DESC:METHOD OF DETECTING CONTAMINATION IN ALGAL CULTURE

FIELD OF THE INVENTION
[0001] The present invention relates to a method of detecting contamination in algal cultures commercially cultivated for environmental and food applications. More particularly, the present invention relates to a method of detecting contamination in algal culture based on the photosynthetic marker, non-photochemical quenching (NPQ) and concentration of ammonia in algal cultures infested with predator cells.

BACKGROUND OF THE INVENTION
[0002] Occurrence of predator such as zooplankton, protozoa has often been reported in large scale algae cultivation. Predation or grazing leads to the complete algal prey cell displacement often leading to the culture crash. This poses an operational challenge for the cultivation of microalgae in open ponds. Predation of algae affects the biomass productivity and as a result overall operation of commercial scale cultivation of microalgae is cost intensive. To prevent algae culture crash, a quick reliable grazer monitoring tools are necessary that potentially serves as early warming measures. Currently very few algae contamination monitoring tools are available, which can provide an early indication of algae culture crash at the large-scale cultivation of algae. Algal prey cells are autotrophic and photosynthetic in nature whereas majority of the predator are heterotrophic. Photosynthetic performance of the algal prey cells can be monitored using ‘chlorophyll a’ fluorescence; which can be recoded using commercially available hand-held devices based on pulse-amplitude modulation principle.

[0003] Chlorophyll a fluorescence is a term used when light is re-emitted by chlorophyll molecules during return from excited to non-excited states and used as indicator of photosynthetic energy conversion in higher plants, algae and cyanobacteria.

[0004] absorbed photons causes molecular vibration in the energy levels and excites chl-a to Chl-a*. De-excitation of Chl-a* can be brought by transfer of electrons in three possible ways. One of the de-excitation pathway is photochemistry, wherein first electrons are transferred to the next acceptor molecules that are arranged in tandem in photosystems embedded in chloroplast. This process contributes to photosynthesis, resulting in release of oxygen. Another de-excitation pathway, is by fluorescence which brings Chl-a* to a ground-state level. The third de-excitation pathway is non-photochemical quenching (NPQ). Photosynthetic measurements such as photosynthetic yield based on photochemistry and NPQ are exhaustively studied under different abiotic stress. However, grazing-specific changes in chlorophyll a fluorescence or related photosynthetic parameters are not yet available.

[0005] Several other stress such as abiotic stress for example intense light and temperature and biotic stress such as grazing can cause prey cells to alter their photosynthetic properties. The onset of grazing-mediated cellular degradation may change the photosynthetic pigment composition hence electron transport capability of prey cells which affects the de-excitation pathway. Such changes at the cellular level can begin as soon as prey cells are exposed to predators.

[0006] Other technical methods such as existing imaging methods such as microscopy, flow cytometry fails to detect early onset of culture contamination since initial load of contaminate is very low. Molecular tools like qPCR are species specific hence unknown contaminants remain undetected. Above mentioned methods are time consuming, offline and needs special infrastructural support.

[0007] Therefore, there is a need to develop a method for early detection of contamination in algae culture that can trace grazing-mediated changes in photosynthetic properties of prey cells as an indicator of predation. Also, there is a need for Quick, reliable and on-site detection tools for efficient algae pond management.

SUMMARY
[0008] The present invention relates to a method of early detection of contamination or predation in algae culture based on the NPQ profiles of algae and concentration of ammonia in the presence of predator cells. The method of detecting contamination in an algae culture comprises the steps of preparing a control and grazing algae culture by inoculating prey cells and a predator in a predetermined concentration, incubating the algae culture under white light and in dark light conditions, sampling the algae culture in light phase of incubation at a regular intervals, periodically calculating concentration of ammonia in the algae culture. The method may further comprise the steps of setting at least one pulse amplitude modulation (PAM) parameter in a photosynthetic apparatus for measuring photosynthetic parameters of prey cells, adding samples of the algae culture to a PAM cuvette along with a small magnetic bead for mixing, recording a plurality of photosynthetic parameters of prey cells in various physiological conditions. The method may further comprise the steps of periodically calculating a non-photochemical quenching (NPQ) profile of prey cells using the plurality of recorded photosynthetic parameters and comparing the NPQ profiles and ammonia concentration of control and grazing culture to detect a possibility of predation or culture crash.

