CC BY
15
Original article / Оригинальная статья DOI: https://doi.org/10.21285/2227-2925-2020-10-3-459-469
Impact of cultivation conditions on xylanase production and growth in Paenibacillus mucilaginosus
© Dung T. Ha*, Albert V. Kanarsky*, Zosia A. Kanarskaya*, Andrei V. Shcherbakov**, Elena N. Shcherbakova**, Andrey V. Pranovich***
*Kazan National Research Technological University, Kazan, Russian Federation **All-Russia Research Institute for Agricultural Microbiology, St. Petersburg, Russian Federation
***Abo Academy, Finland
Abstract: Xylanase is an enzyme that hydrolyses в-1,4 bonds in plant xylan. This enzyme is applied in the byconversion of agro-industrial waste for xylooligosaccharide hydrolysate production to improve digestibility and nutrition value of animal feed, food processing, the utilisation and faster decomposition of crop debris in soil, as well as in cellulose bleaching and other industries. The current trend focuses on using renewable resources, such as agricultural waste, as substitutes for expensive purified xylan in producer screening and xylanase synthesis. This work aimed to determine the impact of Paenibacillus mucilaginosus cultivation conditions on the xylanase production yield. Rice bran ferment lysate along with birch and beech timber xylans were used as a carbon source. Temperature, medium pH, pH correction factors, inoculant incubation time, carbon and nitrogen sources and concentrations were the studied criteria of xylanase biosynthesis and growth in bacteria P. ucilaginosus strain 560. We show that the xylanase biosynthesis and cultivation in P. mucilaginosus strain 560 are more practical and cost-effective with the use of a rice bran ferment lysate-based nutrient medium. Inductors contained in the rice bran ferment lysate improve the xylanase biosynthesis. Calcium ions also facilitate biosynthesis in the studied strain. Cultivation recommendations are: carbon source concentration in medium 0.5% of total reducing substances content; 0.2% carbamide as optimal nitrogen source; calcium hydroxide as an agent for medium pH correction to 6.0±0.2; cultivation temperature 30±1 °С. Under the specified conditions, cultivation of P. mucilaginosus does not require inoculate preprocessing, and a maximal xylanase activity in stationary culture reaches 20 U/mL.
Keywords: rice bran, birch, beech, xylan, Paenibacillus mucilaginosus, culturing, xylanase
Information about the article: Received February 20, 2020; accepted for publication August 31, 2020; available online September 30, 2020.
For citation: Kha TZ, Kanarskiy AV, Kanarskaya ZA, Scherbakov AV, Scherbakova EN, Pranovich AV. Impact of cultivation conditions on xylanase production and growth in Paenibacillus mucilaginosus. Izvestiya Vuzov. Prikladnaya Khimiya i Biotekhnologiya = Proceedings of Universities. Applied Chemistry and Biotechnology. 2020;10(3):459-469. (In English) https://doi.org/10.21285/2227-2925-2020-10-3-459-469
УДК 57.013:57.014
Влияние условий культивирования на продуцирование ксиланазы и рост бактерий Paenibacillus mucilaginosus
Т.З. Ха*, А.В. Канарский*, З.А. Канарская*, А.В. Щербаков**, Е.Н. Щербакова**, А.В. Пранович***
*Казанский национальный исследовательский технологический университет (КНИТУ),
г. Казань, Российская Федерация ** ВНИИ сельскохозяйственной микробиологии (ВНИИСХМ), г. Санкт-Петербург, Российская Федерация, *** Або Академия, Финляндия
Резюме: Ксиланаза - фермент, гидролизующий в-1,4-связи в ксиланах. Этот фермент используется для получения из отходов агропромышленного комплекса гидролизата ксилоолигосахаридов с целью улучшения энергетической ценности и повышения усвояемости кормов для животных, обработки пищевых продуктов, утилизации и ускорения разложения поживных остатков в почве, а также в технологии отбелки целлюлозы и других областях. В настоящее время представляет интерес использование возобновляемых ресурсов, в частности, отходов сельскохозяйственного производства, в качестве субстрата вместо дорогого очищенного ксилана для скрининга продуцентов и выделения ксиланаз. Целью настоящей работы являлось определение влияния условий
культивирования бактерий Paenibacillus mucilaginosus на продуцирование ксиланаз. В качестве источника углерода использовали ферментолизат рисовой шелухи, ксилан, выделенный из березы и бука. Изучено влияние температуры, рН среды, факторов корректировки рН среды, продолжительности инкубации инокулята, источ-ников углерода и азота, и также их концентраций на биосинтез ксиланаз и рост штамма 560 P. mucilaginosus. Установлено, что для биосинтеза кисланазы культивирование штамма 560 P. mucilaginosus перспективно и экономически целесообразно проводить на питательной среде, приготовленной на основе ферментолизата рисовой шелухи. Присутствующие в составе ферментолизата рисовой шелухи индукторы улучшают биосинтез кси-ланаз. Показано положительное влияние ионов кальция на биосинтез ксиланаз у рассматриваемого штамма. Рекомендуемые условия культивирования: концентрация источника углерода в питательной среде по общему количеству РВ - 0,5%; в качестве источника азота целесообразно использовать 0,2% карбамид; при корректировке рН среды до 6,0±0,2 необходим гидроксид кальция; температура культивирования бактерий - 30±1 °С. В указанных условиях культивирования P. mucilaginosus не требуется предварительного приготовления посевного материала, а максимальная активность синтезируемой ксиланазы в стационарной фазе роста бактерий достигает значения 20 ед./мл.
Ключевые слова: рисовая шелуха, береза, бук, ксилан, Paenibacillus mucilaginosus, культивирование, ксиланаза
Информация о статье: Дата поступления 20 февраля 2020 г.; дата принятия к печати 31 августа 2020 г.; дата онлайн-размещения 30 сентября 2020 г.
