Management of waste from a biomass biodigestion to methane and its ecological use in plant production as fertilizers are among important issues in the world agriculture. This problem has become serious because the biogas plants are a prospective branch of energy production, as e.g., increase in electricity production from biogas in 2020 will amount to 125% as compared to 2010. Thus, the production of biogas is increasing and consequently the problem of economic management of the waste from a biomass biodigestion to methane has become very important from economic and environmental point of view. Given the environmental risks and benefits, it is most rational to use these wastes in agriculture (Alburquerque et al., 2012; Jasiulewicz and Janiszewska, 2013; Łagocka et al., 2016), probably to a greater extent than sewage sludge, which may contain toxic substances and should be refined before use in plant crops (Fang et al., 2017; Kołecka et al., 2017). World literature available on these issues, especially concerning agricultural management of waste from corn grains biodigestion to methane, is hard to find and in the majority of cases it refers to the waste produced by specific biogas plants and the raw materials used there (Lukehurst et al., 2010; Nakamura et al., 2014; Lalak et al., 2015).

The problem in the waste application in agriculture is a quick and reliable assessment of the effects of plant fertilization that would enable rational management of this treatment. For this reason, technologists are looking for physiological methods that immediately show the reaction of the plants to different treatments, which enables quick cultivation decisions to be made in the growing season without waiting for the completing plant growth in autumn. One of such checked tests is the evaluation of key for growth enzyme activity (acid and alkaline phosphatase, RNase, nitrate reductase, dehydrogenase), index of chlorophyll content and gas exchange in leaves (net photosynthesis, transpiration, stomatal conductance, intercellular CO₂ concentration (Górnik et al., 2008; Grzesik and Romanowska-Duda, 2014, 2015; Piotrowski et al., 2016; Grzesik et al., 2017a,b). Kalaji M.H. et al. (2014), Kalaji et al. (2018) indicated that chlorophyll fluorescence parameters may be taken into account as an interesting new indicator which can measure most types of plant stress and perhaps their vigor and thus they published a practical information on the hardware, methodology and the hands on application of this technology. Chlorophyll fluorescence measurements allow the recognition of changes in the general bioenergetic state of the photosynthetic apparatus. Moreover, such measurements relate, directly or indirectly, to all stages of light-dependent photosynthetic reactions, including water splitting, electron transport, and pH gradient formation across the thylakoid membrane and ATP synthesis (Kalaji et al., 2012). The chosen fluorescence parameters can give insights into the ability of plants to tolerate environmental stresses and into the extent to which those stresses have damaged the photosynthetic apparatus. By measuring the intensity and nature of this fluorescence, plant ecophysiology can be investigated (Lichtenthaler et al., 1986; Żebrowska and Michałek, 2014). Strasser et al. (2000) suggested six parameters which can be used in screening tests: F₀/F_m, (dV/dt)₀, VJ, V_I, t_Fmax and S_m. They describe that F₀/F_m, (dV/dt)₀ and VJ refer to the structure and function of PSII while V_I, t_Fmax and S_m concern the activity of the electron transport chain (ETC) beyond Q_A. Among parameters of chlorophyll fluorescence, the maximum PSII efficiency, as given by F_v/F_m in dark-adapted leaves is more often used in research (Su et al., 2015) probably because it is more frequently used and commonly available from early pulse amplitude modulated (PAM) fluorimeters (Osinga et al., 2012). The ratio-metric normalization of F_v/F_m also facilitates ease of results interpretation. The F_v/F_m is calculated as (F_m - F_o)/F_m when F_o means a (minimal) fluorescence level of dark-adapted sample when all reaction centers of the photosystem II are open and F_m is a (maximal) fluorescence level of dark-adapted sample when a high intensity pulse has been applied and the all reaction centers of the photosystem II are closed (Kitajima and Butler, 1975).