[0009] The NPQ profile of the prey cells decreases and the concentration of ammonia increases in the grazing culture compared to the control culture in each physiological condition, as predator concentration increases in grazing culture, and such reduction in NPQ levels and increased concentration of ammonia indicate the possibility of predation or culture crash as early as 24 - 48 hours prior to the culture crash.

OBJECT OF THE INVENTION
[0010] The object of the present invention is to provide a quick, reliable and early indication of contamination in algae culture.

[0011] Another object of the present invention is to provide early detection of contamination in algae culture based on the NPQ profiles of algae and ammonia concentration in the presence of predator cells.

[0012] Yet another object of the present invention is to trace grazing-mediated changes in photosynthetic properties of prey cells as an indicator of predation.

BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The detailed description is described with reference to the accompanying figures.

[0014] Figure 1, illustrates a graphical representation of effects of predator infestation levels on NPQ profiles of prey cells.

[0015] Figure 2, illustrates NPQ profiling and ammonia accumulation in grazer-infested algae culture.

DETAILED DESCRIPTION OF INVENTION
[0016] The present invention relates to a method for early and online detection of contamination using pulse-amplitude modulation (PAM) technology to estimate NPQ (non-photochemical quenching) levels. Traditionally PAM measurements are recorded for studying photosynthetic robustness of plant or algae cells in various physiological conditions. NPQ is one of the parameter which can be calculated using PAM data. Technically NPQ values indicate the photo-protective ability of the cell e.g. under high light conditions any excess absorbed photons will be released as heat and can be recorded on PAM.

[0017] NPQ is one of the processes through which excited (state of high energy) chlorophyll (Chl-a*) can return to a ground state. Electron channeling through NPQ typically occurs in order to prevent cellular damage when algae cells are stressed. Therefore, high NPQ values are prominently associated with cells combating any abiotic (light, temperature) or biotic stress.

[0018] The present invention, relates to NPQ profiles of algae culture that changes during the grazing conditions. For this study marine green microalgae including cyanobacteria are used as a prey cell and marine zooplankton species is used as predator. Zooplankton comprise, for example, microscopic protozoans, rotifers, cladocerans and copepods and other aquatic organisms. NPQ levels of grazing culture is found to be indicative 48 h prior to the culture crash regardless of the level of infection (low, moderate, and high). This indicates that NPQ levels can be used as an early indicator of culture crash.

[0019] In addition to NPQ, a high amount of ammonia is accumulated in grazing cultures. Increased concentration of ammonia indicates the presence of predator. Tracing concentration of ammonia along with NPQ levels indicates early onset of predator outbreak in grazing culture. Predator-mediated ammonia secretion is also the probable cause in alterations of NPQ profiles of algal prey.

[0020] Few studies have been reported wherein use of PAM for monitoring wastewater based open pond cultivation of cyanobacteria has been carried out. The contaminating organisms in this study was Diatoms and Scenedesmus spp. which competes with the other algae for nutrients but do no feed on them. Such contaminants are photosynthetically active and hence contributes in PAM reading. On contrary present invention describes NPQ profiles of algae which are being predated by non-photosynthetically active organisms like ciliate, protozoa etc.

[0021] For maximal photochemistry estimation, algal cells are dark adapted to ensure NPQ is zero. This state is an open state where the photosynthetic apparatus can absorb photons at maximal efficiency and use it to drive photochemistry. A weak beam of measuring light (ML) is applied which is too low to drive photochemistry but nonetheless results in a small amount of fluorescence. This is referred as F0 or minimal fluorescence. Next, a saturating pulse (SP) of light is applied for a brief period (usually around 450 to 500 ms) to instantly saturate the photosynthetic apparatus. This is a closed state where maximum fluorescence or Fm is recorded. At this point no NPQ has occurred. Further a series of (AL) actinic light (sufficient to saturate the rate of light harvesting and electron transport – in the present invention 480 ?moles/m2/s) is applied. Cells become light adapted and hence there would be possibility of Chl-a* de-excitation via NPQ or photochemistry. Lastly, actinic (AL) illumination is followed by a saturation pulse (SP) which completely shuts electron flow through the photosynthetic apparatus. If NPQ is creating the maximum fluorescence at this point, Fm' is decreased. Generally, Fm' values are lower than Fm and NPQ is calculated using Stern-Volmer equation (Fm- Fm')-1.