Для цитирования: Ха Т.З., Канарский А.В., Канарская З.А., Щербаков А.В., Щербакова Е.Н., Прано-вич А.В. Влияние условий культивирования на продуцирование ксиланазы и рост бактерий Paenibacillus mucilaginosus. Известия вузов. Прикладная химия и биотехнология. 2020. Т. 10. N 3. С. 459-469. https://doi.org/10.21285/2227-2925-2020-10-3-459-469
INTRODUCTION
The second most abundant renewable natural polysaccharide after cellulose, xylan is a major hemi-cellulose of grain and wood [1]. This complex polysaccharide consists of p-1,4-xylopyranosyl residues chained through p-1,4-glycosidic bonds [2]. Side chains are linked to the backbone with p-(1^3)-glycosidic bonds. Some hydroxyl groups in xylose residues can be acetylated and attached with 4-O-methyl-D-glucuronic acid and L-arabinofuranose [2, 3]. The xylan structure varies depending on the plant tax-on and extraction method [4].
Xylan is abundant in hardwood (15-30%) and conifer timber (7-10%). A high xylan content (about 30%) is found in straw, stems and other parts of annual plants and grasses (cereals, including sorghum, sugar cane cake, stems, cobs and husk of corn) [5]. Hardwood xylan is O-acetyl-4-O-methylglucuro-noxylan. Conifer timber contains arabino-4-O-methyl-glucuronoxylan, which is distinguished from hardwood xylan by the absence of acetyl groups and presence of arabinofuranose branches. Grasses and annual plants usually possess arabinoxylans [6, 7]. Linear unsubstituted xylan was also found in esparto grass [8], tobacco [9] and some sea algae [10] and contains xylopyranosyl residues linked by 1,3-p- and 1,4-p-bonds [10, 11].
Xylans are the main antinutrient components of plant material that hamper nutrient absorption in the gastrointestinal tract of monogastric animals. Xylan is hydrolysed by xylanases (1,4-p-D-xylanases, EC 3.2.1.8) used for xylooligosaccharide (XOS) hy-drolysate production from agricultural waste to improve digestibility and nutritive value of animal feed,
in food processing, in the utilisation and effective decomposition of crop debris in soil, as well as in cellulose bleaching and other industries [12]. Xylanases are the main endoenzymes hydrolysing p-1,4-bonds in xylan, the major hemicellulose polymer [13]. The current trend focuses on the use of renewable resources, such as agricultural waste, as substitutes for expensive purified xylan in producer screening and xylanase synthesis [14]. Fungi are known promising producers of xylanases. However, today's biotechnology opts for bacteria as primary xylanase producers, which are distinguished from mycelial fungi by a higher growth rate and effective production and absorption of carbon from various types of plant matter. Bacteria can also produce xylanase in large volumes and at moderate enzyme purification costs[15].
Paenibacillus bacteria are able to hydrolyse various carbohydrates and produce numerous extracellular enzymes, including xylanase. This xylan-degrading genus includes the species of P. favisporus, P. phyllosphaerae, P. barcinonensis and P. panacisoli [16-19]. Besides the benefits of their enzymatic action, the biomass and other products of Paenibacillus metabolism can supplement feed additives to replenish the diet of farm animals and birds with biologically active substances [20]. Sustainable growth in animal and poultry farming demands for the development of high-quality compound feeds containing enzyme additives. The Russian market is dominated by import fodder enzyme preparations, whereas their domestic production is very limited. The search for new efficient xylanase producer strains in the animal feed industry and the development of scalable fermentation technologies
for the substitution of imported fermented fodder preparations is a pressing issue.
The present work aimed to determine the impact of Paenibacillus mucilaginosus cultivation conditions on the xylanase production yield.
EXPERIMENTAL
Xylanase producer. Strain 560 of bacterium P. mucilaginosus was provided by the Russian Collection of Agricultural Microorganisms (RCAM, All-Russian Research Institute for Agricultural Microbiology, St. Petersburg).
Nutrient growth media. Submerged cultivation of P. mucilaginosus was carried out on the Alexandrov's nutrient medium modified as follows, %: NaCl - 0.02, K2HPO4 - 0.2, MgSC>4.7H2O - 0.05, CaCOa - 0.01, (NH4)2SO4 - 0.1, yeast extract - 0.1 [21].
Rice bran fibre ferment hydrolysate containing 0.5% of the total reducing substances (RS) content was used as a carbon source. The bran was pre-treated with 2.5% sodium hydroxide, with a solid-surface to sodium hydroxide saline ratio of 1:8, treatment temperature was 120±1 °C and treatment time 20 min. The fibre was then rinsed with tap water and exposed for 24 h to the Accellerase 1500 enzyme complex containing exoglucanase, endoglucanase, hemicellulase and beta-glucosidase at 55±1 °C, pH 5.5±0.3. This ferment lysate contained RS available for bacterial growth. Silicon-containing lye segregated from fibre was used as a medium pH correction factor.
