Quantification of algal biofilms colonising building materials: chlorophyll a measured by PAM-fluorometry as a biomass parameter

Abstract

The aim of this study was to quantify algal colonisation on anthropogenic surfaces (viz. building facades and roof tiles) using chlorophyll a (chl a) as a specific biomarker. Chl a was estimated as the initial fluorescence F₀ of ‘dark adapted’ algae using a pulse-modulated fluorometer (PAM-2000). Four isolates of aeroterrestrial green algae and one aquatic isolate were included in this study. The chl a concentration and F₀ showed an exponential relationship in the tested range between 0 and 400 mg chl a m^?2. The relationship was linear at chl a concentrations <20 mg m^?2. Exponential and linear models are presented for the single isolates with large coefficients of determination (exponential: r² > 0.94, linear: r² > 0.92). The specific power of this fluorometric method is the detection of initial algal colonisation on surfaces in thin or young biofilms down to 3.5 mg chl a m^?2, which corresponds to an abundances of the investigated isolates between 0.2 and 1.5 million cells cm^?2.     

Nondestructive determination of leaf chlorophyll content in two flowering cherries using reflectance and absorptance spectra

Leaf chlorophyll quantification is a key technique in tree vigor assessment. Although many studies have been conducted on nondestructive and in-field spectroscopic determination, it is reasonable to develop species-specific chlorophyll indices for accurate determination, because leaf spectra can vary independently of chlorophyll content due to leaf surface and structural differences among species. The present study aimed to develop optimal reflectance and absorptance indices for estimating the leaf chlorophyll content of Cerasus jamasakura (Siebold ex Koidz.) H. Ohba var. jamasakura and Cerasus × yedoensis ‘Somei-yoshino,’ and to examine their performance by comparing them with 46 published chlorophyll indices and SPAD. For 96 and 100 leaf samples, measurements were taken using a spectroradiometer with a leaf-clip attachment and a SPAD-502 chlorophyll meter, and chlorophyll content was determined by extraction with N,N′-dimethylformamide. The optimal leaf chlorophyll indices were then developed systematically by testing eight types of indices. As a result, we confirmed that the optimal chlorophyll indices performed better than any of the published leaf chlorophyll indices or SPAD, giving RMSEs that were approximately twice as good as those for SPAD, and found that the newly proposed index type—a difference and ratio combination type—may be a useful form of chlorophyll content estimation. We also found that absorptance indices achieved equivalent results to reflectance indices despite the hypothesis that absorptance measurement is direct and has more potential. Among the published indices, the reflectance ratio index of Datt [Datt B (1999) Int J Remote Sens 20(14):2741–2759] and the red edge chlorophyll index of Ciganda et al. [Ciganda V, Gitelson A, Schepers J (2009) J Plant Physiol 166:157–167] were effective at estimating the leaf chlorophyll contents of both flowering cherries.     

Linking ice structure and microscale variability of algal biomass in Arctic first-year sea ice using an in situ photographic technique

Microscale photographs were taken of the ice bottom to examine linkages of algal chlorophyll a (chl a) biomass distribution with bottom ice features in thick Arctic first-year sea ice during a spring field program which took place from May 5 to 21, 2003. The photographic technique developed in this paper has resulted in the first in situ observations of microscale variability in bottom ice algae distribution in Arctic first-year sea ice in relation to ice morphology. Observations of brine channel diameter (1.65–2.68 mm) and number density (5.33–10.35 per 100 cm²) showed that the number of these channels at the bottom of thick first-year sea ice may be greater than previously measured on extracted ice samples. A variogram analysis showed that over areas of low chl a biomass (≤20.7 mg chl a m⁻²), patchiness in bottom ice chl a biomass was at the scale of brine layer spacing and small brine channels (∼1–3 mm). Over areas of high chl a biomass (≥34.6 mg chl a m⁻²), patchiness in biomass was related to the spacing of larger brine channels on the ice bottom (∼10–26 mm). Brine layers and channels are thought to provide microscale maxima of light, nutrient replenishment and space availability which would explain the small scale patchiness over areas of low algal biomass. However, ice melt and erosion near brine channels may play a more important role in areas with high algal biomass and low snow cover.     

