In July 2018 Ukrainian team have purchased a new piece of biosencoring equipment – three portative “Fluorotests” (Chlorophyll meters) for NATO SPS G4687 funds. The devices were constructed and manufactured at the Glushkova Institute of Cybernetics, National Academy of Sciences of Ukraine. The purchasing of those devices by NULP and NULES teams allowed researchers to expand objectives of ongoing project to chlorophyll fluorescence as a bioindicator of effects on growth M. x giganteus at contaminated sites for further biomass production. Chlorophyll-a fluorescence induction is a rapid technique for measuring photosynthetic electron transport in plants. Measuring of activity of M. x.giganteus grown at research military and deposit sites allows photosynthesis under stress to be studied in situ and in the laboratory. The concentration and fluorescence of chlorophyll has been measuring for M. x giganteus in situ at Dolyna, Kurkhove military sites and Tokarivka PGR testing/deposit site.
Using of this technique will help to response to question how to mitigate the impact of stress factors while producing miscanthus biomass at contaminated sites.
Cooperation with liasoning institution Zagreb University and end-users from village St.Helena, Croatia
On May 2-9th, 2017 prof. Valentina Pidlisnyuk did a mission trip to Croatia. The goals of the mission were to discuss on-going research on M.xgiganteus growing at the contaminated land and to enrich the deposit Mxgiganteus plantation located at village St.Helena with partners from Agronomy Department at Zagreb University and potential end-uses of the phytotechnology.
On May, 3rd prof. Pidlisniuk met with Dr. Zeljka Zgorelec, ZU. Results of 2 years research on M.giganteus growing at the soil artificially contaminated by heavy metals and 2 years experiment with soil contaminated by metals taken from Bakar, Croatia were discussed. The design of training 2 scheduled at UJEP for November, 7-9 was observed and participation of ZU in this event was discussed. Dr.Zgorelec agreed to do lecturing on quality ensuring of soils and plants analysis. Prof. Pidlisnyuk met with Iva Hrelja, student from the Master program “Agroecology”, Antonija Jonjic and Ana Lijovic, students from Master program in “Organic farming and agrobusiness” and shared results of NATO SPS G4687 project.
During the visit prof.Pidlisnyuk updated on-going 3rd year semi-field experiment with M.xgigateus at the soil taken from Bakar and did sampling.
On May 4-5 the planting of additional deposit plantation at village st.Helena was done. The farmer organization passed the site for UJEP research on cost-sharing base and assisted in field plugging and planting process. The consultation with potential end-users- local farmers regarding planting of energetic crops at the Northern Croatia was done and questions about treatment of M.xgiganteus plantation against weeds was developed. The field was plugged, prepared for planting and 230 rhizomes of M.xgiganteus were planted. Currently the deposit plantation calculated 545 rhizomes. On May, 7 the fence from decorative bushes were planted in order to mark plantation.
On May, 8 prof. Pidlisnyuk made a field trip to Bakar, Rijeka region for additional sampling of metal- contaminated soil. Cost –sharing for that trip was done by potential end-users from village St.Helena.
Establishing field plots at Dolyna, Ukraine
On April 3-5, 2017 the new research plots were established in Dolyna, Ivano-Frankivsk region, Ukraine. The place is former military site used as base of soviet army rockets and possess constantly a negative effect to the health of Dolyna habitats. The local authority is strongly aware of implementing the proposed phytotechnology for cleaning the site and production of biomass. Mayor of the city Volodymir Garazd participated in Training 1 in Lviv on October, 6-7, 2016 and initiated and promoted the establishing of that research Being involved in EU project on alternative energy and energy saving cities is implementing in Ukraine by Association “Energy Efficient Cities of Ukraine he proposed to use the site in Dolyna as demonstration one for governmental officials and professionals interested in alternative energy, including those involved in the EU project.
The establishing was jointly done by NULP and NULES in cooperation with new project’s partners from Ivan Franko Lviv National University and local administration of Dolyna city. Consequently, Co-Director Dr.Pavlo Shapoval, NULP led the establishing at the place assisted by PhD student Vytaliy Stadnik, NULP and PhD Student Artem Medkov, NULES. The design of the plots and scientific assistance was done by Dr.Volodymir Kvak, NULES and Dr. Lubov Byn’o, IFNUL. The local governmental officials: Ruslana Havrulkiv (Deputy Mayor of Dolyna city) and Andrij Yanishevskiy (Head of the Dolyna Soil Station) assisted in paper work and supplying the technical equipment for planting, which was a cost-sharing from the local authority.