Table 1. Experimental design Таблица 1. Планирование эксперимента
Experiment number Substrate concentration, % Temperature, °C pH corrector pH Carbon source Inoculate Incubation time, h Nitrogen source and concentration
1 0.25; 0.5; 0.75; 1 30 Calcium hydroxide 7 Rice bran ferment lysate 24 0.1% (NH4)2SO4 + 0.1% yeast extract
2 Opt. value as in Trial 1 25, 30, 35 Calcium hydroxide 7 Rice bran ferment lysate 24 0.1% (NH4)2SO4 + 0.1% yeast extract
3 Opt. value as in Trial 1 Opt. value as in Trial 2 Calcium hydroxide, sodium hydroxide, Si-containing lye 7 Rice bran ferment lysate 24 0.1% (NH4)2SO4 + 0.1% yeast extract
4 Opt. value as in Trial 1 Opt. value as in Trial 2 Opt. value as in Trial 3 6, 7, 8, 9 Rice bran ferment lysate 24 0.1% (NH4)2SO4 + 0.1% yeast extract
5 Opt. value as in Trial 1 Opt. value as in Trial 2 Opt. value as in Trial 3 Opt. value as in Trial 4 Rice bran ferment lysate, birch xylan, beech xylan 24 0.1% (NH4)2SO4 + 0.1% yeast extract
6 Opt. value as in Trial 1 Opt. value as in Trial 2 Opt. value as in Trial 3 Opt. value as in Trial 4 Opt. value as in Trial 5 0, 12, 24, 36, 48 0.1% (NH4)2SO4 + 0.1% yeast extract
7 Opt. value as in Trial 1 Opt. value as in Trial 2 Opt. value as in Trial 3 Opt. value as in Trial 4 Opt. value as in Trial 5 Opt. value as in Trial 6 Total concentration 0.2%
8 Opt. value as in Trial 1 Opt. value as in Trial 2 Opt. value as in Trial 3 Opt. value as in Trial 4 Opt. value as in Trial 5 Opt. value as in Trial 6 0; 0.02; 0.1; 0.2; 0.3; 0.4% of opt. nitrogen source in Trial 7
Birchwood xylan was also used as a carbon source [22]. Xylan was extracted from birchwood chips (Betula pendula) through oxygen-free steaming at 150-155 °C and 0.60-0.65 MPa excess pressure. Xylan was precipitated from the resulting water extract by intensive vortexing in an ethanol/water solution (85:15) with overnight exposure for complete coagulation. After decantation, xylan precipitate was vacuum sieved with a filter paper (Black ribbon). Eluted xylan was vacuum dried at 40 °C for 48 h.
Beechwood xylan (Cath Roth) was used as a carbon source in comparison assays.
Nutrient medium was autoclaved at 120 °C and 1 atm. Sterile medium was corrected to neutral pH with calcium hydroxide. Cultivation was carried out with 250 mL Erlenmeyer flasks in 100 mL medium stirred continuously at 200 rpm on an ES-20 incubator shaker for 3 days at 30 °C. The flasks were inoculated at 5% relative to medium volume.
Experiments were designed as OFAT (One-Factor-At-a-Time). The OFAT approach was used to study the influence of cultivation conditions (substrate concentration, medium pH, temperature, inoculant incubation time, nitrogen source, nitrogen source concentration, carbon source) on the growth and metabolic yield in P. mucilaginosus bacteria. The method varies one tested factor per trial, while leaving the others constant [23]. The OFAT experimental design is detailed in Table 1.
Trial 1 assessed the xylanase activity and growth parameters of P. mucilaginosus strain 560 under rice bran ferment lysate concentrations varying from 0.25 to 1% total RS. Temperature, pH, inoculate incubation time, nitrogen sources and consumption rate were set constant. The RS concentration in ferment lysate corresponding to the maximal xylanase activity and optimal growth parameters in Trial 1 was fixed downstream in Trial 2, which tested the effect of temperature on bacterial growth and xylanase yield under other fixed parameters (pH, incubation time, nitrogen source and consumption rate). Trials 3-8 are designed likewise by varying one cultivation parameter at a time.
Assessment of growth parameters. Specific growth rate, bacterial biomass generation time and total yield were estimated as recommended in [24]. Biomass was pelleted by centrifugation with a 5418 R Eppendorf microcentrifuge at 12,000 rpm for 10 min. Biomass yield was determined thermogra-vimetrically with an MX-50 moisture analyser.
Assessment of xylanase activity. The xylanase activity and residual RS content of undegraded carbohydrates in supernatant after a 12, 24, 48 and 72-h submerged cultivation of P. mucilaginosus were measured by adding concentrated sulfuric acid in the ratio 1:1. Enzymatic activity was measured as in [25], with certain modifications. Xylanase activity was assessed with a 1% beechwood xylan substrate (1 g of xylan per 100 mL of acetate buffer, pH 6). Enzymatic activity was measured relative to the RS value [26]. The measurement procedure was as follows: 0.12 mL of supernatant with 1.2 mL of substrate were incubated for 1 h at 50 °C followed by the addition of 0.6 mL of 3,5-dinitrosalicylic acid (DNS reagent). In the control, 1.2 mL of substrate was mixed with 0.6 mL of DNS reagent and 0.12 mL of supernatant. Tubes with the substrate, culture medium and DNS reagent were boiled in a water bath for 10 min, cooled down and 6 mL of distilled water was added before optical density measurement at 540 nm. One xylanase activity unit was defined as the amount of enzyme needed to hydrolyse 1 g of substrate (30% of reaction total) to monosugars in 1 h under assumed pH and temperature.
Trials were in the form of biological and analytical assays performed in triplicate, and statistical analyses were performed using the MS Excel 2010 and Prism 7 software.
DISCUSSION
Effect of substrate concentration on growth and xylanase biosynthesis. Cost-efficient and therefore practical sources of carbon are substrates derived from recycled plant matter, such as crop debris and timber stands. Grain husk, straw, bran and wood shavings are typically rich in xylans.
Our trials demonstrate an impact of the substrate concentration, such as rice bran ferment ly-sate, on the growth and secreted xylanase activity in P. mucilaginosus strain 560. Higher substrate con-
centrations corresponded to a higher enzyme activity (Fig. 1, a), lower generation time and increased specific growth rate in bacteria (Table 2). Maximal xylanase activity reached 7.66 U/mL after 24 h of cultivation, with the total rice bran ferment lysate RS value of 0.5%, effective growth conditions and maximal biomass yield of 40%.
Effect of cultivation temperature. In 0.5% ferment lysate medium trials, a cultivation temperature of 30±1 °C facilitated both effective xylanase production and optimal bacterial growth (Fig. 1, b). A higher temperature of 35 °C was associated with a higher specific growth rate and 2-fold reduced generation time compared to culturing at 25 °C, and 1.5-fold reduction compared with 30 °C. However, the biomass yield at 25 or 35 °C diminished compared with culturing at 30 °C (see Table 2).