Relationship between specific leaf area,leaf thickness,leaf water content and <Emphasis Type="Italic">SPAD-502</Emphasis> readings in six Amazonian tree species

The aim of this work was to assess the effect of leaf thickness, leaf succulence (L_S), specific leaf area (SLA), specific leaf mass (W_s) and leaf water content (LWC) on chlorophyll (Chl) meter values in six Amazonian tree species (Carapa guianensis, Ceiba pentandra, Cynometra spruceana, Pithecolobium inaequale, Scleronema micranthum and Swietenia macrophylla). We also tested the accuracy of a general calibration equation to convert Minolta Chl meter (SPAD-502) readings into absolute Chl content. On average, SPAD values (x) increased with fresh leaf thickness (FLT [μm] = 153.9 + 0.98 x, r ² = 0.06**), dry leaf thickness (DLT [μm] = 49.50 + 1.28 x, r ² = 0.16**), specific leaf mass (W_s [g (DM) m⁻²] = 6.73 + 1.31 x, r ² = 0.43**), and leaf succulence (L_S [g(FM)] m⁻² = 94.2 + 1.58 x, r ² = 0.19**). However, a negative relationship was found between SPAD values and either specific leaf area [SLA (m² kg⁻¹) = 35.1 − 0.37 x, r ² = 0.38**] or the leaf water content (LWC [%]= 80.0 − 0.42 x, r ² = 0.58**). Leaf Chl contents predicted by the general calibration equation significantly differed (p<0.01) from those estimated by species-specific calibration equations. We conclude that to improve the accuracy of the SPAD-502 leaf thickness and LWC should be taken into account when calibration equations are to be obtained to convert SPAD values into absolute Chl content.     

Visible foliar injury caused by ozone alters the relationship between SPAD meter readings and chlorophyll concentrations in cutleaf coneflower

The ability of the SPAD-502 chlorophyll meter to quantify chlorophyll amounts in ozone-affected leaves of cutleaf coneflower (Rudbeckia laciniata var. digitata) was assessed in this study. When relatively uninjured leaves were measured (percent leaf area affected by stipple less than 6%), SPAD meter readings were linearly related to total chlorophyll with an adjusted R ² of 0.84. However, when leaves with foliar injury (characterized as a purple to brownish stipple on the upper leaf surface affecting more than 6% of the leaf area) were added, likelihood ratio tests showed that it was no longer possible to use the same equation to obtain chlorophyll estimations for both classes of leaves. Either an equation with a common slope or a common intercept was necessary. We suspect several factors are involved in altering the calibration of the SPAD meter for measuring chlorophyll amounts in visibly ozone-injured leaves, with the most likely being changes in either light absorption or scattering resulting from tissue necrosis.     

Effect of crab shell size on bio-demineralization with lactic acid-producing bacterium, <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> subsp. <Emphasis Type="Italic">tolerans</Emphasis> KCTC-3074

Demineralization (DM) from crab shell (CS) waste was carried out using a lactic acid-producing bacterium, Lactobacillus paracasei subsp. tolerans KCTC-3074 for 7 days at 25, 30, and 35°C. DM rates were 89∼92% and slightly affected by temperature. DM was also performed for four particle-sized shell samples (0.84∼3.35, 3.35∼10, 10∼20, and 20∼35 mm) with 10% inoculum, 5% shell, and 10% glucose at 30°C and 180 rpm for 7 days. It was found out that the shell size had a slight effect on the rate of DM. Negative relationships were found between DM and residual dry weight (r² = 0.960), and between DM and pH (r² = 0.906). Conversely, positive relationships were found between DM and medium protein (r² = 0.696), and between DM and total titratable acidity (r² = 0.630).     

Ribulose-1, 5-bisphosphate carboxylase activity,chlorophyll and protein concentrations in differentPopulus clones

Ribulose-1,5-bisphosphate carboxylase activity (RuBPC), chlorophyll (chl) and protein (prot) concentrations and chlorophyll/protein (chl/prot) ratios were determined in five differentPopulus clones together with their maximal net CO₂ uptake rates (P_max). A classic reference clone (Populus ×euramericana “Robusta” (Dode) Guinier) was compared with four recently selected euramerican and interamerican crossings. Chl/prot ratio and RuBPC activity varied among the different clones, while chl a/chlb ratio showed only a very low coefficient of variation (1.7%) for the five clones. Poplar clone “Robusta” could be distinguished from the recent faster growing clones based on the different biochemical characteristics. A significant correlation was found between both total chl concentration and chl/prot ratio with P_max for the five clones.     

