64 Hmeljarski bilten / Hop Bulletin 30 (2023)
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IMPACT OF MICROBIOLOGICAL PREPARATION ON YIELD AND 
SEED COMPOSITION IN THE INDUSTRIAL HEMP VARIETY FUTURA 
75 - PRELIMINARY RESULTS 
 
Tamara KOROŠEC
1
 and Alenka LEVART
2
 
 
Original sceintific article / Izvirni znanstveni članek 
Received / Prispelo: 25. 10. 2023 
Accepted / Sprejeto: 11. 12. 2023 
 
Abstract  
Hemp cultivation in Slovenia currently takes place on just over a hundred hectares. 
The predominant varieties are those used for seed and seed-flower production 
(Futura 75 and Fedora 17), while smaller areas are dedicated to flower varieties. As 
hemp is promoted as a suitable crop for sustainable agriculture, we aimed to 
investigate whether we could influence the yield by using a microbiological 
preparation that is expected to enhance the utilization of nutrients from the soil, 
thereby reducing the need for conventional fertilization. During the 2022 growing 
season, we applied a commercial microbiological product containing a consortium 
of microbial species with the prevalence of the Pseudomonas putida strain to half of 
the test plots in the field trial. Compared to the control group, the group treated with 
microbiological product exhibited increased height, with indications of differences 
in root characteristics (larger main root mass, shallower roots with a larger main 
root diameter, and greater root ramification). However, there were no differences in 
flower, seed, and stem yields between the two groups. Furthermore, there were no 
variations in the analysis of basic seed nutrients, fatty acid composition, or CBD and 
THC content in the dried inflorescence. Given the prevailing drought conditions in 
2022, it is essential to conduct additional research on tested microbiological 
preparation in order to draw credible conclusions and make recommendations for 
its further use in hemp production. 
Key words: hemp, microbial preparations, nutritional composition, fatty acid 
composition, yield 
 
 
 
 
 
 
1
 Dr., univ. dipl. inž. zoot., Kmetijsko gozdarski zavod Maribor, e-naslov: 
tamara.korosec@kmetijski-zavod.si 
2
 Dr., univ. dipl. inž. kem., Univerza v Ljubljani, Biotehniška fakulteta, e-naslov: 
alenka.levart@bf.uni-lj.si 

Hmeljarski bilten / Hop Bulletin 30 (2023) 
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65 
 
VPLIV UPORABE MIKROBIOLOŠKEGA PREPARATA NA PRIDELEK IN 
SESTAVO SEMEN INDUSTRIJSKE KONOPLJE SORTE FUTURA 75 - 
PRELIMINARNI REZULTATI 
 
Izvleček 
Pridelava konoplje v Sloveniji trenutno poteka le na nekaj več kot sto hektarjih. 
Prevladujejo sorte za pridelavo semena in kombinacije seme-socvetje (Futura 75 in 
Fedora 17) ter na manjših površinah sorte za socvetja, katerih sortiment se iz leta v 
leto spreminja. Ker je konoplja rastlina, ki se promovira kot primerna za trajnostno 
kmetijstvo, smo želeli preverili ali lahko vplivamo na pridelek z uporabo 
mikrobiološkega pripravka, ki bi naj povečal izkoristljivost hranil iz tal in tako 
zmanjšal potrebe po gnojenju. V poljskem poskusu smo v vegetacijski sezoni 2022 
na polovici testnih ploskev aplicirali komercialni mikrobiološki preparat, ki vsebuje 
združbo različnih vrst mikrobov, s prevalenco seva Pseudomonas putida sp. V 
primerjavi s kontrolno skupino je skupina, tretirana s preparatom, zrasla višja, trend 
je nakazoval razlike v karakteristikah korenin (večja masa glavnih korenin, bolj plitke 
korenine z večjim premerom glavne korenine in večjo razvejanostjo in gostoto 
koreninske grude). Pridelek socvetja, semen in stebel se med skupinama ni 
razlikoval. Prav tako ni bilo razlik v sestavi osnovnih hranilnih snovi v semenu, 
maščobno-kislinski sestavi semena in vsebnosti CBD ter THC v biomasi socvetij. 
Zaradi izjemno neugodnih vremenskih razmer v letu 2022 (suše) bi bilo potrebno 
izvesti dodatne poskuse, da bi lahko podali kredibilne zaključke in priporočila za 
uporabo preizkušenega komercialnega mikrobiološkega preparata v pridelavi 
konoplje.  
Ključne besede: konoplja, mikrobni preparat, maščobno kislinska sestava, hranilna 
vrednost, pridelek  
 