Chlorophyll fluorescence has been found as the genetic marker of barley productivity (Planchon et al., 1989). Jalink et al. (1998) and Górnik et al. (2013) indicated that it can be used for assessment of maturation advancement of cabbage and coriander seeds, taking into consideration that their degreening process is directly related with the amount of chlorophyll in seed coats, which was monitored by measuring of maximal efficiency of PSII. Górnik and Lachuta (2017) demonstrated that the changes in maximal photochemical efficiency of PSII (F_v/F_m) in sunflower seedlings was related to their growth, which was reduced by previous chilling and increased by subsequent priming of seeds, sown then after these treatments to obtain seedlings subjected to assessments of physiological activity. Chlorophyll fluorescence was also proposed as a marker of the plant reactions to herbicides mechanism of action (Dayan et al., 2012), cellular damage by glyphosate herbicide in Raphanus sativus L. plants (Silva et al., 2014) and it was used in physiological evaluation of strawberry varieties (Żebrowska and Michałek, 2014). Available literature indicates fragmentarily on changes in maximal efficiency of PSII under the influence of selected plant treatments. Checking the applicability of maximal efficiency of PSII measurements as a marker of plant responses to different fertilization methods requires further extensive research using different cultivation conditions and fertilization methods, as well as comparison to the tests of vigor and plant reaction recognized in literature so far. To the best our knowledge, the use of maximal efficiency of PSII as the marker of energy plant vigor, including sorghum, fertilized with waste from a biomass biodigestion to methane and selected biostimulators, as well as its comparison to a wide range of previously used plant vigor evaluation tests, was not studied yet, despite the urgent need to develop a quick and non-invasive methods for assessing plant response to fertilization and making rapid decisions concerning their cultivation before completing growth.

The purpose of this work was to investigate the suitability of maximal efficiency of PSII, as the marker indicating growth, vigor, energetic value and physiological activity of sorghum fertilized with wastes from a corn grain biodigestion to methane, supplemented with new generation biostimulators, Apol-humus and Stymjod, and its usefulness in monitoring of waste applicability in sorghum crops.

To the best of our knowledge, the investigation concerning the suitability of the maximum PSII efficiency, as the marker indicating vigor, growth, energetic value and physiological activity of sorghum fertilized with wastes from a biomass biodigestion to methane, in a distillery integrated with a biogas plant using corn grains as substrate, have not previously been reported. The performed studies demonstrate that the maximum PSII efficiency, as given by F_v/F_m in dark-adapted leaves of sorghum plants, can be used as a non-destructive marker for defining, the degree of plant development and similarly as other studied tests (gas exchange, index of chlorophyll content and activity of selected enzymes) can indicate the plant vigor, and physiological activity. Changing Fv/Fm values depending on the doses of un-centrifuged and centrifuged waste from a biomass biodigestion to methane used were strictly correlated with the height and mass of Sorghum plants. The linear regression equation makes it possible to predict the yields of sorghum plants based on Fv/Fm. This relationship is very tight. The study exhibit the usefulness of F_v/F_m in evaluation of impact of the waste from a corn grain biodigestion to methane on development and physiological activity of sorghum plants, thus showing their potential as an organic fertilizers. Obtained results indicate that F_v/F_m acts as a stable metric for measuring the impact of waste fertilization on the maximal quantum yield of PSII. Additionally, this parameter responded to the waste doses positively, allowing easy interpretation for practitioners. The same was stated in Banks (2018) studies in which the F_v/F_m responded to drought stress negatively. The found dependences in maximal efficiency of PSII, similarly as these in other laborious studies, closely reflected the effect of fertilization with various doses of waste, biostimulants, cultivation conditions and soil fertility on plant height, biomass yield, gas exchange and enzymatic activity and ash content in sorghum plants, which were determined in separate labor-intensive tests. These dependencies were observed independently these plants have been cultivated in peat substrate in pots or in podzolic soil in field, which showed other fertility, and under different climate in Central and North Poland. The changes in photochemical efficiency of PSII in relation to fertilization methods and sorghum growth, confirmed in other investigations, demonstrated positive impact of all studied waste doses on plant development and showed that the non-centrifuged waste is more effective in growth enhancement than the centrifuged one used in the same dose. Their application in doses of 30 m³ ha^-1 and 40–50 m³ ha^-1 respectively, were most efficient. These changes in F_v/F_m measurements were not in line with chemical analyze results showing that plant fertilization with the waste, did not increased macro- and microelement content in leaves but it decreased only ash content in burnt sorghum biomass, which is important when these plants are produced for energetic purposes. The obtained results confirm research of Shool and Shamshiri (2014) who demonstrated the enhanced F_v/F_m by improved nutritional status of the pistachio seedlings under four water regimes, especially by increasing content of the available phosphorus (P) element in soil. The close link between fluorescence emission and nitrogen fertilization was found also by Corp et al. (2003), Schächtl et al. (2005), Cendrero-Mateo et al. (2016), and Camino et al. (2018). This findings, that chlorophyll is the most widely used proxy for N content were also confirmed by research performed by Herrmann et al. (2010), Homolová et al. (2013), and Camino et al. (2018). In studies of Kalaji M.H. et al. (2014) on maize and tomato, the deficiencies of individual nutrients affected the photochemical processes of photosynthesis, as it was demonstrated by a complex of parameters derived from chlorophyll a fluorescence transient recorded in vivo. They stated that early detection of nutrient deficiency based on chlorophyll fluorescence data might be useful in particular species. Hamann et al. (2018) indicated that in young apple trees of two cultivars submitted to water restriction regimes fluorescence-based indices, related to chlorophyll content and nitrogen balance, promised to be a useful non-destructive tool to estimate physiological status of plants.