Use of a SPAD-502 meter to measure leaf chlorophyll concentration in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The SPAD-502 meter is a hand-held device that is widely used for the rapid, accurate and non-destructive measurement of leaf chlorophyll concentrations. It has been employed extensively in both research and agricultural applications, with a range of different plant species. However, its utility has not been fully exploited in relation to the most intensively studied model organism for plant science research, Arabidopsis thaliana. Measurements with the SPAD-502 meter produce relative SPAD meter values that are proportional to the amount of chlorophyll present in the leaf. In order to convert these values into absolute units of chlorophyll concentration, calibration curves must be derived and utilized. Here, we present calibration equations for Arabidopsis that can be used to convert SPAD values into total chlorophyll per unit leaf area (nmol/cm²; R ² = 0.9960) or per unit fresh weight of leaf tissue (nmol/mg; R ² = 0.9809). These relationships were derived using a series of Arabidopsis chloroplast biogenesis mutants that exhibit chlorophyll deficiencies of varying severity, and were verified by the subsequent analysis of senescent or light-stressed leaves. Our results revealed that the converted SPAD values differ from photometric measurements of solvent-extracted chlorophyll by just ~6% on average.     

SPAD chlorophyll meter reading can be pronouncedly affected by chloroplast movement

Non-destructive assessment of chlorophyll content has recently been widely done by chlorophyll meters based on measurement of leaf transmittance (e.g. the SPAD-502 chlorophyll meter measures the leaf transmittance at 650 and 940 nm). However, the leaf transmittance depends not only on the content of chlorophylls but also on their distribution in leaves. The chlorophyll distribution within leaves is co-determined by chloroplast arrangement in cells that depends on light conditions. When tobacco leaves were exposed to a strong blue light (about 340 μmol of photons m⁻² s⁻¹), a very pronounced increase in the leaf transmittance was observed as chloroplasts migrated from face position (along cell walls perpendicular to the incident light) to side position (along cell walls parallel to the incoming light) and the SPAD reading decreased markedly. This effect was more pronounced in the leaves of young tobacco plants compared with old ones; the difference between SPAD values in face and side position reached even about 35%. It is shown how the chloroplast movement changes a relationship between the SPAD readings and real chlorophyll content. For an elimination of the chloroplast movement effect, it can be recommended to measure the SPAD values in leaves with a defined chloroplasts arrangement.     

Seasonal patterns of pore-water nutrients, benthic chlorophyll a and sedimentary AVS in a macrobenthos-rich tidal flat

Monthly field investigations were carried out at controlled low-tide in an estuarine intertidal sandflat of the Seto Inland Sea (Japan) between January 1995 and April 1996. We assessed the spatial and temporal distribution of pore-water nutrient [NH ₄ ⁺ -N, (NO ₃ ⁻ + NO ₂ ⁻ )-N, PO ₄ ³⁻ -P and Si(OH)₄-Si], chlorophyll a (chl a) and acid-volatile sulphide (AVS) concentrations in the uppermost 0–10 cm sediment horizon, and evaluated their relationships with macrobenthic assemblages. Monthly hydrological data, nutrient and chl a concentrations in low-tide creek water adjacent to the flat were used as a complementary environmental characterisation of the study area. All different pore-water nutrients showed a 10 to > 30-fold variability between different layers and periods. NH ₄ ⁺ -N, PO ₄ ³⁻ -P and Si(OH)₄-Si concentrations were lowest in winter, progressively increased throughout spring and summer, and were highest between September and October, with a major increase at intermediate (4–8 cm) layers. In contrast, (NO ₃ ⁻ + NO ₂ ⁻ )-N concentrations, correlated positively with chl a, peaked in winter and sharply decreased with depth in all different seasons and in most occasions. Depth-integrated NH ₄ ⁺ -N, PO ₄ ³⁻ -P and Si(OH)₄-Si pools were correlated positively with biogenic nutrient flux calculated from the nutrient excretion rates of dominant bivalves Ruditapes philippinarum and Musculista senhousia. These relationships indicated rapid removal pathways of P, Si and N (i.e., every 0.1, 0.24 and 0.34 day, respectively), implying highly dynamic sediments. Based on the mass balance P = F + I, biogenic production (P) vs. diffusive flux (F) estimates suggested a predominant contribution of animal bioirrigation (I) to the upward flux of nutrients. AVS concentrations, correlated negatively with both (NO ₃ ⁻ + NO ₂ ⁻ )-N and chl a, showed a progressive increase from late summer throughout winter, subsequent to that of NH ₄ ⁺ -N, PO ₄ ³⁻ -P and Si(OH)₄-Si. The results indicate that in our study area the spatial and temporal distribution of pore-water nutrients, chl a and AVS in sediments are strongly interlinked and suggest that the metabolic processes (e.g., excretion, bio-deposition) and behavioural activities (e.g., particle mixing, bioirrigation) of abundant macrobenthos play an important role in the year-round biogeochemical processes occurring on this flat.     

