Reuse of Organomineral Substrate Waste from Hydroponic Systems

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Reuse of organo-mineral substrate waste from hydroponic systems as fertilizer in open-field production increases yields, flavonoid glycosides and caffeic acid derivatives of red oak leaf lettuce (Lactuca sativa L.) much more than synthetic fertilizer Dennis Dannehl, Christine Becker, Johanna Suhl, Melanie Josuttis, and Uwe Schmidt J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02328 • Publication Date (Web): 08 Sep 2016 Downloaded from http://pubs.acs.org on September 12, 2016

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Journal of Agricultural and Food Chemistry

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Reuse of organo-mineral substrate waste from hydroponic systems as fertilizer in open-

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field production increases yields, flavonoid glycosides and caffeic acid derivatives of red

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oak leaf lettuce (Lactuca sativa L.) much more than synthetic fertilizer

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Dennis Dannehl*1, Christine Becker2, Johanna Suhl3, Melanie Josuttis4 and Uwe Schmidt1

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Agricultural and Horticultural Sciences, 1Division Biosystems Engineering, Albrecht-Thaer-Weg

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3, 14195 Berlin, Germany;

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Humboldt-Universität zu Berlin, Faculty of Life Sciences, Albrecht Daniel Thaer - Institute of

INRA (French National Institute for Agricultural Research), University Nice Sophia Antipolis,

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CNRS, UMR 1355-7254, Institute Sophia Agrobiotech, 06903 Sophia Antipolis, France;

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Inland Fisheries, Müggelseedamm 310, 12587 Berlin, Germany;

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Department of Biology and Ecology of Fishes, Leibniz-Institute of Freshwater Ecology and

Institute for Product Quality, Wagner-Régeny-Str. 8, 12489 Berlin

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* Corresponding author: E-mail: [email protected] Tel. +49 30209346414 Fax. +49 30209346415

ACS Paragon Plus Environment

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Abstract

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Effects of organic waste from hydroponic system added with minerals (organo-mineral fertilizer)

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and synthetic fertilizer on major polyphenols of red oak leaf lettuce using HPLC-DAD-ESI-MS3

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were investigated. Interestingly, contents of the main flavonoid glycosides and caffeic acid de-

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rivatives of lettuce treated with organo-mineral fertilizer were equal to those synthesized without

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soil additives. This was found although soil nutrient concentrations, including that of nitrogen,

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was much lower without additives. However, lettuce treated with synthetic fertilizer showed a

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significant decrease in contents of caffeic acid derivatives and flavonoid glycosides up to 78.3%

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and 54.2%, respectively. It is assumed that a negative effect of a high yield on polyphenols as

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described in the growth-differentiation balance hypothesis can be counteracted by: i) a higher

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concentration of Mg; ii) optimal physical properties of the soil structure.

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Finally, the organo-mineral substrate waste reused as fertilizer and soil improver resulted in the

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highest yield (+ 78.7%), a total fertilizer saving of 322 kg ha-1 and waste reduction in greenhous-

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es.

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KEYWORDS: flavonoid glycosides, caffeic acid derivatives, anthocyanin, flavonol and flavone

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glycosides, organo-mineral fertilizer, mineral fertilizer, red oak leaf lettuce, substrate waste,

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HPLC-DAD-ESI-MS


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Introduction

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The product quality and ecological aspects can play a major role when foods are purchased by

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the consumer 1. In this context, substrates as growing material in hydroponic systems contribute

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to one of the main waste flow in greenhouse production 2, since these cannot always be recycled

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2-4

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mon substrate used for the cultivation of cucumber, tomato and red pepper in hydroponic sys-

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tems in a wide range of countries 5-8. However, the use of environmentally friendly growing sub-

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strates, which are, for example, biodegradable and provide equally good results in terms of fruit

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quantity and quality as achieved using rock wool substrates, may reduce the waste flow under

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protected growing conditions 4, 9.

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Recently, it was reported that Sphagnum farming enables the production of a renewable organic

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substrate

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cessfully tested for their suitability as replacement for rock wool as substrate in hydroponic to-

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mato production 11. The next step is to find out if this used organic material can be reused as or-

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gano-mineral fertilizer and soil additive in open-field production in order to enhance yields and

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the quality of plants, as well as to reduce the waste flow in greenhouses. It is known that mats of

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Sphagnum biomass after their use in hydroponic tomato production contain abundant amounts of

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essential plant nutrients, such as Ca, K, Mg, N and P 11. This is due to the organic material itself

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and by residues of mineral fertilizers, which were applied during the cultivation of tomato plants.

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Generally, fertilizer can increase the yield of plants

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lizer use can also affect secondary metabolites, especially polyphenols in foods 15. Epidemiolog-

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ical studies suggest that a diet rich in polyphenols can reduce the occurrence of chronic diseases

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16

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fruit and vegetables produced in horticulture. To do this we need detailed information about re-

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sponses of their biosynthesis to different horticultural approaches, e.g., to new fertilization strat-

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egies. Mineral fertilizers have the property to decrease levels of polyphenols, while organic ferti-

. This applies in particular to the rock wool substrate pressed as mats, which is the most com-

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. This cultivated Sphagnum biomass (Sphagnum palustre) pressed as mats was suc-

12-14

. Furthermore, it was reported that ferti-

. Therefore, it seems useful to generally enhance the accumulation of phenolic compounds in


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lizers increase the levels of phenolic compounds, although these results are ranging from positive

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to negative effects in terms of polyphenol levels in organic and conventional vegetables

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Previous studies were conducted with different fertilizer regimes to investigate the effects of

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these on phenolic compounds in different plants. These compounds were increased in tomatoes,

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broccoli and red pepper by the influence of higher levels of Mg, S and Ca, respectively, whereas

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phenolic compounds in red lettuce increased with decreasing N concentrations 19-22.