Two plots (one - at the contaminated land and another - at the control land) were established with the size of 15,4 x22,1 m each, altogether 480 rhizomes were planted. Half of the M.x giganteus rhizomes were treated by PGRs “Stimpo” in order to increase the biomass quality and quantity.
The UJEP miscanthus team meeting
The UJEP miscanthus team meeting was held on January 18, 2019 in order to overview the state of the art with research fields in Czech Republic established within the project, i.e.: Hradčany (visinity of Mimon), Chomutov and Všebořice. NDP Valentina Pidlisnyuk, members of UJEP NATO team: Josef Trögl, Diana Nebeská, Hana Malinská and Jan Černý attended the event. PhD student Hana Burdová, young researcher Jiri Štojdl and PostDoc Karim Al Souki partcipated in meeting as well.
The 2018 data of growing miscanthus at three fields were discussed and perspective for research in 2019 was overviewed. In addition to existed data it is planned to accomplish the detailed analysis of the soil kharacteristics and contaminations at the research fields and to present that data in a form of maps of the fields‘ characteristics. The monitoring of biomentic parameters during growing season and at harvest will be continued with requested picturing. In order to ensure the good growth of miscanthus in Mimon where the plantation will be re-established in spring 2019 is was propose to use soil amendment-biochar, and to investigate the impact of its types (received after municipal waste treatment and after processing of biomass to energy) to miscanthus productivity.
In addition the new research on evaluation content of C13/C12 in the soil at Chomutov and Všebořice miscanthus plantations at 3 depth has been initiated by PostDoc Karim Al Souki in cooperation with PhD student Hana Burdová. The first results will be reported to the team at the end of June, 2019.
New research fields established in the Northern Bohemia, Czech Republic
In spring 2018 two new research fields were established having the following research goals:
- Field testing of the impact of PGRs to M.xgiganteus biomass parameters
- Monitoring of plant stress and it’s influence to biomass quality
- Technological production of miscanthus biomass for further energy testing
- Testing the phytotechnology with M.xgiganteus at the marginal land
Research field at Chomutov
On April 27, 2018 two plots was estabished at the research field of the Crop Research Institute, Chomutov. At the first plot rhizomes were planted according to the scheme used in 2017 (1 m x 1 m, 12 plants in the row). Half of the rhizomes were preliminary treated 12 hours before the planting by PGR Charkor with concentration 4 ml/l. Rhizomes treated by PGR were planted in every second row. The control plants were soaked into the clean water 12 hours before the planting and planted in each first row.
At the second research plot rhizomes were planted using the another scheme: 0,7 m x 0,5 m, 24 plants in row. That scheme is typical for establishing miscantus plantation and was used while establishing research fields in Ukraine. The order of rows with treated/untreated by PGR rhizomes was the same as in the first experiment.
All rhizomes planted at Chomutov were 2 years old and originally from the deposit site located at St.Elena, Croatia.
Research plots at Všebořice
On May 24th and 25th a research M.xgiganteus field was established at the former landfill located in Vseborice, Ústí nad Labem region (marginal land). The rhizomes used for planting were 10 years old and originally from the research plantation of The Silva Tarouca Research Institute of Landscape and Ornamental Gardening (RILOG), Pruhonice, Czech Republic.
The field was divided into the research plots, each of the plot was erected using the following scheme: 0,7 m x 0,5 m, 35 plants in the row, 6 rows; 210 rhizomes were planted in each unit.
Four of the research plots were established with rhizomes preliminary treated by PGRs. In two plots half of the planted rhizomes were treated by PGR Charkor with concentration 4 ml and time of exposure 12 hours. In two other plots half of the rhizomes were treated by PGR Stimpo with concentration 1 ml/l. Rhizomes treated by PGR were planted in every second row. The control plants were soaked 12 hours into the clean water before the planting and planted in each first row.
The rest of the plots established at the research field were planted using not treated rhizomes.
Soil samples were taken from both localities in Chomutov and Všebořice for further nutrient analyses, detection of contaminants and determination of soil microorganisms.
Estabishing a new greenhouse experiment with simulated petroleum contaminated soil
The main goals of the greenhouse experiment are: to access the resistance of Miscanthusxgiganteus while growing at the simulated petroleum contaminated soil and to determine the limited level of soil petroleum contamination when the plant develops well and produces a good biomass.
The soil used for the experiment was taken at the petroleum refinery (Microchem company, Trebon, Czech Republic) and was polluted by different hydrocarbons. That soil was diluted with Mimon soil taken at the military site 1 (2016) which was not polluted by hydrocarbons. The different ratios of petroleum polluted-not polluted soils (0:1, 1:2, 1:1, 2:1, 1:0) were used for the experiment.