Development of a digital image analysis method for real-time estimation of chlorophyll content in micropropagated potato plants

The present work describes a digital image analysis method based on leaf color analysis to estimate chlorophyll content of leaves of micropropagated potato plantlets. For estimation of chlorophyll content, a simple leaf digital analysis procedure using a simple digital still camera was applied in parallel to a SPAD chlorophyll content meter. RGB features were extracted from the image and correlated with the SPAD values. None of the mean brightness parameters (RGB) were correlated with the actual chlorophyll content following simple correlation studies. However, a correlation between the chromaticity co-ordinates ‘r’, ‘b’ and chlorophyll content was observed, while co-ordinate ‘g’ was not significantly correlated with chlorophyll content. Linear regression and artificial neural networks (ANN) were applied for correlating the mean brightness (RGB) and mean brightness ratio (rgb) features to chlorophyll content of plantlet leaves determined through a SPAD meter. The chlorophyll content as determined by the SPAD meter was significantly correlated (RMSE = 3.97 and 3.59, respectively, for linear and ANN models) to the rgb values of leaf image analysis. Both the models indicate successful prediction of chlorophyll content of leaves of micropropagated plants with high correlation. The developed RGB-based digital image analysis has the advantage over conventional subjective methods for being objective, fast, non-invasive, and inexpensive. The system could be utilized for real-time estimation of chlorophyll content and subsequent analysis of photosynthetic and hyperhydric status of the micropropagated plants for better ex vitro survival.     

Seasonal changes in the chlorophyll content and quantum efficiency of the moss Brachythecium rutabulum

Abstract

The concentration of chlorophyll a, b, and total chlorophyll have been monitored on a seasonal basis in Brachythecium rutabulum. Total chlorophyll increases during summer full canopy conditions from 1.70 mg chl g^?1 on 8 May to 11.1 mg chl g^?1 on 11 October. Photosynthetic-illumination curves show that during this period light saturation declines from 200 μmol m^?2s^?1 to 30 μmol m^?2s^?1 by 6 July, and light compensation falls dramatically from 65 μmol m^?2s^?1 to 4 μmol m^?2s^?1. The data also appear to support the conclusion that there is concurrently an increase in the density of photosynthetic units by the end of September.     

Relationships between bacterioplankton, chl a and temperature in Antarctic surface waters in summer

Spatial variations of bacterio- and phytoplankton were studied in order to compare their relationship in open-sea and coastal areas. Sampling was done quasi-synoptically south of the Antarctic Convergence in the Lazarev Sea and in the eastern part of the Weddell Sea during austral mid-summer. Thymidine incorporation rate was on average 1.10 nmol/m³ per hour in the open sea and 4.04  nmol/m³ per hour in the coastal area, bacterial abundance was 4.44 × 10¹¹ and 6.11 × 10¹¹ cells/m³ and chlorophyll a (chl a) was 0.43 and 2.42 mg/m³, respectively. Thymidine incorporation rate and chl a correlated positively in both the open-sea and coastal samples. In the coastal area bacterial numbers also correlated positively with chl a. The scale of spatial resolution was not important for detecting empirical relationships between phytoplankton and bacterioplankton parameters. In the coastal area, the low bacterial biomass in relation to chl a concentration compared to other oceans, indicates that generalised relationships between these parameters are not valid in Antarctic coastal waters. Grazing could not explain the discrepancy. The results suggest a strong coupling between phytoplankton and bacterioplankton. In addition, the results suggest that the bacterial assemblage in the coastal area was psychrophilic and well adapted to the prevailing low temperatures. Received: 18 October 1996 / Accepted: 8 December 1996     