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Major phenolic compounds in red leaf lettuce are caffeic acid derivatives and glycosides of cya-

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nidin, quercetin, as well as luteolin 23. The cyanidin glycoside absorbs photons from the yellow-

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green wavebands and is, thus, responsible for the red appearance of the leaves. To the best of our

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knowledge, a comparison of the effects of equal quantities of nutrients provided by mineral and

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organo-mineral fertilizers on yield and caffeic acid derivatives, as well as flavonoid glycosides

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of red oak leaf lettuce has not been reported before. Therefore, one objective of the present study

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was to investigate the effects of the mentioned fertilizers on the major phenolic compounds in

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red leaf lettuce via HPLC-DAD-ESI-MS³, where the organo-mineral fertilizer was obtained from

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the substrate waste caused by hydroponic tomato production. In this context, the analysis of min-

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erals and the determination of the plant development of red leaf lettuce were also part of this

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study, because it was demonstrated that an increase in yield caused by nitrogen can be accompa-

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nied by a decrease in phenolic compounds 24. Furthermore, changes in physical properties of the

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soil treated with or without mineral and organo-mineral fertilizers were also determined in order

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to draw further conclusions on plant development and accumulations of secondary metabolites of

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red leaf lettuce. Finally, under consideration of all plant responses of red leaf lettuce, it was eval-

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uated whether the application of the organo-mineral fertilizer can be used: i) to reduce an addi-

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tional mineral fertilization in open field production; ii) to decrease the substrate waste in the hy-

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droponic tomato production; iii) to improve the soil structure.

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Materials and Methods


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.

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Crop cultivation and fertilization. The experiments were conducted in open-field produc-

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tion in Berlin (52°28´ N, 13°18´ E), Germany, from 19th June until 24th July 2014. During the

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investigations, the values of temperature, relative humidity, global radiation and carbon dioxide

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were recorded by a weather station on the ground, which are displayed in Figure 1 as weekly

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mean values. Seeds of red oak leaf lettuce (Lactuca sativa L. cv. Eventai RZ; RijkZwaan, De

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Lier, The Netherlands) were sown in rock wool cubes, where the plants were grown in a conven-

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tional greenhouse for five weeks until they had formed four true leaves. Before the plants were

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transferred into the open-field, the experimental area was treated with three different fertilization

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levels: control (no added fertilizer) and two types of fertilizers (one mineral and one organo-

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mineral). Based on the initial state of the soil and according to the fertilizer recommendation for

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lettuce described by Lattauschke 25, the mineral fertilizer was applied as water-soluble fertilizer

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once only before the experiments were started. The organo-mineral fertilizer consisting of the

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dried substrate waste described in detail in the study of Dannehl , et al. 11 and Table 1 was mixed

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with the top soil to a depth of 30 cm, where 1.72 kg substrate waste per square meter were used.

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This volume was exactly the same amount which was previously used to produce two tomato

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plants m-2 ground area in the greenhouse

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organo-mineral fertilizer was supplemented with a water-soluble mineral fertilizer to reach equal

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quantities of nutrients in the soil as those contained in the soil treated with the mineral fertilizer.

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The mineral composition in the soil of all treatments and supplemented minerals to obtain the

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target mineral concentration for each treatment are listed in Table 1. Under consideration of the

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supplemented minerals, fertilizer savings under the use of the organo-mineral fertilizer will be

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calculated and discussed in the results and discussion section. All fertilizer treatments were car-

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ried out in three repetitions with a total of 48 plants per two-square metres plot, where the plots

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were arranged randomly. The plants were uniformly irrigated with an amount of 10 l fresh water

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m-2 applied twice a week.

11

. Immediately after this tillage, the soil treated with


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Assessment of plant growth. Starting in June 2014, the aboveground organs of lettuce were

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randomly harvested once a week for a period of six consecutive weeks in order to obtain data on

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different growth stages. At all harvest dates, nine plants per treatment were weight to measure

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the mean mass of the lettuce head, which was expressed as grams of fresh weight (FW). After-

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wards, we counted and cut the leaves (minimum length = 2 cm), and determined the leaf area per

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lettuce head using an area meter (Model LI-3100 Area Meter, LI-COR; Lincoln, USA). The re-

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sults were expressed as square centimeter (cm-2). Additionally, the plants were dried in a venti-

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lated oven (Heraeus; Hanau, Germany) at 60 °C until a constant weight. The data were used to

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determine the relative growth rate (RGR) per week. RGR was calculated as RGR = (ln W2 – ln

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W1) (t2 – t1)-1, where ln W1 and ln W2 are the means of the natural logarithm-transformed plant

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dry weights at times t1 and t2 according to the description by Hoffmann and Poorter 26. The data

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were expressed as gram per gram and week (g g-1 w-1).

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Sample preparation. A mixed sample from four plants was prepared for each fertilization

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treatment and replicate at the end of the experiments. Only wilted or damaged outer leaves were

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removed. Within 30 minutes after harvesting, the lettuce heads were cut in smaller pieces and an

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aliquot of each sample was used to determine the dry matter. This was obtained by weighing the

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sample before and after drying in a ventilated oven (Heraeus; Hanau, Germany) at 105 °C for

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one day. Afterwards, the dry matter content was calculated by the ratio of the dry mass to the

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fresh mass and is expressed as gram dry matter per 100 g fresh weight. The remaining sample

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material was shock-frozen with liquid nitrogen, kept at -20 °C and afterwards freeze-dried for 48

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hours (Christ Alpha 1-4, Christ; Osterode, Germany). The freeze-dried samples were ground to a

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fine homogenized powder and stored in a desiccator until further analysis of phenolics and min-

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erals.

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Analyses of phenolic compounds. Flavonol and flavone glycosides, as well as caffeic acid

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derivatives were analysed using HPLC-DAD-ESI-MS³ according to the method described by 27.

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Briefly, 0.5 g of the freeze-dried lettuce powder was extracted with 25 ml of aqueous methanol


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(50% MeOH) for 90 min at room temperature. During this time, this mixture of substances was

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kept in motion using a magnetic stirrer and then centrifuged for 15 minutes at 4500 rpm (La-

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bofuge 400 R, Heraeus Instruments, Thermo Disher Scientific; Waltham, USA). The supernatant

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was cleaned using PTFE-syringe filters with a size of 0.25 µm (Roth; Karlsruhe, Germany),

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transferred into glass vials and analysed using HPLC-DAD-ESI-MS3. The anthocyanin extracts

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were prepared similarly, except for a slightly different composition of the extraction solvent and

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shorter extraction time. This means that acidified aqueous methanol (40% MeOH, 10% acetic

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acid) with a pH of 2.6 was used as extraction solvent and the extraction took 15 minutes.