 
1 INTRODUCTION 
 
Hemp, a versatile and environmentally friendly crop, has been gaining significant 
attention for its potential in various industries, including textiles, food, construction, 
and wellness products (Adamovičs and Zēverte-Rivža, 2015). With its ability to grow 
in diverse climates and soils, hemp holds great promise for sustainable agriculture 
(Sunoj Valiaparambil Sebastian et al., 2023).  
 
Hemp production in Slovenia is decreasing. From approximately 500 hectares in 
2015, the hemp cultivation area has decreased to 103.6 hectares in 2023 (Ministry 
for Agriculture, Forestry and Food of Slovenia - personal communication). French 
varieties continue to dominate, with Futura 75 and Fedora 17 being the most 
prevalent. These varieties are primarily grown for seed production (for oil) and for 
combined seed-flower production. There is a growing trend of small-scale 
cultivation, focusing on flower varieties such as Eletta Campana, Enectarol, and 
Midwest. Over the past three years, there has been a significant shift in the 
assortment. Some varieties that were highly represented in 2021 have practically 

66 Hmeljarski bilten / Hop Bulletin 30 (2023)
______________
 
 
disappeared in 2023, including Antal, Carmagnola Selected, and Tiborszallasi 
(Ministry for Agriculture, Forestry and Food of Slovenia - personal communication). 
 
Most hemp production in Slovenia is conducted in an extensive and environmentally 
friendly manner, 18.42 ha also under organic certification (Ministry for Agriculture, 
Forestry and Food of Slovenia - personal communication). Hemp is frequently 
recommended as a crop suitable for resource conservation in agriculture and as a 
plant adapted to climate change. It is typically grown without the use of herbicides, 
can withstand high temperatures and elevated CO
2
 levels in the atmosphere, and is 
relatively drought-resistant (Chandra et al., 2011; Tang et al., 2018; Van der Werf, 
2004). However, to achieve optimal yields it requires high-quality soils rich in organic 
matter, nutrients and irrigation in periods of drought (Bajić et al., 2022).  
 
To reduce the use of mineral fertilizers, promote the mineralization of organic 
fertilizers, and enhance nutrient availability, the use of microbial preparations is 
becoming increasingly popular in agriculture (Jacoby et al., 2017). Microbial 
fertilizers harness the power of beneficial microorganisms to improve soil health 
and enhance plant growth. These microorganisms include nitrogen-fixing bacteria, 
mycorrhizal fungi, and other beneficial microbes that form symbiotic relationships 
with plants. The beneficial Pseudomonas spp. are ubiquitous in soils and 
competitively colonize all compartments of the plant microbiome, including the 
rhizosphere, the surface of aerial organs (phyllosphere), and the inner plant tissues 
(endosphere). The host plants, in turn, benefit from growth- and health-promoting 
effects (Balthazar et al., 2022). By incorporating these beneficial microbes into their 
farming practices, hemp growers, especially those who do not have their own 
organic fertilizers, might not only boost crop yields but also contribute to a more 
sustainable and environmentally friendly agricultural future. 
 
In this context, we decided to test whether a commercial microbiological product 
based on the microbial consortia prevalent in Pseudomonas putida strain improves 
the utilization of nutrients from the organic matter of clover that was incorporated 
into the soil before hemp sowing. This test aimed to assess the impact of microbial 
preparation on biomass yield, plant development, and the nutritional quality of hemp 
seeds. 
 