The effect of medium pH. Xylanase activity and growth in P. mucilaginosus was significantly influenced by the nutrient medium pH (Fig. 2) and pH correction factors. Table 2 shows that noncrystalline silicon-containing lye as a pH corrector facilitates bacterial growth and increases the specific growth rate. However, the use of silicon-containing lye or sodium hydroxide correctors was associated with a lower xylanase activity and reduced biomass yield (Fig. 2, a; see Table 2).
A maximal yield was observed with calcium hydroxide (see Table 2), which is likely explained by the regulatory role calcium ions play in many cellular processes. Calcium is a known stabilising factor in the outer lipopolysaccharide membrane and cellular wall in Gram-negative bacteria [27] and a stimulator of bacterial protein biosynthesis resulting in higher biomass yield and enzyme activity [28]. A maximal xylanase activity of 11 U/mL was observed after a 48 h cultivation of P. mucilaginosus on a calcium hydroxide-corrected medium (see Fig. 2, a), which may be related to the calcium-mediated stabilisation and regulation of the enzyme activity [29-31].
The calcium hydroxide adjustment of the medium's pH from 6 to 9 resulted in the arrest of bacterial growth at pH 9. Adding calcium hydroxide for pH adjustment of 6 to 8 accelerated the specific growth rate and had a 2-fold reduction on generation time (6 h at pH 6 vs. 3 h at pH 8, see Table 2).
This result conforms with another study [32], where the growth of rhizobacteria is shown to be co-affected by pH and calcium ion concentrations. In our trials, a maximal biomass yield of 38% is observed at a neutral medium pH of 7.0 (see Table 2). The maximal xylanase activity reaches 15 U/mL under pH 6.0 following a 48h cultivation on a rice bran ferment lysate medium (Fig. 2, b).
Effect of carbon source. Cultivation trials with various carbon sources demonstrated a peak biomass accumulation (about 60%) with beechwood xylan (see Table 2).
Rice bran ferment lysate was identified as the most effective source of carbon for xylanase produc-
Table 2. Effect of cultivation conditions on growth kinetics in P. mucilaginosus strain 560
Таблица 2. Влияние условий культивирования на кинетические параметры роста штамма 560 бактерий P. mucilaginosus
Parameter Range Specific growth rate, h-1 Generation time, h Biomass yield, %
0.25 0.17±0.05 4.10±0.59 37.68±2.62
Substrate 0.5 0.22±0.03 3.09±0.41 40.11±2.83
concentration, % 0.75 0.26±0.05 2.71±0.25 32.09±2.65
1 0.23±0.05 2.96±0.30 33.13±2.63
25 0.11±0.04 6.45±0.66 18.26±1.52
Temperature, °C 30 0.15±0.03 4.54±0.48 38.48±2.72
35 0.24±0.03 2.86±0.14 26.55±3.02
Calcium hydroxide 0.21±0.02 3.29±0.27 29.23±2.69
pH correction factor Sodium hydroxide 0.31±0.04 2.25±0.27 18.92±1.45
Si-containing lye 0.45±0.04 1.54±0.12 17.20±1.65
6 0.11±0.01 6.89±0.57 19.24±2.02
pH 7 0.15±0.03 4.54±0.18 38.48±2.67
8 0.20±0.05 3.52±0.10 19.91±1.82
Birchwood xylan 0.16±0.02 4.24±0.55 44.57±2.85
Carbon source Beechwood xylan Rice bran ferment lysate 0.20±0.03 0.21±0.02 3.48±0.25 3.29±0.27 59.43±3.02 39.23±2.12
0 0.16±0.03 4.36±0.12 43.30±2.63
Inoculate incubation time, h 12 24 0.15±0.03 0.09±0.01 4.57±0.15 7.47±0.81 34.43±2.70 21.38±2.22
36 0.10±0.01 6.77±0.70 33.75±2.68
48 0.13±0.02 5.55±0.71 56.16±3.08
No nitrogen 0.12±0.01 5.84±0.61 16.68±1.82
NH4NO3 0.19±0.02 3.72±0.22 22.40±2.12
(NH4)2SO4 0.19±0.02 3.63±0.20 25.57±2.07
Yeast extract 0.11±0.01 6.68±0.25 33.16±2.82
Nitrogen source (NH4)2SO4 + yeast extract 0.15±0.01 4.67±0.53 37.10±2.68
Corn extract 0.06±0.01 12.17±0.74 49.31±2.64
Pepton 0.15±0.01 4.68±0.46 29.87±2.12
Carbamide 0.17±0.03 4.08±0.46 15.29±1.92
Betafin 0.23±0.05 3.00±0.47 11.39±1.22
0.02 0.21±0.04 3.25±0.36 29.18±2.27
Carbamide content, % 0.1 0.2 0.16±0.01 0.15±0.01 4.33±0.45 4.51±0.35 18.88±1.62 15.21±1.65
0.3 0.08±0.01 8.84±0.70 10.44±1.60
Е
3 >
■>
О ..
го 5 ш
«я л
го 4 с
> 3
0,25 0,5 0,75 1
Substrate concentration, %
Е
3 >
ti
it tc a e s a
n
>
25 30
Cultivation temperature,
b
35
°C
12 24 48 72
Fig. 1. Effect of rice bran ferment lysate carbohydrate concentration (a) and cultivation temperature (b) on xylanase activity (U/mL) in P. mucilaginosus strain 560
Рис. 1. Влияние концентрации углеводов в ферментолизате рисовой шелухи (а) и температуры культивирования (b) на ксиланазную активность (ед./мл) штамма 560 P. mucilaginosus
9
2
1
0
a
Е
3 >
и та ш
(Л
та с
го >
X
Calcium hydroxide
Sodium hydroxide
Si-containing lye
16 14
3 12
to ro
I
ro с
§ i
10 8
2 5 6
ci
d 5 4
о
* 2
m
s
£
<
0
I .