Studies suggest that the maximal PSII efficiency can be also used as indicator of plant health. In the conducted research, the value of this parameter grew, while when the percentage of all eight isolated pathogenic fungi in relation to the total number of isolates and the percentage of infected plants compared to controls decreased under the influence of all doses of fertilization. These found dependencies, demonstrating a lowest infection of plants showing simultaneously the highest maximal PSII efficiency, confirm usefulness of this chlorophyll fluorescence parameter in plant health status evaluation. The beneficial effect of fertilization on plant health is widely described in the literature. Possibility to use maximum PSII efficiency as indicator of plant health results from the fact that the increasing leaf colonization by pathogenic fungi damages progressively the photosynthetic system, whose activity can be assessed by F_v/F_m, chlorophyll content, gas exchange and other assessments. This is in line with research of Mandal et al. (2009) who demonstrated that downy mildew induced a reduction of chlorophyll content and PSII activity in Plantago ovata. According to Zhori et al. (2015), the non-invasive and rapid measurement of chlorophyll fluorescence induction allows characterizing the photosynthetic capacity of healthy and infected plants and of parts of them directly in the field. This test is highly sensitive not only concerning infection, but also toward other local environmental influences, as it was stated in Euphorbia cyparissias research. Chlorophyll a fluorescence has been satisfactorily used also for monitoring leaf health status in lamb’s lettuce (Ferrante and Maggiore, 2007).