[0022] Figure 1 is a schematic representation of the effects of level of predator infestation on NPQ profiles of prey cells. The present invention related to a method for detection of contamination based on evaluating photosynthetic parameters of prey cells in various physiological conditions. Prey cells are infested with increasing concentration of predators, yielding low, moderate and high level of predator infestations. Low level of predators crashed algae culture after 96 h of infection, moderately infected cultures crashed at 72 h and high level of infestation totally removed prey cells at 48 h. Photosynthetic parameters such as NPQ were recorded during the culture crash as shown in figure 1. NPQ profiles of the prey cells are found to be significantly different, i.e. dropped, in the presence of the predator cells and early signs of predation were recorded well before 48 h of culture crash in all three levels of infection. Effects of high (1:1), moderate (1000:1), low (10000:1) predator infestation levels on NPQ profiles of prey cells are shown in figure 1. The NPQ profiles were recorded on a photosynthetic apparatus (PhytoPAM II) based on the pulse amplitude modulation (PAM) principle to evaluate a plurality of photosynthetic properties of cells through chlorophyll fluorescence.

[0023] NPQ profiling to detect the presence of contamination or predation is carried out by the following steps.

1) Establishing a Control (no predator) and grazing culture by inoculating prey cells and predator strain in a predetermined concentration (selected from 1:1, 100:1, 1000:1, 10000:1 ratio).
2) Incubating the prepared algae culture at 25°C under white light of 50 ?moles/m2/s intensity (12 h dark: light).
3) Sampling 5 ml algae culture every 24 h in light phase of incubation and incubate algae cell suspension (100000 cells/ml) in dark for 15 mins prior to the measurement.
4) Carrying the following settings on a photosynthetic apparatus (Phyto-PAM),
Measuring light – 2 ?moles/m2/s
Saturation pulse intensity – 260 ?moles/m2/s
Saturation pulse duration – 400 ms
Actinic light intensity - 480 ?moles/m2/s
Actinic light duration – 600 s (at 20 s intervals)
5) Adding 4 ml sample in clean PAM cuvette and putting a small magnetic stirring bead in the cuvette to allow mixing.
6) Pressing gain for to obtain optimal signal from samples of variable cell density.
7) Clicking on “SAT-Pulse” for saturation pulse and view pulse to ensure stable signal. At the point F0 (minimal fluorescence) and Fm (maximum fluorescence after dark adaptation) would be recorded.
8) Clicking on “AL” actinic light. Allow readings to be taken every 20 s under actinic light conditions and stop until three consecutives stable Fm readings are obtained. It typically takes 400-500 s to get three constant readings. These stable values are Fm’ (Light-adapted maximum fluorescence).
9) Calculating NPQ using Stern-Volmer equation (Fm- Fm’)-1.
10) Comparing NPQ profiles of control and grazing culture to detect possibility of predation or culture crash.

[0024] Ammonia estimation to detect the presence of contamination or predation, is carried out by the following steps.

1) Establishing a Control (no predator) and grazing culture by inoculating prey and predator in a predetermined concentration (selected from 1:1, 100:1, 1000:1, 10000:1 ratio).
2) Incubating at 25°C under white light of 50 ?moles/m2/s intensity (12 h dark: light).
3) Sampling 5 ml algae culture every 24 h in light phase of incubation.
4) Incubating samples with phenol, sodium nitroprusside and sodium citrate, sodium hydroxide and sodium hypochlorite
5) Recording absorbance of the samples by using a spectrophotometer
6) Reading the absorbance at 640 nm to deduce total ammonia concentration with a reference of absorbance of ammonium sulfate as standard.
7) Comparing the ammonia accumulation in control and grazing culture to detect possibility of predation or culture crash

Increased concentration of ammonia indicates possibility of predation or culture crash in comparison to control culture.

[0025] Experimental Results:
1. A change in the NPQ value and ammonia value indicates the possibility of predation 48 h prior to the culture crash.
2. NPQ profiles are studied under various prey predator concentration, abiotic stress such as high light, temperature and salinity.. NPQ levels significantly drops as cultures proceed to crash.
3. The possible cause of decrease in NPQ levels was investigated. Results suggest that predator releases ammonia (determined using spectrophotometric quantitation) which is assimilated by prey cells during photosynthesis. Uptake of ammonia at high concentration abolishes the proton gradient across the thylakoid membrane and therefore increases the size of the reduced plastoquinone pool (one of the electron carriers in the photosynthetic apparatus) and causes a decrease in NPQ levels.
4. Decreasing NPQ levels of prey (inside predator gut) are also observed using single cell microscopy PAM.