Il
12 24 48 72
6 7 8 9
рН
Е
3 >
c a e
1Л
a n
al ly
X
a b
Fig. 2. Effect of pH correction factors (a) and acidity (b) on xylanase activity (U/mL) in P. mucilaginosus strain 560
Рис. 2. Влияние факторов корректировки рН среды (а) и кислотности среды (b) на ксиланазную активность (ед./мл) штамма 560 бактерий P. mucilaginosus
14
Е 12 3 >10 ti
ict 8
a
3 6
a n al
yl 4
X
2 0
12 24 48 72
Birchwood xylan
Beechwood xylan
Rice bran ferment lysate
0 12 24 36 48 Inoculate incubation time, h
b
Fig. 3. Effect of xylan source (a) and incubation time (b) on xylanase activity (U/mL) in P. mucilaginosus strain 560
Рис. 3. Влияние источника ксилана (а) и продолжительности инкубации инокулята (b) на ксиланазную активность (ед./мл) штамма 560 бактерий P. mucilaginosus
tionin bacteria P. mucilaginosus strain 560 (Fig. 3, a). This observation may relate to the presence of specific inductors produced in the partial hydrolysis of ferment lysate, which boost the xylanase biosynthesis as reported by [33] in experiments examining fungal xylanase production in Aspergillus and Trichoderma. As shown in [34], specific inductor substances play a key role in xylanase biosynthesis regulation. Among those are xylose, xylobiose, xylooligo-saccharides, xylose heterodisaccharides and their positional isomers formed during the enzymatic hydrolysis of rice bran fibre. Inoculate incubation time under the trial conditions was found to have an insignificant effect on bacterial growth and xylanase production (Fig. 3, b; see Table 2). Accordingly, the inoculum pre-treatment is not required for experimental assays.
Effect of nitrogen source and concentration. Trials with various nitrogen sources demonstrated a maximal xylanase activity after a 48 h cultivation with carbamide supplemented medium (Fig. 4, a). A carbamide content of 0.2% increased the xylanase activity by 2-4 times compared to the control (no nitrogen) (Fig. 4, b). Under optimal cultivation conditions, the maximum xylanase activity in the stationary phase reached 20 U/mL, giving a 2-fold increase compared to P. campinasensis BL11 cultured under optimal conditions on a lye-treated rice bran-straw lysate medium (xylanase 10.5 U/mL) [35].
A combined application of ammonium sulphate and yeast extract in the medium increased the P. mucilaginosus biomass yield (see Table 2).
a
Е 3
,yti
>
it tc a e s a n
.2
>
х
20 18 16 14 12 10 8 6 4 2 0
1 - без азота 2- №ШОз
3 - (NN4)2804
4 - карбамид
5 - дрожж.экстракт
6 - №)2304 + дрожж. экстракт
7 - кукурузный экстракт
8 - пептон
9 - бетафин
20
Е 3
it tc a e
¡§10 n
.2
>
х
¿1
J
12 24 48 72
1 2 3 4 5 6 7 Источник азота
89
без азота
0.02 0.1 0.2 0.3 0.4 Концентрация карбамида, %
b
Рис.
Fig. 4. Effect of nitrogen source (a) and concentration (b) in medium on xylanase activity (U/mL)
in P. mucilaginosus strain 560
4. Влияние источника азота на ксиланазную активность (ед./мл) штамма 560 бактерий P. mucilaginosus: а - источник азота; b - концентрация источника азота в питательной среде
CONCLUSIONS
We identify the rice bran ferment lysate-based nutrient medium as optimal for the xylanase production in P. mucilaginosus strain 560. Calcium supplementation positively affects bacterial growth and xylanase biosynthesis. Recommended cultivation conditions are: carbon source concentration in the
medium of 0.5% of total RS content; 0.2% carbamide as the optimal nitrogen source; calcium hydroxide as medium pH corrector to 6.0±0.2; cultivation temperature 30±1 °C. These conditions do not require inoculate pre-treatment of P. mucilaginosus strain 560, and a maximal xylanase activity reaches 20 U/mL in stationary culture.
REFERENCES
1. Collins T, Gerday C, Feller G. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiology Reviews. 2005;29(1):3-23. https://doi.org/10.1016/j.femsre.2004.06.005
2. Sedlmeyer FB. Xylan as a by-product of bio-refineries: characteristics and potential use for food applications. Food Hydrocolloids. 2014;25(8):1891-1898. https://doi.org/10.1016ZJ.F00DHYD.2011.04. 005
3. Shanthi V, Roymon MG. Isolation and screening of alkaline thermostable xylanase producing bacteria from soil in Bhilai Durg region of Chhattis-garh, India. International Journal of Current Microbiology and Applied Sciences. 2014;3(8):303-311.
4. Habibi Y, Vignon MR. Isolation and character-rization of xylans from seed pericarp of Argania spi-nosa fruit. Carbohydrate Research. 2005;340(7):14 31-1436. https://doi.org/10.1016/j.carres.2005.01. 039
5. Singh S, Madlala AM, Prior BA. Thermomy-ces lanuginosus: properties of strains and their he-micellulases. FEMS Microbiology Reviews. 2003;27 (1):3-16. https://doi.org/10.1016/S0168-6445(03) 00018-4
6. Kulkarni N, Shendye A, Rao M. Molecular and biotechnological aspects of xylanases. FEMS Mi-
crobiology reviews. 1999;23(4):411-456. https://doi. org/10.1111/j.1574-6976.1999.tb00407.x
7. Fengel D, Wegener G. Wood: Chemistry. Ultrastructure. Reactions. Berlin. Walter de Gruyter, 1984 (Russ ed.: Fengel D, Vegener G. Drevesina. Khimiya. Ul'trastruktura. Reaktsii. Moscow: Lesnaya promyshlennost': 1988. 511 p.)
8. Chanda SK, Hirst EL, Jones JKN, Percival EGV. 262. The constitution of xylan from esparto grass (Stipa tenacissima, L.). Journal of the Chemical Society (Resumed). 1950:1289-1297. https:// doi.org/10.1039/JR9500001289
9. Eda S, Ohnishi A, Kato K. Xylan isolated from the stalk of Nicotiana tabacum. Agricultural and Biological Chemistry. 1976;40(2):359-364.