Calibration of the Minolta SPAD-502 leaf chlorophyll meter

Use of leaf meters to provide an instantaneous assessment of leaf chlorophyll has become common, but calibration of meter output into direct units of leaf chlorophyll concentration has been difficult and an understanding of the relationship between these two parameters has remained elusive. We examined the correlation of soybean (Glycine max) and maize (Zea mays L.) leaf chlorophyll concentration, as measured by organic extraction and spectrophotometric analysis, with output (M) of the Minolta SPAD-502 leaf chlorophyll meter. The relationship is non-linear and can be described by the equation chlorophyll (mol m^–2)=10(^M0.265), r ²=0.94. Use of such an exponential equation is theoretically justified and forces a more appropriate fit to a limited data set than polynomial equations. The exact relationship will vary from meter to meter, but will be similar and can be readily determined by empirical methods. The ability to rapidly determine leaf chlorophyll concentrations by use of the calibration method reported herein should be useful in studies on photosynthesis and crop physiology.Abbreviations Chl- chlorophyll - M- SPAD-502 meter value     

The use of dialysis culture in a study of chlorophyll budget in a brackish lagoon,Japan

Growth rates of the entire phytoplankton community of a brackish lagoon in northeastern Japan were estimated by measuring increasing chlorophyll a content in dialysis bags during the summer and early autumn of 1986. The chlorophyll a contents of lagoon water fluctuated between 20 and 200 mg m^–3. At lower densities of phytoplankton (20–50 mg chl. a m^–3), growth rates (the rate of increase of chlorophyll a) exceeded 1 turnover per day, while at higher densities (more than 50 mg chl. a m^–3), the growth rate decreased rapidly. Tidal exchanges of chlorophyll a showed net exports of chlorophyll a from the lagoon to adjacent waters. The exchange rate of chlorophyll a was estimated to be 0.65 d^–1. At about 140 mg m^–3 of chlorophyll a concentration, the increase of chlorophyll in the lagoon water compensated for tidal export. Only a small proportion of primary production was consumed by zooplankton in the lagoon. There were also net exports of ammonium and phosphate from the lagoon. Nutrient flux from sediment exceeded the phytoplankton requirement and was the major source of the ammonium and phosphate exports from the lagoon. The low inorganic N/P atom supply ratio in the lagoon suggests that nitrogen is a major nutrient limiting phytoplankton growth.     

Predicting concentration of total phosphorus and chlorophyll a in a lake with short hydraulic residence time

The relationship between total phosphorus and chlorophyll a concentration was determined for Skinner Lake, Indiana over an annual cycle in 1978–79. Total nitrogen:total phosphorus ratios in the epilimnion ranged from 19 to 220 suggesting a phosphorus-dependent algal yield in the epilimnion. Approximately 90% of annual TP loading reached the lake via streamflow, and 93% of this entered during snowmelt and spring-overturn periods. At that time incoming water flushed the lake 2.4 times. Atmospheric loading accounted for 1.4% of annual TP load. Internal hypolimnetic TP loading occurred during summer stratification. Mean [chl a] for the ice-free period was 15.15 mg m^–3, within the range expected for eutrophic lakes.The 1978–79 data were used in conjuction with the Vollenweider & Kerekes (1980) model to produce a model specific for the Skinner Lake system. The model predicted mean epilimnetic total phosphorus and chlorophyll a concentrations from mean total phosphorus concentration in inlet streams and from lake water residence time during the period of spring overturn and summer stratification. The Skinner-specific model was tested in 1982 and it closely predicted observed mean epilimnetic [TP] and [chl a] during the ice-free period. This study shows that variability in lake models which average data over an annual period can be reduced by considering lake-specific seasonal variation in hydrology and external TP loading.     

Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development

Photosynthetic organisms exhibit a green color due to the accumulation of chlorophyll pigments in chloroplasts. Mg-protoporphyrin IX chelatase (Mg-chelatase) comprises three subunits (ChlH, ChlD and ChlI) and catalyzes the insertion of Mg²⁺ into protoporphyrin IX, the last common intermediate precursor in both chlorophyll and heme biosyntheses, to produce Mg-protoporphyrin IX (MgProto). Chlorophyll deficiency in higher plants results in chlorina (yellowish-green) phenotype. To date, 10 chlorina (chl) mutants have been isolated in rice, but the corresponding genes have not yet been identified. Rice Chl1 and Chl9 genes were mapped to chromosome 3 and isolated by map-based cloning. A missense mutation occurred in a highly conserved amino acid of ChlD in the chl1 mutant and ChlI in the chl9 mutant. Ultrastructural analyses have revealed that the grana are poorly stacked, resulting in the underdevelopment of chloroplasts. In the seedlings fed with aminolevulinate-dipyridyl in darkness, MgProto levels in the chl1 and chl9 mutants decreased up to 25% and 31% of that in wild-type, respectively, indicating that the Mg-chelatase activity is significantly reduced, causing the eventual decrease in chlorophyll synthesis. Furthermore, Northern blot analysis indicated that the nuclear genes encoding the three subunits of Mg-chelatase and LhcpII in chl1 mutant are expressed about 2-fold higher than those in WT, but are not altered in the chl9 mutant. This result indicates that the ChlD subunit participates in negative feedback regulation of plastid-to-nucleus in the expression of nuclear genes encoding chloroplast proteins, but not the ChlI subunit.Haitao Zhang and Jinjie Li contributed equally to this work     

Reinvestigation of the chlorophyll distribution among the chlorophyll-proteins and chlorophyll-protein complexes of Hordeum vulgare L.

Solubilization of barley (Hordeum vulgare L.) thylakoid membranes with sodium dodecylsulphate plus sodium deoxycholate with or without Triton X-100 and subsequent fractionation in the polyacrylamide gel electrophoresis system described in this paper resulted: (1) in the resolution of the chlorophyll-proteins and chlorophyll-protein complexes commonly known as CP1a, CP1, LHCP¹, LHCP², CPa and LHCP³; (2) in the highly increased stability of CP1 and CP1a, as judged by their chlorophyll content, (3) at the expense of the free pigment concentration (4) which could be reduced to a negligible amount. Some 40% of the total chlorophyll contained in the mature higher plant thylakoid membrane is associated with CP1 and CP1 a and as already suggested before [19] no significant amount of free chlorophyll occurs in vivo.Abbreviations chl chlorophyll - CP1 P₇₀₀-chla-protein - CPa P680-chla-protein - DOC sodium deoxychlolate - FC free chlorophyll - LHCP light-harvesting chlorophyll a/b-protein - PAGE(S) polyacrylamide gel electrophoresis (system) - SDS sodium dodecylsulphate - TX-100 Triton X-100     

基于叶绿素荧光参数的小麦叶片叶绿素相对含量估算方法

叶绿素含量是植物学和农业相关研究领域常用的生理指标.叶绿素含量和叶片光合功能密切相关,但是现有的叶绿素含量的测定方法无法实现叶绿素含量和光合功能的同步测定和关联分析.为解决该问题,本研究通过测定35个小麦品种旗叶的SPAD值和叶绿素荧光诱导动力学曲线,分别使用不同时间的快速叶绿素荧光动力学曲线的荧光值,以及33个常用荧...     

A non-destructive determination of leaf chlorophyll in Vitis vinifera

A portable leaf greenness meter (SPAD-501) has been used to provide a rapid and non-destructive measurement of leaf chlorophyll in Vitis vinifera. Leaf extracted chlorophyll was related linearly to SPAD readings. It is suggested that separate linear equations should be developed for each cultivar so as to maximise the accuracy of estimating leaf chlorophyll content as a function of SPAD readings.