. Cells grown at pH8.0 aerated at 6 dm³ h^?1 exhibited external CA activity of 5 units mg^?1 Chl once the dissolved inorganic carbon (DIC) was reduced to 300 mmol m^?3, and this increased to 30 units mg^?1 Chl over a period of 3d while the DIC dropped to 30mmol m^?3. Cells aerated at 180 dm³ h^?1 showed a similar trend in CA activity, although the onset was delayed by 1 d and the DIC did not drop below 300 mmol m^?3. Cells grown at pH 7.8 near an air equilibrium DIC of 300 mmol m^?3had no detectable external CA activity. It is probable that it is the CO² supply to the cell, and not total DIC or HCO^?₃ which controls external CA activity. Cells grown at pH 5.0 had no detectable activity, although they reduced the CO² concentration to 0.6 mmol m^?3. The loss of CA upon transfer of air-grown cells to 10 mmol mol^?1 CO² took place over 48 h and was light dependent, while the loss upon transfer from alkaline pH to acid pH look place over 12 h and was independent of light. The effects of pH are independent of the response to CO₂.     

A delta13C-based carbon flux model for the hydrothermal vent chemoautotrophic symbiosis Riftia pachyptila predicts sizeable CO(2) gradients at the host-symbiont interface

The chemoautotrophic symbiosis Riftia pachyptila has extremely 13C-enriched delta13C values. Neither isotopic discrimination by the RubisCO enzyme of their bacterial endosymbionts, nor the delta13C value of CO2 at their hydrothermal vent habitat, suffice to explain biomass delta13C values in this organism, which range from - 9 to - 16 per thousand. However, these 13C-enriched delta13C values are consistent with the presence of 13C-enriched CO2 within the symbiont cytoplasm. Such a 13C-enriched pool of CO2 is expected when the rate of CO2 fixation by RubisCO, which fixes 12CO2 more rapidly than 13CO2, approaches the rate of exchange between intracellular and extracellular CO2 pools. Rapid CO2 fixation rates will also generate concentration gradients between these two pools. In order to estimate the size of these concentration gradients, an equation was derived, which describes the delta13C of tubeworm biomass in terms of the size of the CO2 gradient between the hydrothermal vent environment and the symbiont cytoplasm. Using mass balance equations for CO2 exchange and fixation by the symbionts and the tubeworm host, this model predicts that a CO2 concentration gradient of up to 17-fold between the symbiont cytoplasm and the environment is sufficient to explain even the most 13C-enriched R. pachyptila biomass. This model illustrates how both physical and enzymatic factors can act to influence the delta13C of intracellular CO2, which, in turn, highlights the danger of assigning a carbon fixation pathway to an autotroph based solely on its biomass delta13C value.     

Pelagic and benthic net production of dissolved inorganic carbon in an unproductive subarctic lake

1. Both the pelagic and benthic net dissolved inorganic carbon (DIC) productions were measured in situ on four occasions from June to September 2004, in the unproductive Lake Diktar-Erik in subarctic Sweden. The stable isotopic signal ( δ ¹³C) of respired organic material was estimated from hypolimnion water data and data from a laboratory incubation using epilimnion water.
2. Both pelagic and benthic habitats were net heterotrophic during the study period, with a total net DIC production of 416 mg C m⁻² day⁻¹, of which the pelagic habitat contributed approximately 85%. The net DIC production decreased with depth both in the pelagic water and in the sediments, and most of the net DIC production occurred in the upper water column.
3. Temporal variations in both pelagic and benthic DIC production were small, although we observed a significant decrease in pelagic net DIC production after the autumn turnover. Water temperature was the single most important factor explaining temporal and vertical variations in pelagic DIC production. No single factor explained more than 10% of the benthic net DIC production, which probably was regulated by several interacting factors.
4. Pelagic DIC production, and thus most of the whole-lake net production of DIC, was mainly due to the respiration of allochthonous organic carbon. Stable isotope data inferred that nearly 100% of accumulated DIC in the hypolimnion water had an allochthonous carbon source. Similarly, in the laboratory incubation using epilimnion water, c. 85% of accumulated DIC was indicated to have an allochthonous organic carbon source.     

万峰湖水库溶解性无机碳来源及时空变异特征

溶解性无机碳(DIC)的通量和形式在水生态系统的生物地球化学循环中起关键作用,是碳收支的重要组成部分。通过分析万峰湖水库库区水体理化参数、DIC和稳定碳同位素(δ¹³C_DIC)特征,揭示了DIC的行为和来源。结果表明: 1)在表水层,整个库区pH变化较保守,均呈弱碱性。硝酸盐氮(NO₃^--N)有最大变异系数,具有高度的时空变异性。由于稀释效应的存在,电导率(EC)、二氧化碳分压(pCO₂)和DIC的最低值均出现在夏季高径流量阶段。在水柱面上,夏季氧化还原电位(Eh)和NO₃^--N随水深增加无显著变化,其余指标均变化明显,且在温跃层变异程度最大。两季节的水温(T)、pH和Eh均随水深增加而降低,pCO₂则与之相反。EC、总碱度(TA)和DIC在夏季随水深增加而降低,冬季变化梯度较小。两季节的DIC与pH、Eh呈负相关,与EC、pCO₂呈正相关。2)夏季DIC为2.66～4.9 mmol·L^-1,而冬季为3.38～4.52 mmol·L^-1。水体热分层期间,DIC和δ¹³C_DIC在温跃层的变化梯度最大,DIC与δ¹³C_DIC在夏季表水层呈正相关。两个季节水柱面上及冬季表水层的DIC和δ¹³C_DIC均呈负相关,但冬季DIC和δ¹³C_DIC值随水深变化趋势不明显。3)夏季δ¹³C_DIC较高,为-7.71‰～-1.38‰,表明碳酸盐矿物的溶解占优势。冬季δ¹³C_DIC为-16.93‰～-9.44‰,显著低于夏季且范围更宽,生物源CO₂的输入和有机质矿化是主要来源。δ¹³C_DIC在不同季节和水深均差异显著,一方面是碳的来源不同;另一方面归因于碳来源的相对贡献比例的变化。     

Growth-dependent stable carbon isotope fractionation by basidiomycete fungi: delta(13)C pattern and physiological process

We grew 11 basidiomycetes in axenic culture to characterize their physiological capacities to fractionate stable C isotopes. Generally, delta(13)C values of the fungal biomass were (i) enriched in (13)C relative to the growth medium, (ii) variable among the isolates, and (iii) dependent on the growth rate and growth stage of the fungi. We found a multiphasic dynamic of fractionation for Cryptoporus volvatus and Marasmius androsaceus during various growth stages. The first phase, P1, corresponded to the exponential growth stage and was characterized by an increasing enrichment in (13)C content of the fungal biomass relative to the growth medium ranging between 4.6 and 6.9 per thousand. The second phase, P2, exhibited a continual depletion in (13)C of the fungal biomass, with the delta(13)C values of the fungal biomass asymptotically returning to the delta(13)C value of the growth medium at inoculation. The expression of the various fractionation phases was dependent on the amount of low-concentration micronutrients and growth factors added to the growth medium. The onset of P2 occurred at reduced concentrations of these elements. All of the sugars in the growth medium (sucrose, maltose, and glucose) were utilized for growth, indicating that the observed fractionation was not an artifact derived from the preferential use of (13)C-rich maltose, which was found at low concentrations in the growth medium. In this study, we establish a framework with which to explore the impact of physiological fractionations by fungal interfaces on natural distributions of stable C isotopes.     

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)