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УДК 543.39:547.979.7
DOI: 10.24411/9999-002А-2018-10024
МНОГОВОЛНОВЫЕ УРАВНЕНИЯ ДЛЯ ОПРЕДЕЛЕНИЯ КОНЦЕНТРАЦИЙ ХЛОРОФИЛЛОВ И БАКТЕРИОХЛОРОФИЛЛОВ В 90% ВОДНОМ АЦЕТОНЕ
М.Ю. Горбунов
Институт экологии Волжского бассейна РАН, Тольятти, Россия e-mail: myugor1960@gmail.com
Аннотация. На основе вновь определенных величин молярных коэффициентов экстинкции представлены многоволновые уравнения для определения бактериохлорофиллов a и d при их совместном присутствии с хлорофиллами фитопланктона.
Ключевые слова: фитопланктон, аноксигенные фототрофные бактерии, фотосинтетические пигменты, спектрофотометрическое определение, 90% ацетон.
MULTI-WAVELENGTH EQUATIONS FOR CHLOROPHYLLS AND BACTERIOCHLOROPHYLLS DETERMINATION IN 90% AQUEOUS ACETONE
M.Yu. Gorbunov
Institute of Ecology of the Volga River Basin of the Russian Academy of Sciences, Togliatti, Russia
e-mail: myugor1960@gmail.com
Annotation. Multi-wavelength equations for determination of bacteriochlorophylls a and d at their presence together with chlorophylls of phytoplankton developed on the basis of re-determined molar extinction coefficients are presented.
Key words: phytoplankton, anoxygenic phototrophic bacteria, photosynthetic pigments, spectrophotometry determination, 90% acetone.
The number of stratified lakes with anaerobic hypolimnion increased due to anthropogenic eutro-phication and probably will grow even stronger due to global warming. Anoxia in the bottom layers of stratified lakes leads to the accumulation of reduced inorganic compounds in the near-bottom layers, first of all sulfides, or reduced compounds of iron and manganese. In the presence of light, such conditions are favorable for growth of anoxygenic phototrophic bacteria (APB), which constitute the main component of the phototrophic community of meta- and hypolimnion in most of such lakes. Along with anoxygenic pho-totrophic bacteria, essential components of the upper part of the anaerobic layers of lakes are oxygenic phototrophs. Therefore, bacteriochlorophylls (Bchls) of anoxygenic phototrophs are always coincide in the chemocline with algal chlorophylls (Chl), and first of all Chl a.
Various modifications of the multi-wavelength spectrophotometric methods are now and will likely remain in near future the most practical way of routine pigment determination. For example, three-wavelength formulas for 90% acetone extract, proposed by Jeffrey and Humfrey in 1975 are generally accepted for determining the concentration of chlorophylls in freshwater hydrobiology. Similar equations
for simultaneous determinations of Chl a and bacteriochlorophylls are used less frequently. Most used of them are equations for Chl a and Bchls d+e determination in 99.5% acetone extracts suggested by Overmann and Tilzer in 1989. Zykov and Rogozin have developed their own equations for 90% acetone. We have also proposed 4-wavelenght equations for extracts in 90% acetone, but they based partially on estimated instead of directly measured extinction coefficients. All these equations did not taken into account the influence of Bchl a. It can be measured directly by its absorbance at 770-775 nm, but in high concentration it could influence spectra at 600-700 nm, so in the cases of dense populations of purple bacteria, other determinations should be corrected for its presence.
Here, I present corrected equations for measuring some combinations of photosynthetic pigments frequently coexisting in the chemocline zone of lakes. As a reference, the specific absorption coefficients in 90% aqueous acetone were used for chlorophylls, and the ones of bacteriochlorophylls in 100% acetone. Based on these data new values of molar and specific absorption coefficients in 90% acetone in the absorption maxima of chlorophylls and bacteriochlorophylls were obtained.
In principle, these results allow to derive full multiwavelength equations for all seven studied pigments (Chls a-c, Bchl a, Bchl c-e). However, due to similarity of spectra of Chl a and Bchl c, and of the triad Chl b-Bchl d-Bchl e, these equations will have extremely low accuracy because of the "small difference of large quantities" errors. However, it is possible to offer several particular multiwavelength formulas that allow determining some subsets of the photosynthetic pigments.
If only three key pigments, Chl a, Bchl a and Bchl d are present in the sample, their concentrations in extract (in nM L-1) may be calculated as
[Chl a]= -7.20 A'653.5 + 16.56 AV5 - 1.32A'772 [Bchl d] = 17.33 A'653.5 - 9.15 A'663.5 - 0.43 A'772 [Bchl a] = - 0.01 A'663.5 + 15.65 A'772 where A'X is the extinction of the sample at the wavelength of X nm minus extinction at 850 nm.
If green algae or euglenids are present in significant amounts, the concentration of Chl b must be taken into account, and the equations are as follows:
[Chl a]= 3.50 A'647 - 9.66 A'653.5 + 16.96 A'663.5 - 1.42AV/2 [Chl b]= 41.13 A'647 - 28.90 A'653.5 + 4.68 A'663.5 - 1.17A'tt2 [Bchl d] = -20.09 A'647 + 31.45 A'653.5 -11.44 A'663.5 + 0.14 A'772
The equation for Bchl a here and below remains the same and so does not repeated.