2 MATERIALS AND METHODS 
 
2.1 Field trial  
 
A randomized block design (3 blocks x 2 treatments) to assess the impact of 
microbial preparation on hemp yield in a field experiment conducted at GERK PID 
964486 (WGS 84 46°22'58,59" N 15°44'19,71" E) was employed during the 2022 
growing season. The randomized block design was chosen to account for the 
expected spatial variability and to reduce the potential bias associated with soil 
heterogeneity. The chemical analysis of soil showed a pH of 6.09, 42.5 mg/100g 
P
2
O
5
, 31.5 mg/100 g K
2
O and 4.13% of humus. 

Hmeljarski bilten / Hop Bulletin 30 (2023) 
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67 
 
Cultivated field had a prior crop of black clover, which was ploughed under in spring 
before the field preparation for the hemp sowing. In this experiment, half of the 
designated plots received an application of a commercial microbiological consortia 
preparation (MICROB). The total number of microorganisms is declared as minimum 
of 4.10
9
 CFU/cm
3
. The consortia consist of various microbial specimens like 
Azotobacter chroococcum, Azospirillum brasilense, Bacillus megaterium and 
prevalent Pseudomonas putida. The general objective of this preparation is to 
expedite the decomposition of post-harvest residues, straw, and organic matter 
within the soil matrix, concomitant with enhancing the assimilation of soil-borne 
nutrients. Conversely, the remaining half of the plots remained untreated, serving as 
the control group for comparison (CONTROL). All applications were administered 
during the evening hours, with subsequent seedbed preparation and the actual 
sowing performed post-application. No ordinary fertilizers were used. 
 
The chosen hemp variety for seed production was Futura 75. Sowing density was 35 
kg/ha. Sowing was conducted on 12
th
 of June 2022 using a conventional grain 
seeder with inter-row spacing of 25 cm (every other row closed). Test plots 
measured 4x4 m each, samples were taken from one square meter in each plot.  
 
2.2 Data Collection 
 
Data collection involved monitoring crop growth parameters, pests, diseases and 
weeds throughout the growing season. The sampling was carried out on 26
th
 of 
September 2022 after 98 days from sowing. We collected the plants manually from 
1 m
2 
in each block and measured plant height, seed, stem, main root and 
inflorescence yield, diameter of stems and main root, CBD content, basic nutrient 
content and fatty acid composition of seeds. Weeds were identified and their mass 
was determined. The seed yield in manual sampling was much higher than in harvest 
with combine harvester. Ten days after the samples were taken, the whole field was 
mechanically harvested for seed, namely. The yield of mechanically harvested seed 
was only 30 % of the manually harvested (calculated per hectare). This loss can be 
contributed to mechanical disturbances (seeds falling down) and bird consumption.  
 
2.3 Fatty acid analysis of hemp seeds 
 
Fatty acid analysis of hemp seeds was performed in laboratory of Department of 
Animal Science, Biotechnical Faculty, University of Ljubljana. Hemp seeds were 
ground using laboratory homogenizer Grindomix (Retsch). The fatty acid 
composition of seeds was analysed using a gas chromatographic method after the 
in-situ transesterification of lipids, as described previously by Park and Goins (1994). 
Briefly, fatty acids seed samples (approximately 0.5 g) were transmethylated using 
0.5 M NaOH in methanol followed by 14% BF
3
 in methanol. Fatty acid methyl esters 
were extracted using hexane. For separation of fatty acid methyl esters, an Agilent 
6890 gas chromatograph equipped with an DB-FATWAX UI (30 m × 0.32 mm i.d. × 
0.25 μm, Agilent, Santa Clara, CA) and flame-ionization detector was used. 
Instrument was calibrated using Nu-Check-Prep (Elysian, MN) GLC reference 

68 Hmeljarski bilten / Hop Bulletin 30 (2023)
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standards 411, 85, 68A and 674. Total fatty acids in seeds were calculated according 
to Ministry of Agriculture, Fisheries and Food (1998) by multiplying fatty acid 
proportion with lipid content in samples.  
 