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The system used for analyses consists of an Agilent HPLC series 1100 Ion Trap (Agilent, Wald-

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bronn, Germany). The compounds were separated on a Prodigy column (ODS 3, 150 × 3 mm, 5

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µm, 100 Å; Phenomenex, Aschaffenburg, Germany) with a security guard C18 (ODS 3, 4 × 3

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mm, 5 µm, 100 Å) at 30 °C using a water/acetonitrile (ACN) gradient and a flow rate of 0.4 ml

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min-1.

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As such, Eluent A was 0.5% acetic acid in water (Merck, Darmstadt, Germany) and eluent B was

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100% ACN (J.T. Baker, Deventer, The Netherlands). Gradient 1 was used for flavonol and fla-

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vone glycosides as well as caffeic acid derivatives and gradient 2 for anthocyanins. Gradient 1

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held the following percentages of eluent B: 7 - 9% for 10 min, 9 - 12% for 20 min, 12 - 15% for

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55 min, 15 - 50% for 5 min, 50% isocratic for 5 min, 50 - 7% for 5 min and 7% isocratic for

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3 min. Using the same eluents, gradient 2 was applied as follows: 10 - 50% for 10 min,

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50% isocratic for 10 min, 50 - 10% for 5 min and 10% isocratic for 5 min.

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Flavonol and flavone glycosides, as well as caffeic acid derivatives were detected in the mass

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spectrometer using an Agilent series 1100 MSD (ion trap) with an ESI interface as ion source in

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the negative mode. The anthocyanin glycosides, however, were qualified in the positive mode.

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DAD was used for quantification of caffeic acid derivatives, flavonol and flavone glycosides, as

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well as anthocyanidin glycosides at wavelengths of 330 nm, 350 nm and 520 nm, respectively.

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All results were expressed as mg 100 g-1 FW.


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Analyses of nutrients. To determine the nutrients in red oak leaf lettuce, an aliquot of 0.5 g

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of the freeze-dried sample was weighed into deionized containers. The microwave digestion,

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which was carried out as a preparation for determining the amount of nutrients in the samples,

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was described in detail by Dannehl, et al. 28. Afterwards, the analysis of the elements in the di-

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gestion solution was conducted via inductively coupled plasma-optical emission spectrometry

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(ICP-OES) using an ICP Emission Spectrometer (iCAP 6300 Duo MFC, Thermo; Waltham,

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USA). The ICP-OES operated with 1150 W RF power and a nebulizer gas flow of 0.55 L min-1,

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where argon was used as a plasmogen and carrier gas. The analyses were performed using a

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cross-flow nebulizer (MIRA Mist, Thermo Scientific; Cambridge, England) and from a radial

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view for Ca, K, Mg and P. In terms of each element, a single-standard solution (Carl Roth

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GmbH; Karlsruhe, Germany) of 1000 mg L-1 was used to prepare the reference solutions in 1.4

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mol L-1 HNO3. The calibration curves were generated with the following reference solutions:

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blank 1.4 mol L-1 HNO3; 0.5-300 mg L-1 of K; 0-100 mg L-1 of Ca; 0-50 mg L-1 of Mg and P.

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The respective element in the digestion solutions was measured in duplicate at the following

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wavelength: K = 766.5 nm; Ca = 317.9 nm; Mg = 279.1 nm; P = 213.6 nm.

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However, an aliquot of 0.3 g of the freeze-dried sample was used to quantify the contents of

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carbon and nitrogen in red oak leaf lettuce using an elemental analyser (vario MAX, Elementar

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Analysensysteme GmbH; Hanau, Germany) and according to DIN-ISO-10694

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13878 30. The modified method was described in detail by Dannehl, et al. 28. The contents of all

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chemical elements detected in red oak leaf lettuce were expressed as g 100 g-1 FW.

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29

and DIN-ISO-

Physical properties of the soil. To evaluate the physical properties of the soil treated with

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different fertilizer, 100 cm3 metal rings were slowly pushed into the soil before planting. Three

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samples were randomly taken from each fertilizer treatment and repetition (n = 9). Afterwards,

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the samples were saturated with water for 24 hours and then weighed to record the initial value.

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The water-saturated samples were placed on a layer of synthetic sand in a sandbox (Sandbox,

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Eijelkamp; Giesbeek, The Netherlands), where different suction points (pF = 0 and pF = 1.8)


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were successively applied using the negative pressure method in order to calculate the total pore

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space (TPS), air capacity (AC) and the field capacity (FC) as defined by Scheffer and

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Schachtschabel 31 and Hartge and Horn 32. As such, the corresponding gravimetric water content

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was multiplied with the bulk density to obtain the volumetric water content for each sample at

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the mentioned suction points. The volumetric water content of the water-saturated sample at a

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suction point of pF = 0 is defined as TPS, whereas the volumetric water content calculated for

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samples exposed to a suction point of pF = 1.8 is equal to the FC. AC is calculated as the differ-

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ence between TPS and FC, where all variables are expressed as vol%.

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Statistical analysis. Differences in physical properties of the soils caused by different ferti-

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lizer strategies and the effects of these on plant growth characteristics, yields, minerals and sec-

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ondary metabolites (caffeic acid derivatives and flavonoid glycosides) of red oak leaf lettuce

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were evaluated using analysis of variance (ANOVA) with SPSS, package version 19.0. The

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normal distribution of the data was proved using Kolmogorov-Smirnov test, where the results

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obtained did not oppose the evaluation by factorial ANOVA. Significant differences were calcu-

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lated using Tukey-tests at a significance level of p < 0.05. Different small letters describe signifi-

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cant differences. The mean variability was indicated using standard deviation, which was illus-

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trated by ± in tables or bars in figures.

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Results and Discussion

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Plant growth, minerals of red lettuce and soil properties. One week after planting, fresh

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weight, leaf area and number of leaves of lettuce heads were still low and did not differ signifi-

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cantly (Fig. 2 a,b,c). However, both fertilizer treatments strongly affected the mentioned growth

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characteristics during the following phases of plant development. With the exception of the

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fourth and the fifth week after planting, the fresh weight of lettuce heads was significantly in-

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creased in the mineral and the organo-mineral fertilizer treatments compared to the control (Fig.