The obtained results indicate that maximum PSII efficiency may be an rapid and not-destructive marker of sorghum plant vigor and can be profitable in fast evaluation of plant reactions to waste from a corn grain biodigestion to methane. It can be alternative or supplementary test in relation to labor-intensive and destructive studies of enzymatic activity or laboratory evaluation of chlorophyll content, which were useful in assessing the beneficial effects of various treatments on growth and physiological activity of plants. What’s more, this test is more useful in agriculture because it shows the total reaction of plants to specific treatments while others, such as chemical analysis, only results of some phenomena occurring in the tissues. In the performed research, the used maximal PSII efficiency measurements were comparable to activity of several enzymes (acid and alkaline phosphatase, RNase, dehydrogenase), index of chlorophyll content and gas exchange in leaves (net photosynthesis, transpiration, stomatal conductance and intercellular CO₂ concentration), which were useful in assessment of physiological activity in plants and were recommended to be a markers of plant development. It was shown in research on China aster and tomato (Badek et al., 2014, 2016) and in studies showing stimulatory impact of algae on Virginia fanpetals, corn and willow growth and biostimulants on apple seed germination (Grzesik et al., 2009; Grzesik and Romanowska-Duda, 2014, 2015; Grzesik et al., 2015; Grzesik et al., 2017a,b) and sewage sludge on energy plants biomass yield (Romanowska-Duda et al., 2010). It is believed that maximum PSII efficiency measurement can be used to monitor parameters that depend on the content and activity of chlorophyll, and thus it can be an indicator of plant growth, gas exchange, activity of certain enzymes, biomass yield and the amounts of elements which depends on the functioning photosynthetic apparatus. The limiting factor of this parameter application for monitoring plant development can be a low temperature and insufficient nutrient supplementation that negatively affect the activity of the photosynthetic apparatus activity (Ostrem et al., 2018). F_v/F_m can be affected by numerous environmental factors, such as high-light, extreme temperature, CO₂, drought, and can change diurnally. Too high light and temperature are considered to be the main factors responsible for the decrease of F_v/F_m at midday in terrestrial plants and in macrophytes. These changes are caused by either down-regulation or photodamage of photosystem II as a response to high irradiances (Jiang et al., 2018). In the conducted research too high temperature (well above 30°C in sun) and strong insolation and a too low air humidity (about 50%) could limit the activity of the photosynthetic system, which may cause the lower values of gas exchange parameters and photochemical efficiency of PSII (Banks, 2018). Drought is damaging when combined with other stressors, including high light intensities which cause an imbalance in the reaction centre leading to oxidative damage and photoinhibition (Goltsev et al., 2012).

Suggestions to use a maximal efficiency of PSII as a marker of sorghum growth, under influence of waste and biostimilants, are in line with research of Jalink et al. (1998) and Górnik et al. (2013) who proposed to use it in Brassica oleracea and coriander seeds as a non-destructive indicator for their maturity, taking into consideration that degreening process is directly related with the chlorophyll content in seed coats, which was monitored by the studied chlorophyll fluorescence parameter. Dayan et al. (2012) suggested chlorophyll fluorescence as a marker for herbicide mechanisms of action, while Silva et al. (2014) as an indicator of cellular damage by glyphosate herbicide in Raphanus sativus L. plants. Żebrowska and Michałek (2014) stated, that considerably differentiated phenotypic values of chlorophyll fluorescence parameters, including F_v/F_m, observed in the analyzed two cultivars of strawberry, were resulted from the specific response of photosynthetic apparatus, dependent on genotype to environmental conditions.

The found possibility to use maximal efficiency of PSII for rapid assessment of plant reactions to studied fertilizers become more important because amount of waste, produced by distilleries integrated with biogas plants, is rapidly growing around the world and thus its management and preferably ecological use as fertilizers for plant crops, have become one of important issues developed in the world. The tested non-centrifuged waste contained more macro- and microelements, essential for plant growth, than the centrifuged one in the unit of volume. The use of non-centrifuged waste in plant production is more justified since the costly centrifugation process is eliminated. Moreover, it contains more macro- and micronutrients thus slightly lower doses can be used to stimulate plant growth as compared to the centrifuged waste. All these measurements and findings under different climate conditions of central and northern Poland indicate, that the non-centrifuged waste from a corn grain biodigestion to methane can be used as a fertilizer in sorghum crops, solving the serious problem of its utilization and storage which is expensive and dangerous for environment. Moreover, the use of this waste in crop fertilization seems to be safer than of sewage sludge, that may contain toxic substances and thus should be refined before use in agriculture (Fang et al., 2017; Kołecka et al., 2017). The waste from corn grain biodigested to methane can be used in agriculture as fertilizers, similarly as waste from slurry digested to methane, which effectively reduce greenhouse gas emissions (Nakamura et al., 2014). However, the contents of nutrients in the studied waste was smaller (depending on the macronutrient) than in cattle manure and lower than in the waste from a maize residue biodigestion to methane in a biogas plant (Alburquerque et al., 2012; Łagocka et al., 2016), both recommended also as natural fertilizers in agriculture.