2.4 Proximate analysis of hemp seeds 
 
The relative amounts of protein (AOAC Official method 954.01), lipid (AOAC Official 
method 954.01), water (AOAC Official Method 934.01), and ash (AOAC Official 
method 942.05) in seed samples were estimated by proximate analysis in the 
laboratory of Department of Animal Science, Biotechnical Faculty, University of 
Ljubljana. Carbohydrate content in seeds was calculated by difference (100 – water 
– lipids – proteins – ash). 
 
2.5 Cannabinoid analysis of inflorescence 
 
Cannabinoid content was measured by Spectral Fingerprints laboratories (SFP 
d.o.o., Medenska cesta 4a, 1000 Ljubljana, Slovenia) with gas chromatography with 
flame ionization detector (GC-FID full spectrum_v1.0). 
 
2.6 Statistical Analysis 
 
The two-way t-test was performed using SAS STAT software to compare the means 
of the MICROB and CONTROL group. The analysis included the calculation of the t-
statistic, degrees of freedom, and the corresponding p-value. Prior to conducting the 
t-test, several assumptions were checked, including normality of the data 
distribution, homogeneity of variances, and independence of observations. 
Normality was assessed using the Shapiro-Wilk test. The data of fatty acid 
composition was transformed by arcus sinus of square root to ensure normal 
distribution.  
 
3 RESULTS AND DISCUSSION 
 
The average daily temperatures throughout the hemp growing season ranged 
between 10 and 29°C, with three heatwaves (June, July and August) during which 
the average daily temperature did not drop below 20°C. The longest heatwave period 
lasted from mid-July to mid-August (Table 1). In comparison, the thirty-year (1981‒
2010) average daily temperatures for the mentioned months were 18.6°C, 20.4°C 
and 19.6°C, respectively (ARSO, 2023). Simultaneously, in 2022 there were extended 
periods without precipitation, with the longest one occurring from mid-July to early 
August and the last one in the first half of October (ARSO, 2023). The total amount 
of participation in 2022 at the nearby meteorological station was 742.4 mm (Table 
1), which is 190 mm less than the thirty-year average. The hemp crop visually 
appeared to be in better condition than the adjacent corn crops, which showed 
significant damage due to drought. 

Hmeljarski bilten / Hop Bulletin 30 (2023) 
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69 
 
Table 1: Average and maximum temperatures, sunshine hours, and precipitation levels at the 
meteorological station at the airport »Edvarda Rusjana Maribor« (lon=15.6818, lat=46.4797, 
height=264m) (ARSO, 25.10.2023).  
 
2022 Ave. T 
[°C] 
Ave. max 
T [°C] 
Abs. 
max T 
[°C] 
Sunlight 
duration [h] 
Precipitation 
[mm] 
Jan 0.8 6.6 14.2 137 32.3 
Feb 4.2 10.8 16.2 164.3 12.3 
Mar 4.4 12.1 22.7 229.4 5.6 
Apr 9.5 15.2 22.9 213.7 87.7 
May 17.3 23.1 29.7 236.3 69.2 
Jun 21.8 27.2 33.7 296.7 73.3 
Jul 22.6 29.2 35.7 312 53.7 
Aug 21.7 28.4 35.2 240.5 78.8 
Sep 15.3 20.8 28.4 154.3 162.7 
Oct 13.1 20 23 186.7 17.2 
Nov 6.7 11.2 23.9 84.2 55.6 
Dec 2.1 5.3 16.5 61.3 94 
Year 11.6 17.5 35.7 2316.4 742.4 
 