4 parallel pots with 6 kg of soil in each were prepared for each selected ratio. M.xgiganteus rhizomes were planted in 3 pots and one control pot was left without rhizomes. The rhizomes used for planting were originally from the same plant in order to ensure a genetic consistency and to avoid the effect of different stress tolerance.
The impact of soil petroleum contamination to plant’s biometric parameters, stress response and structure of soil microbial communities will be monitored during vegetation season.
Construction of fences at research sites in Hradčany airport (Mimoň)
On April 3-4, 2018 group of UJEP PhD students and employees visited research sites located at the former military airport Hradčany near Mimoň. They build a fence around both research plots where Miscanthus plants were planted in spring and autumn 2017. The reason was to protect them from herbivores which could damage biomass. It was a necessary step towards further successful Miscanthus field research. Fields were also marked with signs with information about the G4687 project and research activities.
Enrichment of the research field plots in Mimon, Czech Republic
The research plots located in Mimon (contaminated and control) were enriched in November by planting 300 M.xgiganteus rhyzomes, from those 200 rhyzomes were planted at the contaminated site and 100 rhyzomes - at the control site. The experiment will permit to detect differences in growing the crop planted at the spring and at the fall as well as to research the peculiarities of soil microbial communities for both plantations established at the different seasons.
On October 31 both fields were prepared for planting by plugging. Preliminary the orientation plan of planting was created for both field and localities were measured for planting. Rhyzonmes were planted on November 7 (at the contaminated site) and on November 9 (at the control site) with assistance of participants of the Training 2.
Monitoring mission to the research sites at Czech Republic
On August, 17-18 prof. Pidlisnyuk and assoc.prof. Trogl made a control mission to the research sites in Mimon (contaminated and control plots) and deposit site in Chomutov.
The both plots in Mimon show the good shooting rate of Miscanthus xgiganteus about 60%, and plantations are not much littering by weeds. The plantations were established in June because of delay in permission for planting and overcame the extremely hot temperature waves were in Czech Republic in July and August.
On August 18 prof. Pidlisnyuk and assoc.prof. Trogl visited the deposit site planted at the Agricultural research station in Chomutov and met with Ing.Honzuk, head of department at the unit. The necessary work on maintaining plantation and cleaning it from weeds was discussed, also ways of treatment the MxG plantation by PGRs in 2018 was overviewed.
New experiment with miscanthus growing at the oil-contaminated soil at UJEP
On July, 19 the new experiment was established at UJEP with growing miscanthus at the artificially oil-contaminated soil. The idea run from the fact that during sampling in spring 2017 one locality was found at the research site in Mimon contaminated by oil products. The source of the contamination was caused by previous release of oil-products into the soil by soviet troops while living airport in 90th. Such contaminated localities were cleaned up in 1996-2006 during implementation of projects supported by Czech Ministry of the Environment and other governmental agencies based at oxidation of oil-products inside the soil depth. However, the locality found is still remained contaminated.
The experiment has been designed in a way, that miscanthus seedling first produced at the regular agricultural soil during 2 month and further re-planted to the pots. The clean soil for the experiment was artificially contaminated by oil in the different proportions referred to the real concentration of contaminants at Mimon site. One seedling of miscanthus was planted in each pot which has 11 kg of differently contaminated soil.
The miscanthus growth will be monitored during the vegetation season and content of oil concentration will be measured.
Field experiment in Hradčany airport – Mimoň, Czech Republic
Two experimental fields were settled in Hradčany former military airport near city Mimoň on June 1 2017. One locality is slightly contaminated with residuals of organic compounds as consequence of activities of Soviet army and the second one is control without hydrocarbon contamination.
Main goals of the experiment are:
- Research of biomass production on military affected soil
- Research of phytoremediation process
- Research of influence of plant growth regulator on phytotechnology
Both localities were prepared by ploughing and 92 rhizomes were planted into each locality, half of them was treated by Stimpo plant growth regulator.
Semi-field experiment at UJEP, Czech Republic
First year experiment was founded at UJEP starting from May, 2016. The design of the experiment was developed during meeting of Co-Directors in Kyiv, Ukraine in Aril and is unified for teams from UJEP, NULES and IBPP. The experiment consists of two main directions: research of phytoremediation process and research of biomass production.
The contaminated soil was taken from former military site, located at Mimon, North Bohemia. The site used to be a place for soviet air-force location Hradcany. The negotiation was done at the Ministry of Defense of Czech Republic and branchof Military Forest at Mimon for receiving an access to the site for sampling. The soil was taken from the site and analyzed for contamination by heavy metals and residues of organic substances.