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2 a). However, there is an interesting detail here. Although the initial nutrient concentration of

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both fertilizer treatments was the same, similar growth characteristics were only observed during


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the first two weeks, whereas these drifted further and further apart in the following growth stag-

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es. Six weeks after planting, the fresh weight of the lettuce heads exposed to the mineral and

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organo-mineral fertilizer was significantly increased by 37.4% and 78.7%, respectively

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(Fig. 2 a), while the leaf area was increased by 22.7% and 43.7%, respectively (Fig. 2 b), com-

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pared to the control plants. As the treatments did not result in large differences regarding the

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number of leaves (Fig. 2 c), the mentioned increases in head mass are probably due to the larger

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leaf area.

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Generally, it was further found that the weekly relative growth rates of lettuce plants exposed to

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organo-mineral fertilizer were mostly higher in comparison to the other fertilizer treatments (Fig.

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3). The dimension of differences in RGR can be described as follows: plants treated with organo-

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mineral fertilizer > mineral fertilizer > control. This especially applies to the RGR calculated

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from the 1st to the 2nd, 2nd to the 3rd and 4th to the 5th week, where the differences in RGR of let-

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tuce plants caused by organo-mineral and mineral fertilizer ranged between 0.06 and 0.14 g g-1w-

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1

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pared to the control plants, plants treated with organo-mineral and mineral fertilizer achieved an

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increase in RGR by up to 0.34 and 0.21 g g-1w-1, respectively, when the above-mentioned calcu-

247

lation dates were considered. However, the RGR calculated from the 3rd to the 4th week is an

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exception here, because the RGR of control plants showed a significant increase by 0.20 and

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0.25 g g-1w-1 in terms of the organo-mineral and mineral fertilizer, respectively.

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Although the lettuce plants showed different growth patterns, no significant differences in dry

251

matter were found under consideration of different fertilization strategies (Fig. 4). This means

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that the fresh and dry weight of plants grown under different conditions increased in equal pro-

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portions. Nevertheless, it can be noted that the dry matter of all plants decreased over time from

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approximately 9.4 to 6.2 g 100 g-1.

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It is quite clear that the higher values for the lettuce head mass and other growth characteristics

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were caused by the fertilizer treatments. Similar results were found at a higher nutrient supply

. A significant difference was only found during plant growth from the 4th to the 5th week. Com-


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19

given to green lettuce and wheat as shown by Becker, et al.

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Based on the higher nutrient supply, we assume that an increased nutrient uptake promoted plant

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physiological processes, resulting in a higher carbon fixation and nitrate assimilation rate, and

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consequently result in a higher biomass production. In this context, a high correlation between

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RGR and photosynthesis was found by Poorter 33, whereas no correlation was demonstrated be-

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tween RGR and dry matter as shown by Poorter and Remkes 34. Both studies support our find-

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ings. However, the higher RGR of control plants obtained from week three to week four might

264

be caused by a lower leaf density and an associated lower self-shading of leaves resulting in a

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higher light use efficiency during these growth stage. Similar was found by Poorter

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context, we have found that the RGR-responses in terms of all treatments were delayed by two

267

weeks under consideration of the mean global radiation (comparison Fig. 1 and Fig. 3).

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In contrast to Becker, et al. 19, it was found that the total nitrogen concentration in lettuce leaves

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increased only moderately at a higher nitrogen level in the soil when the results are calculated on

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a fresh weight basis of 100 g (Table 2). The same result can be generated by calculating on dry

271

weight basis since the dry matter content of the lettuce heads did not differ significantly (Fig. 4).

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Compared to the control and the mineral treatment, the organo-mineral treatment resulted in

273

higher nutrient concentrations in lettuce leaves, meaning that these had a higher nutritional value

274

(Table 2). In detail, significantly higher contents were found for the elements K (+ 8.2%), Ca (+

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11.5%) and Mg (+ 20%) compared to control plants (Table 2). Interestingly, nearly the same

276

differences were obtained between mineral and organo-mineral treated plants in favour of the

277

organo-mineral fertilizer treatment, although the same initial rates of nutrients in the soil were

278

given (Table 1). It might be possible that the organo-mineral fertilizer acted as a depot fertilizer

279

based on complexing the minerals with chemical components contained in the Sphagnum based

280

organic material. A further explanation could be the physical properties of the treated soils.

281

Compared to the control and the mineral fertilizer treatments, the volume of TPS, AC and FC

282

calculated for the organo-mineral fertilizer treated soil was significantly increased by approxi-


and Gao, et al.

14

257

, respectively.

33

. In this

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mately 10 vol%, 6 vol% and 4 vol%, respectively (Table 3). This means that the higher volume

284

in TPS and FC may have been advantageous for plant growth due to a higher availability of wa-

285

ter-soluble nutrients for plants grown under organo-mineral fertilizer conditions. Considering the

286

soil parameters, the control soil and the mineral treated soil, however, are exposed to a higher

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risk for leaching of important nutrients 31. Furthermore, the volume of AC in the soil caused by

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organo-mineral fertilization can be classified as very high, whereas that of the other both treat-

289

ments can be evaluated as high 35. This indicates that plant roots affected by organo-mineral fer-

290

tilizer were exposed to more oxygen, which can result in a more pronounced root development

291

followed by an enhanced nutrient uptake and hence an accelerated plant growth. On the other

292

hand, it could also be possible that an increased microbial biomass resulting from earthworm

293

activity

294

growth indirectly. There is very substantial body of evidence demonstrating that microorganisms

295

are capable of producing growth regulators, e.g., auxins, which can positively influence growth

296

of plants grown in soil enriched with organic material13, 37.