Hemp plants have grown significantly higher in the MICROB group (Table 2). Also, 
the yield of main roots showed a trend to be higher in the MICROB group (Table 3). 
The main roots in MICROB group tend to be shorter, with higher percentage of high 
ramification (28.3 % vs 19.7 % in CONTROL, data not shown) and wider main root 
diameter (Table 2). On the contrary, the results show numerically higher yield of 
aboveground biomass in the CONTROL group, however with no statistical 
significance (Table 3). This is probably connected with higher density of plants in 
this group. The density of plants was very variable, pointing out either problems with 
seeding or germination. Pseudomonas putida is a plant growth promoting 
rhizobacteria (PGPR) that establishes commensal relationships with plants. The 
interaction involves a series of functions encoded by core genes which favour 
nutrient mobilization, prevention of pathogen development and efficient niche 
colonization (Molina et al., 2020). PGPRs were capable to improve growth and 
development, secondary metabolite accumulation and antioxidant capacity or Finola 
variety under glasshouse conditions (Pagnani et al., 2018). Pseudomonas sp. 
inoculation also increased the photosynthetic rate at the vegetative and reproductive 
stages, and the harvest index of a hemp (Lyu et al., 2019). Inoculation with other 
microorganisms, for example Trichoderma harzianum, positively affected root 
density, inflorescence number, fresh weight moisture and compactness of 
inflorescence and cannabidiol (CBD) content in Fedora 17 and Felina varieties 
(Kakabouki et al., 2021). Slow-release potassium fertiliser with added soil 
microorganisms increased stalk diameter, height, shoot dry weight and the contents 
of tetrahydrocannabinol (THC) and similar cannabinoids (De Prato et al., 2022). The 
problem in microbial effect on hemp yield might be in the periods of drought, which 
could negatively affect the microbial activity. Pseudomonas putida has very low 

70 Hmeljarski bilten / Hop Bulletin 30 (2023)
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tolerance to air desiccation in comparison to other bacteria. Desiccated state has a 
decreased cell size, retracted cytoplasm and damaged membranes which can be 
repaired after prolonged rehydration or rehydration in the presence of plant root 
exudates (Pazos-Rojas et al., 2019). On the other hand, Azotobacter 
chroococcum strains, also present in the used microbial preparation, exhibited high 
tolerance to drought stresses and could alleviate the negative effects exerted by 
abiotic stress in tomato plants (Viscardi et al., 2016).  
 
The conclusion of this preliminary trial is that in the following field trials, the 
microbial identification and activity should be measured alongside other parameters 
to confirm the effect of microbial preparations on plant production.  
 
Table 2: Measurements of crop density and hemp plant height on microbially treated and 
untreated field areas (mean ± std err)  
 
 
Plant density 
(1000 
plants/ha) 
Plant 
height (cm) 
ɸ of stem 
(mm) 
ɸ of 
central 
root 
(mm) 
Root length 
(mm) 
CONTROL 550.0 ±23.0 129.0±3.2 6.05±0.14 5.96±0.35 201.10±3.00 
MICROB 463.3±117.2 144.8±3.9 7.15±1.00 7.11±0.57 189.20±5.81 
P-value 0.538 0.037 0.385 0.052 0.166 
 
Table 3: Yield (kg dry matter per hectare) of hemp biomass on microbially treated and 
untreated field areas (mean ± std err) 
 
 Yield of 
inflorescence 
(kg DM/ha) 
Yield of 
seeds (kg 
DM/ha) 
Yield of 
stems (kg 
DM/ha) 
Yield of roots 
(kg DM/ha) 
CONTROL 2000.6±450.4 1765±141.6 3600±236.8 235.0±20.7 
MICROB 1598.7±361.0 1487±70.5 3492±419.8 396.3±49.0 
P-value 0.297 0.179 0.836 0.064 
 
We also sent the samples to the lab for informative cannabinoid testing, although 
the hemp in this case was grown for the seed production and cannabinoids were of 
no commercial interest. This is why we just made a joint sample from all three plots 
in each treatment/group. The percentages of CBD and Δ9-THC in both tested groups 
were identical, 0.82 % and 0.03 % of dried inflorescence mass, respectively. Thus, 
the CBD yield per hectare was 16.40 kg in CONTROL and 13.03 kg in MICROB group. 
Statistical analysis was not performed as we took only one average sample from all 
three plots from each group. The CBD content of our samples was higher if we 
compare it to the results of Burgel et al. (2020), where Futura 75 reached 0.15 % of 
CBD in inflorescence, yielding together 3,9 kg of CBD per hectare. The CBD content 
can be affected by several factors (weather conditions, daily light, fertilisation, time 
of harvest, stress etc.) thus CBD content of Futura 75 variety can vary from 0.14 % 

Hmeljarski bilten / Hop Bulletin 30 (2023) 
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71 
 
to 2.2 % in the time of harvest (Folina et al., 2020; Hillig & Mahlberg, 2004; Pavlovic 
et al., 2019). From other cannabinoids and terpenes measured only 
cannabichromene (CBC) was detected in small percentage (0.04 % in both samples), 
all others were below detection level.  
 