The control soil was taken from the agricultural land located in 3 km from the site. The contaminated and control soils were mixed in different proportion, put in pots. 2 Miscanthusxgiganteus rhizomes were planted in each pot.
The monitoring of biomass growing is providing constantly and was finished as leaves become deep yellow and dry, on November, 16, 2016.
During visit to UJEP prof. Octaviana Trijillo, Northern Arizona University and University of Arizona ( USA) on August,1st the peculiarities of phytotechnologies developed were discussed and decision was proposed to present the approach with using second generation crops for phytoremediation at the Department of Soil, Water and Environmental Sciences University of Arizona. Prof. Trijillo was presented the semi-field research on phytotechnology at UJEP.
Using microbiology indicators: phospholipid fatty acid and enzymes for assessment changing in military contaminated soil’s ecosystem during application of phytotechnology with Miscanthus x giganteus
The efficiently of the proposed approach has to be assessed and constantly monitored in order to develop an advanced phytotechnology. The progress may be assessed by three main approaches:
- assess of phytoremediation efficiently by analyzing translocation factors
- assess the physiological parameters during phytotechnology application: i.e. plant growth, volume of biomass, photosynthetic pigments, and antioxidant enzymes
- assess the microbial indicators of soil ecosystem during process of phytotechnology application.
There is a luck of data regarding impact to microbial community of rhizospheres, its dynamic and role in the process of phytoremediation. In particular it is necessary to have that data during growing of second generation biofuel crop Miscanthusxgiganteus at the military contaminated soils taking into account the need for assessing such phytotechnology.
The main goal of the research is to explore the microbial community in a contaminated soil close to Miscanthus x giganteus rhizospheres and in the treated soil, it’s changing during vegetation seasons and to calculate microbiological indicators in order to improve the overall process of phytotechnology with Miscanthus x giganteus phytotechnoly to be applied for the former military sites in-situ.
The research is going on with the soils taken from the military sites at Sliac, Slovakia; Mimon, Czech Republic and with the abandoned soil from Bakar, Croatia.
Nematology cooperation establishment with KSU
Assoc. Prof. Tatyana Stefanovska had meeting with the nematode specialist in Kansas State University Tim Todd. The meeting objective was to discuss the possibility of cooperation in nematodes as indicators of soil health researches in the frame of NATO SPS # G4687 project. Tim Todd had a solid research background in nematode ecology and had experience in study of contribution of nematodes to the structure and function of the soil, as well as nematode community responses to environmental disturbance. He was involved in collaborative research in remediation with using nematodes as indicators during bioremediation (more microbial than plant) of petroleum and brine contamination.
Tatyana Stefanovska visited the Tim Todds’ Nematode laboratory and observed the methods of nematodes isolation in nematode ecology study. It was agreed to incorporate the study of nematodes as indicators of soil quality change to the ongoing researches of miscanthus biomass Miscanthus x giganteus production in Fort Riley Military contaminates site which is carried out by KSU team of NATO SPS G4687. The researches will be done in cooperation with leaded by Tatyana Stefanovska nematology group.
Photo 1. Tatyana’s Stefanovska Visit of Tim Todd’s Nematology Lab.
Visit of partners from liasoning institution to NULES
In October, 9-17 2017 representatives of NATO G4687 liaisoning institution- Warmia and Mazury University in Olsztyn/Nematology Lab Dr. Andrzej Skwiercz and Dr. Franciszek Kornobis from the Department of Zoology, Research Institute of Plant Protection, Poland visited Department of Entomology at NULES, Ukraine. The visit of Polish scientists had two purposes:
- to deliver Crossed-course in Nematology (Lectures/Laboratory exercises) to students specialized in Plant protection and to young researchers involved in G4687;
- to conduct laboratory researches focused at sampling and isolation the nematodes from different soil samples where M. giganteus was grown, i.e: model pot experiment, deposit field at NULES, contaminated military sites in Dolyna (Ukraine) and Mimon (Czech Republic), research plot of the Institute of Bioenergy crop and sugar beet (Ukraine).
Polish scientists delivered two lectures to Bachelor and Master students at the Faculty of Plant Protection, Biotechnology and Ecology, NULES. The lectures had a goal to give students information about nematodes studying at the different soils in which M. giganteus growth.
The emphasis was made on the importance of nematodes as potential viruses’ vectors which cause the decrease of biomass while growing energy crops on agricultural land, abandoned sites including military ones.