10. Barry VC, Dillon T. Occurrence of xylans in marine algae. Nature. 1940;146(3706):620-620. https://doi.org/10.1038/146620a0
11. Percival EGV, Chanda SK. The xylan of Rhodymenia palmate. Nature. 1950;166(4227):787-788. https://doi.org/10.1038/166787b0
12. Kaur A, Singh A, Patra AK, Mahajan R. Cost-effective scouring of flax fibers using cellulase-free xylano-pectinolytic synergism from a bacterial isolate. Journal of Cleaner Production. 2016;131:107-111. https://doi.org/10.1016/jjclepro.2016.05.069
5
0
a
13. Akin DE. Plant cell wall aromatics: influence on degradation of biomass. Biofuels, Bioproducts and Biorefining. 2008;2:288-303. https://doi.org/10. 1002/bbb.76
14. Kaur A, Mahajan R, Singh A, Garg G, Shar-ma J. A novel and cost effective methodology for qualitative screening of alkalothermophilic cellulase free xylano-pectinolytic microorganisms using agricultural wastes. World Journal of Microbiology and Biotechnology. 2011;27:459-463. https://doi.org/10. 1007/s11274-010-0457-9
15. Subramaniyan S, Prema P. Biotechnology of microbial xylanases: Enzymology, molecular biology and application. Critical Reviews in Biotechnology. 2002;22(1):33-64. https://doi.org/10.1080/0738855 0290789450
16. Velázquez E, de Miguel T, Poza M, Rivas R, Rosselló-Mora R, Villa TG. Paenibacillus favisporus sp. nov., a xylanolytic bacterium isolated from cow faeces. International Journal of Systematic and Evolutionary Microbiology. 2004;54(1):59-64. https//doi. org/10.1099/ijs.0.02709-0
17. Rivas R, Mateos PF, Martínez-Molina E, Velázquez E. Paenibacillus phyllosphaerae sp. nov., a xylanolytic bacterium isolated from the phyllo-sphere of Phoenix dactylifera. International Journal of Systematic and Evolutionary Microbiology. 2005; 55(2):743-746. https://doi.org/10.1099/ijs.0.63323-0
18. Sánchez MM, Fritze D, Blanco A, Spröer C, Tindall BJ, Schumann P, et al. Paenibacillus barci-nonensis sp. nov., a xylanase-producing bacterium isolated from a rice field in the Ebro River delta. International Journal of Systematic and Evolutionary Microbiology. 2005;55(2):935-939. https://doi.org/ 10.1099/ijs.0.63383-0
19. Ten LN, Baek SH, Im WT, Lee M, Oh HW, Lee ST. Paenibacillus panacisoli sp. nov., a xylanolytic bacterium isolated from soil in a ginseng field in South Korea. International Journal of Systematic and Evolutionary Microbiology. 2006;56(11):2677-2681. http://dx.doi.org/10.1099/ijs.0.64405-0
20. Rafikova GF, Loginova EV, Melentev AI, Loginov ON. Biological basis of microbial fodder additive. Patent RF, no. 2 662 931, 2018. (In Russian)
21. Li X, Yang SH, Yu XC, Jin ZX, Li WD, Li L, et al. Construction of transgenic Bacillus mucilaginosus strain with improved phytase secretion. Journal of Applied Microbiology. 2005;99(4):878-884. https://doi.org/10.1111/j.1365-2672.2005.02683.x
22. Von Schoultz S, AB BLN-WOODS LTD. Method for extracting biomass. U.S. Patent Application. 14/413,409. 2015. https://doi.org/10.1080/000 31305.1999.10474445
23. Czitrom V. One-factor-at-a-time versus designed experiments. The American Statistician. 1999;53(2):126-131.
24. Maier RM. Environmental microbiology (second edition). Chapter 3. Bacterial growth. Elsevier
Inc. 2009. Р. 37-54. https://doi.org/10.1016/B978-0-12-370519-8.00003-1
25. Bailey M, Biely P, Poutanen K. Interlaborato-ry testing of methods for assay of xylanase activity. Journal of Biotechnology. 1992;23(3):257-270. https://doi.org/10.1016/0168-1656(92)90074-J
26. Morozova YuA., Skvortsov EV, Alimova FK, Kanarsky AV. Biosynthesis of xylanases and cellu-lases by fungi Trichoderma on the post-alcohol bard. Vestnik Kazanskogo tekhnologicheskogo uni-versiteta = Bulletin of Kazan Technological University. 2012;15(19):120-122. (In Russian)
27. Dias FE, Okrend H, Dondero NC. Growth-promoting activity of spent sulfite liquor for Sphaero-tilus natans growing in a continuous-flow apparatus. Applied Microbiology. 1968;16(2):276-278. https: //doi.org/10.1128/aem.16.2.276-278.1968
28. Patrauchan MA, Sarkisova S, Sauer K, Franklin MJ. Calcium influences cellular and extracellular product formation during biofilm-associated growth of a marine Pseudoalteromonas sp. Microbiology. 2005;151 (9):2885-2897. https://doi.org/10. 1099/mic.0.28041-0
29. Lamed R, Setiter E, Bayer EA. Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum. Journal of Bacteriology. 1983;156(2):828-836. https://doi.org/ 10.1128/JB.156.2.828-836.1983
30. Grepinet O, Chebrou MC, Beguin P. Nucleotide sequence and deletion analysis of the xylanase gene (xynZ) of Clostridium thermocellum. Journal of Bacteriology. 1988;170(10):4582-4588. https://doi. org/10.1128/jb.170.10.4582-4588.1988
31. Ratanakhanokchai K, Kyu KL, Tanticharoen M. Purification and properties of a xylan-binding endoxylanase from alkaliphilic Bacillus sp. strain K-1. Applied and Environmental Microbiology. 1999; 65(2):694-697. https://doi.org/10.1128/AEM.65.2.69 4-697.1999
32. Howieson JG, Robson AD, Abbott LK. Calcium modifies pH effects on acid-tolerant and acid-sensitive strains of Rhizobium meliloti. Australian Journal of Agricultural Research. 1992;43(3):765-772. https://doi.org/10.1071/AR9920765
33. Haltrich D, Nidetzky B, Kulbe KD, Steiner W, Zupancic S. Production of fungal xylanases. Biore-source Technology. 1996;58(2):137-161. https://doi. org/10.1016/S0960-8524(96)00094-6