36

in the soil treated with organo-mineral fertilizer may have influenced the lettuce

297

Flavonoid glycosides and caffeic acid derivatives. In our HPLC-DAD-ESI-MS3 analyses of

298

phenolic compounds in red oak leaf lettuce, we identified two quercetin glycosides, one luteolin

299

glycoside, one cyanidin glycoside and several caffeic acid derivatives. Based on the control

300

plants, the main phenolic compound was quercetin-3-O-(6”-O-malonyl)-glucoside followed by

301

chicoric acid (di-O-caffeoyltartaric acid), chlorogenic acid (5-O-caffeoylquinic acid), cyanidin-

302

3-O-(6”-O-malonyl)-glucoside, luteolin-7-O-glucuronide, quercetin-3-O-glucuronide,

303

caffeoylmalic acid, isochlorogenic acid (di-O-caffeoylquinic acid) and caftaric acid (caffeoyltar-

304

taric acid) (Table 4). The order for phenolic compounds in red leaf lettuce in all treatments was

305

more or less the same. All listed compounds for red leaf lettuce were previously reported by

306

DuPont, et al. 38 and Llorach, et al. 23.

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With exception of caffeoylmalic acid and cyanidin-3-O-(6”-O-malonyl)-glucoside, all identified

308

flavonoid glycosides and caffeic acid derivatives in red leaf lettuce were significantly decreased


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by influence of the mineral fertilizer treatment when these phenolic compounds were compared

310

with those accumulated in control plants (Table 4). In this context, the contents of quercetin-3-O-

311

(6”-O-malonyl)-glucoside, chicoric acid (di-O-caffeoyltartaric acid), chlorogenic acid (5-O-

312

caffeoylquinic acid), luteolin-7-O-glucuronide, quercetin-3-O-glucuronide, isochlorogenic acid

313

(di-O-caffeoylquinic acid) and caftaric acid (caffeoyltartaric acid) were decreased by 53.0%,

314

73.2%, 65.3%, 35.4%, 54.2%, 78.3% and 52.9%, respectively. At the first glance, these results

315

suggest that all the mentioned compounds were decreased at an increased N level as shown by

316

other studies 19, 24, 39. However, the non-significant results in terms of the C/N ratio in the plants

317

from different fertilization strategies (Table 2) did not confirm the C/N ratio-theory, which

318

states that the accumulation of polyphenols might simply be driven by the carbon/nitrogen ratio

319

in plants, resulting in a general shift to carbon based metabolites under N deficiency 40. Rather,

320

the results were closely related to the growth-differentiation balance hypothesis (GDBH), when

321

comparing the control and the mineral fertilizer treatment in terms of the lettuce head mass and

322

flavonoids, as well as caffeic acid derivatives of red leaf lettuce (Figure 2 A, Table 4). The

323

GDBH suggests that the accumulation of biomass and secondary metabolites are negatively cor-

324

related under a high nutrient availability

325

GDBH were found when the organo-mineral fertilizer treatment was included in these compari-

326

sons (Figure 2 A, Table 4). Despite a higher lettuce head mass elicited by this treatment, the re-

327

sults show no significant differences in all analysed polyphenols in red leaf lettuce subjected to

328

control and organo-mineral fertilizer. This was observed, even though the initial nutrient concen-

329

tration in the soil, including that of N, caused by the latter treatment was much higher and, on the

330

other hand, equal to the mineral fertilizer treatments. Therefore, we state that the GDBH cannot

331

completely explain the observed changes of polyphenol concentrations in red lettuce. It might be

332

possible that the accumulation of polyphenols was not decreased because other nutrients were

333

present in higher concentrations, e.g., Mg, which was increased in lettuce grown under organo-

334

mineral fertilizer conditions (Table 2). Fanasca, et al. 20 found higher concentrations of phenolic

41

. Interestingly, contradictory results in terms of the


13


Page 14 of 29

335

acids to be accompanied by higher Mg concentrations in tomatoes, as also shown in red lettuce

336

heads in the present study. The authors attributed this to the high Mg demand for glutamine syn-

337

thetase activity which is an important enzyme that regulates ammonia assimilation within the

338

chloroplasts. The importance of nitrogenous compounds such as phenylalanine in the pathway of

339

phenolic compounds is widely recognized 15. This may explain the increasing concentrations of

340

flavonoids and phenolic acids in red leaf lettuce in favour of the soil treated with organo-mineral

341

fertilizer compared to those obtained with mineral fertilizer.

342

Furthermore, it is supposed that a reduction of flavonoids and caffeic acid derivatives at a high

343

fresh weight of red leaf lettuce can also be avoided by changes in soil properties. It was shown

344

that adding organo-mineral fertilizer as a soil supplement was capable to create optimal proper-

345

ties of the soil structure, which can generally result in higher secondary metabolites as reported

346

by Woese, et al. 42 and Wang and Lin 43. These improved soil properties, including a higher vol-

347

ume of TPS, FC and AC, were caused by the incorporated organic material. The application of

348

organic material can also cause a greater earthworm population

349

acids that can contribute to the promotion of synthesis of phenolic compounds, especially that of

350

flavonoids 44.

351

Which of the mentioned parameters were mainly responsible for changes in flavonoids and caf-

352

feic acid derivatives in red lettuce has to be investigated in more detail.

353

Comparing organic and mineral fertilizer, Sinkovic, et al.

354

the phenolic profiles in leaves of chicory. However, Vinha, et al.

355

significantly higher concentrations of flavonoids in organically grown tomatoes compared to

356

conventionally grown ones. The changes in metabolites caused by organically farmed fields were

357

accompanied by either less yields as reported by Vinha, et al. 45 or higher yields as demonstrated

358

by Mitchell, et al. 46, where explanations for these results are rare. Although an organo-mineral

359

fertilizer instead of a pure organic fertilizer was used in the current study, the results presented

360

are in line with the last mentioned study.

18

36

, which can produce humic

found no significant influence on


45

and Mitchell, et al.

46

found

14

Page 15 of 29


361

Moreover, no significant differences between all fertilizer treatments were found when the me-

362

tabolite cyanidin-3-O-(6”-O-malonyl)-glucoside was considered (Table 4). This anthocyanin can

363

be increased by increasing photosynthetic photon flux densities and temperature (up to 22 °C), as

364

well as nitrogen deficiency as shown by Becker, et al. 47, Josuttis, et al. 48, and Becker et al. 19,

365

respectively. No matter under what fertilization conditions, all lettuce plants were exposed to the

366

same temperature and light conditions during the experiments (Fig. 1) and their tissue had the

367

same nitrogen content (Table 2), which may be why no differences in cyanidin-3-O-(6”-O-

368

malonyl)-glucoside occurred.