Totally 35 undergraduate and graduate students and young researchers from the faculty of Plant protection, Biotechnologies and Ecology attended the Nematology crossed-course. They actively participated in classroom and laboratory sessions, and asked several questions regarding the role of nematodes in soil food web and how soil contamination may influence the nematode community compositions. The Crossed-course on Nematology developed by Dr. Skwiercz and Dr. Kornobis will be used by Associate professor Stefanovska for updating the course “Technology of beneficial organisms rearing and their use for biological control of pests” (first year Master study) and by Associate professor Kava for updating course “Agrozoology” (first year Bachelor study).
Dr. Skwiercz and Dr.Kornobis trained NULES G4687 young researchers Dr. Kava, Dr. Kvak and PhH student Medkov in procedure of isolation nematodes from soil samples using different methods, including centrifugation.
Polish scientists along with PPD Dr. Tatyana Stefanovska studied the fauna of nematodes at Miscanthus x giganteus grown at the contaminated/non-contaminated soil as well as impact of parasitic nematodes on biomass reduction, that research are within the frame of G4687 project.
NPD Director prof. Valentina Pidlisnyuk attended the lectures being with mission trip to Lviv-Kyiv, Ukraine. She met with NULES team and representatives of liaison institutions for discussion results of ongoing research in Ukraine and Poland. The questions about chapters to the planning Book to be created within the project was overviewed and possibility to organize a professional meeting at WMU Lab in Gdyna on indicators of soil health using nematodes was conferred. She also observed the new equipment (centrifuge and binocular) purchased within G4687 for providing research on nematology indicators.
On-going research at Kurakhove
Yellow blue trap installed for monitoring of aerial-flying insects of M.x g.
On July 6-8, 2017 Dr.Volodymur Kvak (NULES) visited the experimental sites in Kurakhovе, Donetsk region. Research site is located at the place of former military activities and was appeared in 2015 during temporary occupation of that territory by Russian Military troops. Control site is located at the agricultural field in suburb. Research and control sites were established with assistance of the State Enterprise "Center for Certification and Examination of Seeds and Planting Material" (CCESPM), potential end-user of the phytotechnology. Dr. Kvak inspected established plots of M.xg., installed the insect traps and took soil samples for analysis.
It was observed that Mxg planted in autumn have shown better establishment results than those planted in spring. Due to the long spring drought (two months without precipitation), plants are currently in stress state and some already dried up.
Dr. Kvak had a meeting with CCESPM representatives Mykola Bilyi and Elvira Paschenko, potential end-users of the phytotechnology. They considered the idea to increase drought tolerance of Mxg by frequent watering and using amendments.
NULES field research at Kurakhove, Eastern Ukraine
Field research on growing Miscanthusxgigateus at the former military land contaminated by heavy metals is going on in Kurachhove site (Donetzka region). Soil is heavily polluted by heavy metals due to the former antiterrorist operation was there in 2015 year and industrial activities.
The research is focused on testing the impact of time planting and treatment of rhizomes by PGRs. To the biomass production. In addition, growing process of different varieties of Miscanthus x giganteus is monitoring. The plants planted in the fall 2016 were hibernated successfully. Spring planting in 2017 also gave a satisfactory results of seedling appearance and establishment. Since the site is located in the zone where precipitation is limited the special attention is given to the water availability to the crop.
Along with NULES team members of the Donetsk’ branch of State Enterprise on certification and expertise of seeds and plant materials have been assisted in research voluntarily.
Manual removal of weeds at the research site, May 2017.
Enrichment of research at NULES
Semi-field and field experiment on growing Miscanthus x giganteus at the contaminated military soils taken from military sites in Ukraine have been continuing at the Laboratory of Phytotechnologies and Biocontrol, NULES. The second year experiment with miscanthus growing at the soil taken from Kurakhovo was updated and is currently monitored. The miscanthus deposit field established in 2016 was enriched. Since 2017 the research objectives were extended to testing study effect of PGRs to plant’s better adaptation to stress factors while growing crop at the contaminated soils. The research in laboratory at NULES is complementary to experiment carrying out at UJEP. For helping with this new field of research a new young researcher Mgr. Artem Medkov joint NULES NATO team recently: he started PhD study in this topic under supervising of assoc. prof. Tatyana Stefanovska. Artem has an experience in effectiveness of PGRs while applied to the different crops including energy plants when they were grown at the agricultural soil. The topic of his dissertation thesis is focused at peculiarities of biomass quality and quantity when Miscanthusx gigateus is treated by PGRs ( application was done to rhizomes and will be done to leaves). That research field is within G4687 objective on improving the biomass quality.
Master student Van Shuay has started research with supervising of assoc. professor Tatyana Stefanovska. His thesis will be focused at herbivorous diversity at Miscanthus x giganteus growing at the soil taken from Dolyna (former military site).