[0026] Referring to figure 2, illustrated is a schematic showing NPQ profiling and ammonia accumulation in grazer-infested algae culture. It is observed that NPQ levels of prey cells reduce as predator concentration increases in microalgal cultures, a significant drop in NPQ levels can be reported as early as 48 hours prior to the culture crash, that is, total removal of the microalgal prey cells. Further, NPQ drop in predator-infested culture is due to ammonia secreted by predator is also reported.

[0027] Photosynthetic measurements such as NPQ can be utilized to monitor the health of an algal culture in both open and closed cultivation platform. Said measurement can be used to determine and prevent culture crash. Recording NPQ profiles of algae pond with ammonia level monitoring can be used as an early indicator of predation, would help in anticipating possibility of predation hence, appropriate preventive measures can be deployed before significant biomass loss is observed.

[0028] The above description along with the accompanying drawings is intended to describe the preferred embodiments of the invention in sufficient detail to enable those skilled in the art to practice the invention. The above description is intended to be illustrative and should not be interpreted as limiting the scope of the invention. Those skilled in the art to which the invention relates will appreciate that many variations of the described example implementations and other implementations exist within the scope of the claimed invention.
,CLAIMS:We Claim:

1. A method of detecting contamination or predation in an algae culture, the method comprising the steps of:
preparing a control and grazing algae culture by inoculating prey cells and a predator cells in a predetermined concentration;
incubating the algae culture under 12 h white light and 12 h dark light conditions;
sampling the algae culture in light phase of incubation at a regular intervals;
periodically calculating concentration of ammonia in the algae culture;
setting at least one pulse amplitude modulation (PAM) parameter in a photosynthetic apparatus for measuring photosynthetic parameters of prey cells;
adding samples of the algae culture to a PAM cuvette along with a small magnetic bead for mixing;
recording a plurality of photosynthetic parameters of prey cells in various physiological conditions;
periodically calculating a non-photochemical quenching (NPQ) profile of prey cells using the plurality of recorded photosynthetic parameters; and
comparing the NPQ profiles and ammonia concentration of control and grazing culture to detect a possibility of predation or culture crash;
wherein the NPQ profile of the prey cells decreases and the concentration of ammonia increases in the grazing culture compared to the control culture in each physiological condition, as predator concentration increases in grazing culture, and such reduction in NPQ levels and increased concentration of ammonia indicate the possibility of predation or culture crash as early as 48 hours prior to the culture crash.

2. The method as claimed in claim 1, wherein periodically calculating concentration of ammonia in the algae culture comprises the steps of:
incubating samples of algae culture with phenol, sodium nitroprusside and sodium citrate, sodium hydroxide and sodium hypochlorite; and
reading an absorbance of samples at 640 nm by using a spectrophotometer, to deduce total ammonia concentration with a reference of absorbance of ammonium sulfate as standard.

3. The method as claimed in claim 1, wherein the plurality of photosynthetic parameters of prey cells is recorded by periodically applying a saturating pulse from the photosynthetic apparatus to the algae culture under actinic light conditions.

4. The method as claimed in claim 1, wherein prey cells and the predator are inoculated in one of concentration comprising 1:1, 100:1, 1000:1 and 10000:1 ratio.

5. The method as claimed in claim 1, wherein the prey cell comprises marine green microalgae comprising cyanobacteria, and the predator comprises zooplankton.

6. The method as claimed in claim 1, wherein the algae culture is incubated under white light of 50 ?moles/m2/s intensity.

7. The method as claimed in claim 1, wherein during various physiological conditions, prey cells are infested with low, moderate and high level of predators.

8. The method as claimed in claim 1, wherein the plurality of photosynthetic parameters of prey cells is recorded after incubating the culturing solution in dark condition.

9. The method as claimed in claim 1, wherein the plurality of photosynthetic parameters of prey cells comprise a minimal fluorescence (F0) and a maximum fluorescence (Fm), Light-adapted maximum fluorescence (Fm’) of prey cells evaluated through chlorophyll fluorescence.

10. The method as claimed in claim 1 and claim 9, wherein the non-photochemical quenching (NPQ) profile of prey cells is calculated using:
NPQ = (Fm- Fm’)-1.