369

Evaluation of organo-mineral fertilizer. Besides the positive effects in terms of plant char-

370

acteristics, accumulations of secondary metabolites and soil improving properties, it should be

371

noted that substrate waste (Sphagnum palustre biomass) from hydroponic systems can be reused

372

for mineral fertilizer savings in open-field production. Based on supplemented nutrients for red

373

lettuce leaf production as given in Table 1, it was calculated that 54.2 kg N, 71.7 kg P, 139.7 kg

374

K and 56.4 kg Mg can be saved per hectare when the substrate waste from hydroponic systems is

375

reused as organo-mineral fertilizer.

376

Under consideration of the whole organo-mineral substrate cycle, it can be further derived that

377

one of the main waste flow in greenhouses can be reduced. When Sphagnum palustre biomass is

378

used as growing substrate for hydroponic systems in greenhouses and reused as organo-mineral

379

fertilizer in open-field production, approximately 112.5 m3 of rock wool waste per ha and year

380

can be avoided. This corresponds to an average primary energy demand of 111.4 GJ, which is

381

required to produce the mentioned amount of rock wool. These results were calculated according

382

to the results reported by Pieters, et al.

383

consequently the environment can be relieved.

384

In conclusion, our results represent advantages for producers, consumers and the environment.

385

The producer can achieve higher yields without losing health promoting plant compounds in red

386

oak leaf lettuce and can improve physical properties of the soil when they are using organo-

4

and Brandhorst, et al.


49

, and show that landfills and

15


Page 16 of 29

387

mineral substrate waste from hydroponic tomato production as fertilizer. This could be one small

388

step to ensure the food supply with high-quality products for the growing world’s population and

389

to conserve the soil. Furthermore, the integration of organo-mineral substrate waste into fertiliza-

390

tion processes can be seen as depot fertilizer and contribute to a reduction in mineral fertilizer

391

applications in open-field production and of accrued substrate waste caused by greenhouse pro-

392

duction. Calculations further showed that huge amounts of energy required for rock wool pro-

393

duction can be saved. These facts release the environment and can influence the decision-making

394

of consumers to prefer this food product.

395

Beyond these advantages, it can be further concluded that the growth-differentiation balance

396

hypothesis was partly contradicted by our results because no differences in secondary metabo-

397

lites were found between the control and the organo-mineral fertilizer treatments, even though

398

the yield was much higher triggered by the organo-mineral fertilized soil. A negative effect on

399

polyphenols may have been counteracted by: i) a higher concentration of Mg; ii) optimal physi-

400

cal properties of the soil structure.

401

Finally, the hypothesis that a greater earthworm population caused by the application of organo-

402

mineral fertilizer produced more humic acids resulted in an increase in growth development and

403

contents of flavonoids and phenolic acids of red oak leaf lettuce has to be investigated in more

404

detail.

405

Acknowledgements

406

We would personally like to thank all the gardeners and technical assistants of the Division Bio-

407

systems Engineering.

408

Abbreviations

409

Q3Gc, quercetin-3-O-glucuronide; L7Gc, luteolin-7-O-glucuronide; Q3MG, quercetin-3-O-(6”-

410

O-malonyl)-glucoside; Cy3MG, cyanidin-3-O-(6”-O-malonyl)-glucoside; FW, fresh weight;

411

DW, dry weight; MeOH, methanol; ACN, acetonitrile; HPLC-DAD-ESI-MS³, high-performance

412

liquid chromatograph with diode array detector coupled via electrospray ionization interface to a


16

Page 17 of 29


413

mass spectrometer; ICP-OES, inductively coupled plasma-optical emission spectrometry; TPS,

414

total pore space; AC, air capacity; FC, field capacity; Ca, calcium; K, potassium; Mg, magnesi-

415

um; N, nitrogen; C, carbon; P, phosphorus; S, sulphur

416

References

417

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15. Treutter, D., Managing Phenol Contents in Crop Plants by Phytochemical Farming and Breeding-Visions and Constraints. Int. J. Mol. Sci. 2010, 11, 807-857. 16. Dillard, C. J.; German, J. B., Phytochemicals: nutraceuticals and human health. J. Sci. Food Agric. 2000, 80, 1744-1756. 17. Faller, A. L. K.; Fialho, E., Polyphenol content and antioxidant capacity in organic and conventional plant foods. J. Food Compos. Anal. 2010, 23, 561-568.

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18. Sinkovic, L.; Demsar, L.; Znidarcic, D.; Vidrih, R.; Hribar, J.; Treutter, D., Phenolic

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profiles in leaves of chicory cultivars (Cichorium intybus L.) as influenced by organic and

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19. Becker, C.; Urlić, B.; Špika, M. J.; Kläring, H. P.; Krumbein, A.; Baldermann, S.; Ban, S.

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glycosides, caffeic acid derivatives, and sucrose while losing chlorophylls, ß-carotene and

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22. Vallejo, F.; Tomas-Barberan, F. A.; Garcia-Viguera, C., Effect of climatic and sulphur

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23. Llorach, R.; Martinez-Sanchez, A.; Tomas-Barberan, F. A.; Gil, M. I.; Ferreres, F.,

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Food Chem. 2008, 108, 1028-1038.

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24. Radi, M.; Mahrouza, M.; Jaouad, A.; Amiot, M. J., Influence of mineral fertilization

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(NPK) on the quality of apricot fruit (cv. Canino). The effect of the mode of nitrogen supply.

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Agronomie 2003, 23, 737-745.

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25. Lattauschke, G. Salate im Gewächshaus - Hinweis zum umweltgerechten Anbau und

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26. Hoffmann, W. A.; Poorter, H., Avoiding bias in calculations of relative growth rate. Ann. Bot. 2002, 80, 37-42.

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27. Becker, C.; Klaering, H. P.; Kroh, L. W.; Krmbein, A., Temporary reduction of radiation

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does not permanently reduce flavonoid glycosides and phenolic acids in red lettuce. Plant

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Physiol. Biochem. 2013, 72, 154-160.