34. Kulkarni N, Shendye A, Rao M. Molecular and biotechnological aspects of xylanases. FEMS Microbiology Reviews. 1999;23(4):411-456. https: //doi.org/10.1111/j.1574-6976.1999.tb00407.x
35. Ko C-H, Lin Z-P, Tu J, Tsai C-H, Liu C-C, Chen H-T, et al. Xylanase production by Paenibacillus campinasensis BL11 and its pretreatment of hardwood kraft pulp bleaching. International Bio-deterioration & Biodegradation. 2010;64(1):13-19. https://doi.org/10.1016/j.ibiod.2009.10.001
1. Collins T., Gerday C., Feller G. Xylanases, xylanase families and extremophilic xylanases // FEMS Microbiology Reviews. 2005. Vol. 29. Issue 1. P. 3-23. https://doi.org/10.1016/j.femsre.2004.06. 005
2. Sedlmeyer F.B. Xylan as a by-product of biorefineries: characteristics and potential use for food applications // Food Hydrocolloids. 2014. Vol. 25. Issue 8. P. 1891-1898. https://doi.org/10.1016/J. F00DHYD.2011.04.005
3. Shanthi V., Roymon M.G. Isolation and screening of alkaline thermostable xylanase producing bacteria from soil in Bhilai Durg region of Chhattisgarh, India // International Journal of Current Microbiology and Applied Sciences. 2014. Vol. 3. Issue 8. P. 303-311.
4. Habibi Y., Vignon M.R. Isolation and characterization of xylans from seed pericarp of Argania spinosa fruit // Carbohydrate Research. 2005. Vol. 340. Issue 7. P. 1431-1436. https://doi.org/10.1016/ j.carres.2005.01.039
5. Singh S., Madlala A.M., Prior B.A. Thermo-myces lanuginosus: properties of strains and their hemicellulases // FEMS Microbiology Reviews. 2003. Vol. 27. Issue 1. Р. 3-16. https://doi.org/10. 1016/S0168-6445(03)00018-4
6. Kulkarni N., Shendye A., Rao M. Molecular and biotechnological aspects of xylanases // FEMS Microbiology reviews. 1999. Vol. 23. Issue 4. Р. 411-456. https://doi.org/10.1111/j.1574-6976.1999. tb00407.x
7. Фенгел Д., Вегенер Г. Древесина. Химия. Ультраструктура. Реакции / пер. с англ. А.В. Оболенской, З.П. Ельницкой; под ред. А.А. Лео-новича. М.: Лесная промышленность, 1988. 511 с.
8. Chanda S.K., Hirst E.L., Jones J.K.N., Perci-val E.G.V. 262. The constitution of xylan from esparto grass (Stipa tenacissima, L.) // Journal of the Chemical Society (Resumed). 1950. Р. 1289-1297. https://doi.org/10.1039/JR9500001289
9. Eda S., Ohnishi A., Kato K. Xylan isolated from the stalk of Nicotiana tabacum // Agricultural and Biological Chemistry. 1976. Vol. 40. Issue 2. Р. 359-364.
10. Barry V.C., Dillon T. Occurrence of xylans in marine algae // Nature. 1940. Vol. 146. Issue 3706. Р. 620-620. https://doi.org/10.1038/146620a0
11. Percival E.G.V., Chanda S.K. The xylan of Rhodymenia palmate // Nature. 1950. Vol. 166. Issue 4227. Р. 787-788. https://doi.org/10.1038/1667 87b0
12. Kaur A., Singh A., Patra A.K., Mahajan R. Cost-effective scouring of flax fibers using cellulase-free xylano-pectinolytic synergism from a bacterial isolate // Journal of Cleaner Production. 2016. Vol. 131. Р. 107-111. https://doi.org/10.1016/jjclepro.20 16.05.069
13. Akin D.E. Plant cell wall aromatics: influence
:СКИЙ СПИСОК
on degradation of biomass // Biofuels, Bioproducts and Biorefining. 2ÜÜ8. Vol. 2. Р. 288-3Ü3. https: //doi.org/1Ü.1ÜÜ2/bbb.76
14. Kaur A., Mahajan R., Singh A., Garg G., Sharma J. A novel and cost effective methodology for qualitative screening of alkalothermophilic cellu-lase free xylano-pectinolytic microorganisms using agricultural wastes // World Journal of Microbiology and Biotechnology. 2Ü11. Vol. 27. Р. 459-463. https://doi.org/1 Ü.1ÜÜ7/s11274-Ü1Ü-Ü457-9
15. Subramaniyan S., Prema P. Biotechnology of microbial xylanases: Enzymology, molecular biology and application // Critical Reviews in Biotechnology. 2ÜÜ2. Vol. 22. Issue 1. Р. 33-64. https://doi. org/1Ü.1Ü8Ü/Ü738855Ü29Ü78945Ü
16. Velázquez E., de Miguel T., Poza M., Rivas R., Rosselló-Mora R., Villa T.G. Paenibacillus favisporus sp. nov., a xylanolytic bacterium isolated from cow faeces // International Journal of Systematic and Evolutionary Microbiology. 2ÜÜ4. Vol. 54. Issue 1. Р. 59-64. https//doi.org/1Ü.1Ü99/ijs.Ü.Ü27 Ü9-Ü
17. Rivas R., Mateos P.F., Martínez-Molina E., Velázquez E. Paenibacillus phyllosphaerae sp. nov., a xylanolytic bacterium isolated from the phyllo-sphere of Phoenix dactylifera // International Journal of Systematic and Evolutionary Microbiology. 2ÜÜ5. Vol. 55. Issue 2. Р. 743-746. https://doi.org/1Ü.1Ü 99/ijs.Ü.63323-Ü
18. Sánchez M.M., Fritze D., Blanco A., Spröer C., Tindall B.J., Schumann P., et al. Paenibacillus barcinonensis sp. nov., a xylanase-producing bacterium isolated from a rice field in the Ebro River delta // International Journal of Systematic and Evolutionary Microbiology. 2ÜÜ5. Vol. 55. Issue 2. Р. 935939. https://doi.org/1Ü.1Ü99/ijs.Ü.63383-Ü
19. Ten L.N., Baek S.H., Im W.T., Lee M., Oh H.W., Lee S.T. Paenibacillus panacisoli sp. nov., a xylanolytic bacterium isolated from soil in a ginseng field in South Korea // International Journal of Systematic and Evolutionary Microbiology. 2ÜÜ6. Vol. 56. Issue 11. Р. 2677-2681. http://doi.org/1Ü.1Ü99/ ijs.Ü.644Ü5-Ü
20. Пат. № 2662931, Российская Федерация, C12N 1/2Ü, C12N 9I00. Биологическая основа микробной кормовой добавки I Г.Ф. Рафикова, Логинова Е.В., Мелентьева А.И., Логинов О.Н.; заявл.22.02.2017; опубл. 31.07.2018.