The use of this formula is justified only at close concentrations of Chl b and Bchl d, since all multi-wavelength formula give high errors for minor pigments.
If chromophyte algae are present instead of Chl-b-containing, the following system may be recommended:
[Chl a]= 0.24 A'630 - 7.22 A'653.5 + 16.53 A'663.5 - 1.33A'tt2 [Chl c]= 40.04 A'630 - 3.67 A'653.5 - 4.48 A'663.5 - 2.23A'772 [Bchl d] = -1.22 A'630 + 17.44 A'653.5 -9.01 A'663.5 - 0.36 A'772
In fact, the presence of Chl c has little effect on extinction at the Chl a and Bchl d maxima, therefore the equations for their calculation only slight differ from the three-wavelength ones. As for determination of Chl c itself, the remark on Chl b is fully applicable in this case.
Finally, if the presence of both Chl b and Chl c containing algae could not excluded, the equations transformed into the following set:
[Chl 0]= -0.32 A'630 + 3.62 A'547 - 9.72 A'653.5 + 17.01 A'663.s - 1.41A^ [Chl b]= -6.80 A'630 + 43.72 A'647 - 30.10 A'653.5 + 5.74 A'663.5 - O.86AV72
[Chl c]= 42.57 A'630 -16.25 A'647 + 7.52 A'653.5 - 6.61 AV5 - 1.91A^ [Bchl d] = 2.02 A'630 - 20.86 A'647 + 31.80 A'653.5 - 11.75 A'663.5 + 0.05 A'772
These equations should be applied with great caution, except for the samples with minor Bchls concentrations.
In all these equations, in the absence of significant amounts of Bchl a (for example, in the samples without purple bacteria, where Bchl (c+d+e)/Bchl a ~ 100), the item including A'772 may be omitted; Bchl a concentration in most cases may be calculated as [Bchl a] = 15.65 A'772.
The equations for the concentration of "brown" Bchl e instead of "green" Bchl d may also be offered. However, it is indiscreetly to assume the absence of Bchl d without thorough microscopic confirmation, as "brown" forms of Chlorobiaceae frequently coexist with the green ones and intense color of brown APB dominates the weak yellow-green color of Chl-c or d-containing species.
Another set of equations is the system for four bacteriochlorophylls that may be useful, for example, in APB enrichments where chlorophylls are presumably absent:
[Bchl c] = -6.65 A'647 - 1.86 A'653.5 + 16.99 A'664.4 - 1.37 A'772 [Bchl d]= -59.21 A'647 + 57.69 A'653.5 - 9.82 A'664.4 + 0.86 A'772 [Bchl e]= 100.34 A'647 - 69.54 A'653.5 + 2.98 A'664.4 - 2.21 A'772 [Bchl a]=15.65 A'772
Instead of Bchl e red maximum at 652.5 nm, these equations utilized the wavelength of 647 nm; this allow to lower some coefficients and therefore somewhat reduce the errors due to small difference of large quantities, but makes an increased demands on the accuracy of spectrophotometer wavelength setting.
In conclusion, I would like to discuss the effectiveness of extraction. Both 100% and 90% acetone have clear advantages as solvents for chlorophyll spectroscopy, but they are poor extragent of some green algae, and, more important, of many cyanobacteria. Other solvents were proposed, i.e. alcohols and aprotic polar solvents. In microbiological practice of bacteriochlorophylls determinations, the system ace-tone-methanol 7:2 is traditionally used. Recently, Ritchie recommended similar system with absolute ethanol in place of methanol for determination of Chls a and b and Bchl a. All of these systems seem to surpass the acetone-water mixtures as extracting solvents. The work on developing multi-wavelength equations for some of these solvents is currently in progress.
УДК 579.26
DOI: 10.24411/9999-002А-2018-10025
АНОКСИГЕННЫЕ ФОТОТРОФНЫЕ БАКТЕРИИ В РАЗНОТИПНЫХ ВОДОЕМАХ ВОЛЖСКОГО БАССЕЙНА М.Ю. Горбунов, М.В. Уманская
Институт экологии Волжского бассейна РАН, Тольятти, Россия e-mail: myugor1960@gmail.com
Аннотация. Структура микробного сообщества области хемоклина стратифицированных водоемов Среднего Поволжья определяется типом гиполимнической аноксии, которая зависит от того, какие восстановленные соединения накапливаются в гиполимнионе. В докладе обсуждаются особенности развития аноксигенных фототрофных бактерий в эвксинных и сидеротрофных озерах региона.
Ключевые слова: стратифицированные озера, Среднее Поволжье, Chlorobiaceae, Chromatiaceae, Chloroflexaceae.
ANOXIGENIC PHOTOTROPHIC BACTERIA IN DIFFERENT TYPES OF WATER BODIES OF THE VOLGA BASIN M.Yu. Gorbunov, M.V. Umanskaya
Institute of Ecology of the Volga River Basin of the Russian Academy of Sciences, Togliatti, Russia
e-mail: myugor1960@gmail.com
Annotation. The microbial community structure of the chemocline communities of the stratified water bodies of Middle Volga region is determined by the type of hypolimnetic anoxia, which depends on the reduced compounds accumulating in the hypolimnion. Features of the development of anoxigenic photo-trophic bacteria in euxinic and ferrugenous lakes are discussed.