Table 4: The proximate analysis of hemp seeds (g per kg of dry matter (mean ± std err)) 
 
(g/kg DM) CONTROL MICROB P-value 
Crude fibre 275.87±4.73 279.15±3.97 0.446 
Crude ash 58.46±0.97 60.95±0.68 0.112 
Crude fat 302.73±4.74 296.66±2.36 0.336 
Crude protein 265.0.5±3.39 266.37±0.94 0.741 
N-free extract 98.73±2.59 96.88±5.20 0.771 
 
Proximate analysis showed no differences between the nutrient seed composition 
of two test groups (Table 4). The approximate hemp crude fat, protein and fibre 
content was around 30 %, 27 % and 28 %, respectively. The fatty acid profile of the 
seeds also showed a typical Futura 75 profile with no differences between 
microbially treated or untreated group (Table 5). The oil extracted from hemp seeds 
is highly nutritious because it is rich in polyunsaturated fatty acids (mostly linoleic 
acid, α-linolenic acid, oleic acid, and some rare fatty acids like γ-linolenic acid and 
stearidonic acid), some of which are essential and must be acquired from diet 
(Alonso-Esteban et al., 2023; Leizer et al., 2000). There are some studies which 
demonstrate that the use of Pseudomonas spp. inoculants increased seed yield, oil 
yield and/or composition in desirable fatty acids in other oilseed crops including 
sesame, sunflower, flax, soybean, canola, and corn gromwell (Balthazar et al., 2022). 
To our knowledge, this was not yet demonstrated for hemp. Additionally, more 
research is still needed to investigate the largely unknown mechanisms resulting in 
these alterations of seed oil content and composition by microbes.  
 
Table 5: Fatty acid profile of hemp seed (g per 100 g of total fatty acids (mean ± std err)) 
 
FA (g/100 g)* CONTROL MICROB P-value 
SFA  9.46±0.15 9.43±0.07 0,872 
MUFA 10.95±0.23 10.8±0.16 0,637 
PUFA 79.58±0.38 79.75±0.23 0,719 
n6 PUFA 56.63±0.26 56.68±0.16 0,871 
n3 PUFA 20.38±0.42 20.53±0.22 0,777 
n6/n3 ratio 2.78±0.07 2.76±0.03 0,835 
γ linolenic acid 2.56±0.23 2.54±0.04 0,951 
Stearidonic acid 0.96±0.09 0.96±0.002 0,966 
*For assessing normality of the data, the data was transformed by arc sinus square root 
transformation. The data presented in the table is means as gram per 100 grams of fat.  

72 Hmeljarski bilten / Hop Bulletin 30 (2023)
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4 CONCLUSION 
 
Unfortunately, based on preliminary one year experiment we cannot confirm any 
effective influence of used microbial consortia commercial preparation on 
measured parameters except plant height and a trend in changes of some root 
parameters. There are many possible reasons for such outcome, however none of 
them was confirmed. We did not test the viability of microbes in the preparation, 
neither did we have the possibility to measure the microbial populations in the soil 
during the vegetation season. Due to the drought conditions, various microbes in the 
consortia could be differently affected due to their different susceptibility to dry 
conditions. The microbes are not necessarily destroyed by drought, but can become 
temporary less active (depending on the species). However, there is still scientific 
evidence that even in drought stress conditions, plants benefit from microbial 
consortia applications to soil. We can conclude that the effectiveness of various new 
microbial commercial preparations should be regularly evaluated in field trials as 
outdoor field experiments are lacking and these preparations will gain more and 
more popularity in the future. 
 
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