Demonstrated by maximal efficiency of PSII and other applied assessment tests, the stronger stimulating impact of the combined treatments on plant development could be caused by macro- and microelements contained in waste and by the presence of humic acids and chitosan polymers in Apol-humus, and by iodine, humic acids and nutrients in Stymjod, which additionally increased plant health, as their producers indicate. The chemical structure and stimulating impact of humic acids on plant development under normal and stress conditions was well described in literature (Calvo et al., 2014; Fahramand et al., 2014). The positive effect of chitosan on plant health, physiological activity and growth under different stress conditions was demonstrated in vines (Górnik et al., 2008) and in several other horticulture plants (Pichyangkura and Chandchawan, 2015; Bonilla and Sobral, 2016; Bistgani et al., 2017; Mukta et al., 2017; Pereira et al., 2017; Muxika et al., 2017). Kashyap et al. (2015) presented the positive impact of chitosan on production and protection of a wide number of crops and methods of its application in sustainable agriculture. The positive impact of Stymjod, applied alone and together with waste, on sorghum could be caused not only by, humic acids and elements but also by iodine. Iodine had the positive impact on the cyto-morphological changes in tomato and cabbage plants, as it was discovered by Jeznach (2011) after application of Biojodis, which was the preliminary form of Stymjod, contained similar elements, but was less absorbed by plants. Application of Biojodis to plants increased diameter of phloem and xylem tissues. Moreover, the well-formed single-layer epidermis bordered directly with one layer of collenchyma’s cells, and the parenchyma tissue greater than in control cells, was observed. Application of iodine resulted also in the predominance of open stomata which could increase gas exchange in leaves. Iodine stimulated also the physiological processes in cabbage, which were more resistant to stressed field conditions and increased the quantity of several macro- and microelements in leaves (Jeznach, 2011). In the stored carrot roots, not fertilized in field with N, treatment with iodine increased P, K, and Ca content and reduced Fe accumulation (Smoleń et al., 2011). Application of Biojodis also alleviated the negative influence of unfavorable temperature and drought on growth and physiological activity of corn plants (Piotrowski et al., 2016) and Virginia fanpetals (Grzesik et al., 2009). In lettuce, the winter and summer application of iodate (IO₃^-) or iodide (I^-) increased quantity of these elements in leaves (Voogt et al., 2010). The mentioned processes found in vegetable crops, could stimulate also the growth of sorghum by increasing transportation of nutrients from reached by waste soil through the wider xylem cells, increased the plant growth and enhanced photosynthetic system activity, assessed by maximal efficiency of PSII, however, research in this area was not carried out.

The performed studies demonstrate that the maximum PSII efficiency, as given by F_v/F_m in dark-adapted leaves of sorghum plants, can be used as a non-destructive marker for defining, the degree of sorghum plant development. Similarly as other studied tests (gas exchange, index of chlorophyll content, activity of selected enzymes, mycological and chemical analyzes) it can be used to monitor the plant growth, physiological activity, infestation by pathogenic fungi, and ash content in burnt sorghum biomass. The presented maximum PSII efficiency assessments, confirming other physiological test measurements, shows prospects of ecological use of the waste from a corn grain biodigestion to methane in sorghum crops as fertilizers, together with the new generation soil improver Apol-humus and the nano-organic-mineral fertilizer, Stymjod, as the alternative to artificial fertilizers which pollute the environment. Thus, the maximum PSII efficiency can be seen as useful marker helping management of the studied waste as economically and environmentally friendly natural stimulants, if they are used in defined doses and in agreement with national, legal regulations on the safe application of fertilizers. Confirmed that the linear regression equation makes it possible to predict the yields of sorghum plants based on Fv/Fm. This relationship is very tight.

ZR-D carried out the experiments, processed the experimental data and wrote part of the research dealing with maximal efficiency of PSII, and enzyme activity. MG conducted the research, processed the experimental data, and wrote the part of article on plant development and the gas exchange. RJ conducted experiments on the health of plants, their infestation by pathogenic mycoflora and the quality and quantity of pathogenic microflora infesting plants. All authors provided critical feedback, made contributions to analysis and interpretation of data, discussed the results, contributed to the writing of the manuscript, and gave final approval of the version to be published.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.