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28. Dannehl, D.; Huyskens-Keil, S.; Wendorf, D.; Ulrichs, C.; Schmidt, U., Influence of

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29. DIN-ISO-10694, Soil quality - Determination of organic and total carbon after dry combustion (elementary analysis) (ISO 10694:1995). 1995. 30. DIN-ISO-13878, Soil quality - Determination of total nitrogen content by dry combustion ("elemental analysis") (ISO 13878:1998). 1998. 31. Scheffer, F.; Schachtschabel, P., Lehrbuch der Bodenkunde. 15 ed.; Spektrum Akademischer Verlag GmbH: Heidelberg, 2002. 32. Hartge, K. H.; Horn, R., Die physikalische Untersuchung von Böden. Ferdinand Enke Verlag: Stuttgart, Germany, 2009; Vol. 4.

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physiological consequences. SPB Academic Publishing bv: The Hague, The Netherland, 1990; p

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45-68.

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34. Poorter, H.; Remkes, C., Leaf-area ratio and net assimilation rate of 24 wild-species differing in relative growth rate. Oecologia 1990, 83, 553-559.

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35. AG-Boden, Bodenkundliche Kartieranleitung. 4 ed.; Schweizerbart: Stuttgart, 1996.

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management: Effects on soil biology. Soil Sci. Soc. Am. J. 2000, 64, 1651-1659. 37. Frankenberger Jr, W. T.; Arshad, M., Phytohormones in soils: microbial production and function. Marcel Dekker Inc.: New York, 1995.

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38. DuPont, S. M.; Mondin, Z.; Williamson, G.; Price, K. R., Effect of variety, processing

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and storage on the flavonoid glycoside content and composition of lettuce and endive. J. Agric.

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39. Awad, M. A.; de Jager, A., Relationships between fruit nutrients and concentrations of flavonoids and chlorogenic acid in 'Elstar' apple skin. Sci. Hortic. 2002, 92, 265-276.


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Blumwald, E.; Ruiz, J. M., Response of carbon and nitrogen-rich metabolites to nitrogen

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deficiency in PSARK::IPT tobacco plants. Plant Physiol. Biochem. 2012, 57, 231-237.

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41. Glynn, C.; Herms, D. A.; Orians, C. M.; Hansen, R. C.; Larsson, S., Testing the growth-

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differentiation balance hypothesis: dynamic responses of willows to nutrient availability. New

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Phytol. 2007, 176, 623-634.

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42. Woese, K.; Lange, D.; Boess, C.; Bogl, K. W., A comparison of organically and

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conventionally grown foods - Results of a review of the relevant literature. J. Sci. Food Agric.

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43. Wang, S. Y.; Lin, H. S., Compost as a soil supplement increases the level of antioxidant

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compounds and oxygen radical absorbance capacity in strawberries. J. Agric. Food Chem. 2003,

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51, 6844-6850.

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44. Theunissen, J.; Ndakidemi, P. A.; Laubscher, C. P., Potential of vermicompost produced

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from plant waste on the growth and nutrient status in vegetable production. International Journal

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of the Physical Sciences 2010, 5, 1964-1973.

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45. Vinha, A. F.; Barreira, S. V. P.; Costa, A. S. G.; Alves, R. C.; Oliveira, M. B. P. P.,

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Organic versus conventional tomatoes: Influence on physicochemical parameters, bioactive

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compounds and sensorial attributes. Food and Chemical Toxicology 2014, 67, 139-144.

531

46. Mitchell, A. E.; Hong, Y. J.; Koh, E.; Barrett, D. M.; Bryant, D. E.; Denison, R. F.;

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Kaffka, S., Ten-year comparison of the influence of organic and conventional crop management

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practices on the content of flavonoids in tomatoes. J. Agric. Food Chem. 2007, 55, 6154-6159.

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47. Becker, C.; Klaering, H. P.; Schreiner, M.; Kroh, L. W.; Krumbein, A., Unlike quercetin

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glycosides, cyanidin glycoside in red leaf lettuce responds more sensitively to increasing low

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radiation intensity before than after head formation has started. J. Agric. Food Chem. 2014, 62,

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6911–6917.


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538

48. Josuttis, M.; Dietrich, H.; Patz, C. D.; Krueger, E., Effects of air and soil temperatures on

539

the chemical composition of fruit and agronomic performance in strawberry (Fragaria x ananassa

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Duch.). J. Hort. Sci. Biotech. 2011, 86, 415-421.

541

49. Brandhorst, J.; Spritzendorfer, J.; Gildhorn, K.; Hemp, M., Dämmstoffe aus

542

nachwachsenden Rohstoffen. In Fachagentur Nachwachsende Rohstoffe e.V., 4 ed.; FNR, Ed.

543

Druckerei Weidner: Rostock, 2012.

544 545

Figure captions

546 547

Fig. 1 Weekly mean values of temperature, relative humidity and carbon dioxide during the pro-

548

duction of red oak leaf lettuce.

549 550

Fig. 2 Effects of different fertilizer treatments on fresh weight (A), leaf area (B) and number of

551

leaves (C) of red oak leaf lettuce during different growth stages. Data represent average values of

552

three biological replicates per treatment (n = 3 with 3 plants per replicate). Different small letters

553

indicate significant differences calculated using Tukey-tests at p < 0.05. Bars display the stand-

554

ard deviation.

555 556

Fig. 3 Effects of different fertilizer treatments on relative growth rates (RGR) of red oak leaf

557

lettuce under consideration of a harvest interval of one week. Data represent average values of

558

three biological replicates per treatment (n = 3 with 3 plants per replicate). Different small letters

559

indicate significant differences calculated using Tukey-tests at p < 0.05. Bars display the stand-

560

ard deviation.

561 562

Fig. 4 Influence of fertilizer treatments on dry matter of red oak leaf lettuce. The data represent

563

average values of three biological replicates per fertilizer treatment (n = 3 with 3 plants per repli-


22

Page 23 of 29


564

cate). Different small letters suggest significant differences calculated using Tukey-test proce-

565

dure at a significance level of p < 0.05. Bars represent the standard deviation.

566 567 568 569 570

Tables

571

Table 1. The mineral composition in the soil of all treatments and supplemented minerals Fertilizer treatment (g m-2)* Supplemented minerals (g m-2) Element Control Mineral Organo-mineral Control Mineral Organo-mineral N 2.41 24.00 24.00 0 21.59 16.17 P 17.10 24.35 24.35 0 7.25 0.08 K 24.89 55.00 55.00 0 30.11 16.14 Mg 16.50 22.30 22.30 0 5.8 0.16

572 573 574 575 576 577 578 579 580 581 582 583 584

*Target mineral concentration in the soil

g 100 g-1 FW

Table 2. Nutrient composition of red oak leaf lettuce cultivated with different fertilizer strategies Fertilizer strategy Unit Element Control Mineral Organo-mineral

585 586 587 588 589 590 591

K Ca Mg P C N C/N ratio

0.329 ± 0.003 a 0.087 ± 0.004 a 0.015 ± 0.001 a 0.031 ± 0.003 a 2.751 ± 0.132 a 0.203 ± 0.032 a 13.730 ± 1.695 a

0.330 ± 0.004 a 0.088 ± 0.003 a 0.015 ± 0.001 a 0.032 ± 0.005 a 2.685 ± 0.076 a 0.238 ± 0.018 a 11.350 ± 1.056 a

0.356 ± 0.006 b 0.097 ± 0.004 b 0.018 ± 0.001 b 0.033 ± 0.004 a 2.779 ± 0.041 a 0.244 ± 0.032 a 11.553 ± 1.791 a

The concentrations of nutrients in red oak leaf lettuce represent average values of three biological replicates per fertilizer treatment (n=3 with 4 plants per replicate). Different small letters indicate significant differences according to the Tukey-test procedure at a significance level of p < 0.05. The sign ± represent the standard deviation.


23


592 593

594 595 596

Page 24 of 29

Table 3. Physical properties of soils subjected to different fertilizer strategies Total pore space Air capacity Field capacity (vol%) (vol%) Fertilizer strategy (vol%) Control 41.5 ± 3.3 a 16.7 ± 3.0 a 24.8 ± 1.1 a mineral 39.2 ± 3.3 a 14.5 ± 3.7 a 24.7 ± 1.7 a Organo-mineral 51.0 ± 5.0 b 22.7 ± 2.9 b 28.4 ± 2.5 b The data represent mean values of nine samples per fertilizer treatment (n=3 with 3 soil samples per replicate) collected before planting. All values were tested using Tukey-test, where different small letters indicate significant differences (p < 0.05). The symbol ± is given as standard deviation.

597 598 599 600 601

Table 4. Concentrations of flavonoid glycosides and phenolic acids in red oak leaf lettuce sub-

602

jected to different fertilizer strategies.

mg 100 g-1 FW

Unit

603 604 605 606 607 608 609 610 611 612 613

Metabolite

Control

Phenolic acids Caffeoyltartaric acid 5-O-caffeoylquinic acid Caffeoylmalic acid Dicaffeoyltartaric acid Dicaffeoylquinic acid

0.85 ± 0.09 b 18.21 ± 2.63 b 1.58 ± 0.15 ab 20.59 ± 4.96 b 1.57 ± 0.57 b

Fertilizer strategy Mineral

Organo-mineral

0.40 ± 0.07 a 6.31 ± 2.87 a 1.06 ± 0.42 a 5.51 ± 2.22 a 0.34 ± 0.29 a

0.69 ± 0.17 ab 20.61 ± 1.54 b 1.96 ± 0.13 b 19.00 ± 4.90 b 2.09 ± 0.41 b

Flavonol and flavone glycosides Q3Gc 3.95 ± 0.93 b L7Gc 13.55 ± 1.54 b Q3MG 32.65 ± 7.05 b

1.81 ± 0.60 a 8.75 ± 1.21 a 15.18 ± 3.31 a

3.86 ± 0.84 b 13.42 ± 2.82 b 32.79 ± 4.23 b

Anthocyanidin glycoside Cy3MG 17.14 ± 3.19 a

13.48 ± 4.73 a

14.28 ± 5.28 a

The concentrations of phenolic compounds of red oak leaf lettuce represent average values of three biological replicates per fertilizer treatment (n=3 with 4 plants per replicate). Different small letters indicate significant differences calculated using Tukey-test procedure at a significance level of p < 0.05. The sign ± represent the standard deviation. Q3Gc, quercetin-3-O-glucuronide; L7Gc, luteolin-7-O-glucuronide; Q3MG, quercetin-3-O-(6”-O-malonyl)glucoside; Cy3MG, cyanidin-3-O-(6”-O-malonyl)-glucoside.

614


24

Page 25 of 29


Weekly mean values of temperature, relative humidity and carbon dioxide during the production of red oak leaf lettuce. Figure 1 258x166mm (300 x 300 DPI)



Effects of different fertilizer treatments on fresh weight, leaf area and number of leaves of red oak leaf lettuce during different growth stages. Data represent average values of three biological replicates per treatment (n = 9). Different small letters indicate significant differences calculated using Tukey-tests at p < 0.05. Bars display the standard deviation. Figure 2 258x467mm (300 x 300 DPI)


Page 26 of 29

Page 27 of 29


Figure 3. Effects of different fertilizer treatments on relative growth rates (RGR) of red oak leaf lettuce under consideration of a harvest interval of one week. Data represent average values of three biological replicates per treatment (n = 3 with 3 plants per replicate). Different small letters indicate significant differences calculated using Tukey-tests at p < 0.05. Bars display the stand-ard deviation. 258x166mm (300 x 300 DPI)



Figure 4. Influence of fertilizer treatments on dry matter of red oak leaf lettuce. The data represent aver-age values of three biological replicates per fertilizer treatment (n = 3 with 3 plants per repli-cate). Different small letters suggest significant differences calculated using Tukey-test proce-dure at a significance level of p < 0.05. Bars represent the standard deviation. 257x166mm (300 x 300 DPI)


Page 28 of 29

Page 29 of 29


Graphical Abstract/TOC 250x187mm (300 x 300 DPI)


Reuse of Organomineral Substrate Waste from Hydroponic Systems

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