21. Li X., Yang S.H., Yu X.C., Jin Z.X., Li W.D., Li L., et al. Construction of transgenic Bacillus muci-laginosus strain with improved phytase secretion // Journal of Applied Microbiology. 2ÜÜ5. Vol. 99. Issue 4. Р. 878-884. https://doi.org/1Ü.1111/j.1365-2672.2ÜÜ5.Ü2683.x
22. Von Schoultz S., AB BLN-WOODS LTD. Method for extracting biomass. U.S. Patent Application. 14/413,4Ü9. 2Ü15. https://doi.org/1Ü.1Ü8Ü/ÜÜÜ3 13Ü5.1999.1 Ü474445
23. Czitrom V. One-factor-at-a-time versus de-
signed experiments // The American Statistician. 1999. Vol. 53. Issue 2. Р. 126-131.
24. Maier R.M. Environmental microbiology (second edition). Chapter 3. Bacterial Growth. Elsevier Inc. 2009. Р. 37-54. https://doi.org/10.1016/B97 8-0-12-370519-8.00003-1
25. Bailey M., Biely P., Poutanen K. Interlabora-tory testing of methods for assay of xylanase activity // Journal of Biotechnology. 1992. Vol. 23. Issue 3. Р. 257-270. https://doi.org/10.1016/0168-1656(92) 90074-J
26. Морозова Ю.А., Скворцов Е.В., Алимова Ф.К., Канарский А.В. Биосинтез ксиланаз и целлю-лаз грибами рода Trichoderma на послеспиртовой барде // Вестник Казанского технологического университета. 2012. Vol. 15. N 19. Р. 120- 122.
27. Dias F.E, Okrend H., Dondero N.C. Growth-promoting activity of spent sulfite liquor for Sphaero-tilus natans growing in a continuous-flow apparatus // Applied Microbiology. 1968. Vol. 16. Issue 2. Р. 276-278. https://doi.org/10.1128/aem.16.2.276-278. 1968
28. Patrauchan M.A., Sarkisova S., Sauer K., Franklin M.J. Calcium influences cellular and extracellular product formation during biofilm-associated growth of a marine Pseudoalteromonas sp. // Microbiology. 2005. Vol. 151. Issue 9. Р. 2885-2897. https://doi.org/10.1099/mic.0.28041 -0
29. Lamed R., Setiter E., Bayer E.A. Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum // Journal of Bacteriology. 1983. Vol. 156. Issue 2. P. 828-836. https://doi.org/10.1128/JB.156.2.828-836.1983
30. Grepinet O., Chebrou M.C., Beguin P. Nu-
Contribution
Dung T. Ha, Albert V. Kanarsky, Zosia A. Kanarskaya, Andrei V. Shcherbakov, Elena N. Shcherbakova, Andrey V. Pranovich carried out the experimental work, on the basis of the results summarized the material and wrote the manuscript. All authors have equal author's rights and bear equal responsibility for plagiarism.
Conflict interests
The authors declare no conflict of interests regarding the publication of this article.
The final manuscript has been read and approved by all the co-authors.
INFORMATION ABOUT THE AUTHORS
Dung T. Ha,
Postgraduate Student, Department of Food Biotechnology, Kazan National Research Technological University,
cleotide sequence and deletion analysis of the xylanase gene (xynZ) of Clostridium thermocellum // Journal of Bacteriology. 1988. Vol. 170. Issue 10. Р. 4582-4588. https://doi.org/10.1128/jb.170.10.4582-4588.1988
31. Ratanakhanokchai K., Kyu K.L., Tan-ticharoen M. Purification and properties of a xylan-binding endoxylanase from alkaliphilic Bacillus sp. strain K-1 // Applied and Environmental Microbiology. 1999. Vol. 65. Issue 2. Р. 694-697. https://doi. org/10.1128/AEM.65.2.694-697.1999
32. Howieson J.G., Robson A.D., Abbott L.K. Calcium modifies pH effects on acid-tolerant and acidsensitive strains of Rhizobium meliloti // Australian Journal of Agricultural Research. 1992. Vol. 43. Issue 3. Р. 765-772. https://doi.org/10.1071/AR99 20765
33. Haltrich D., Nidetzky B., Kulbe K.D., Steiner W., Zupancic S. Production of fungal xylanases // Bioresource Technology. 1996. Vol. 58. Issue 2. Р. 137-161. https://doi.org/10.1016/S0960-8524(96) 00094-6
34. Kulkarni N., Shendye A., Rao M. Molecular and biotechnological aspects of xylanases // FEMS Microbiology Reviews. 1999. Vol. 23. Issue 4. Р. 411-456. https://doi.org/10.1111/j.1574-6976.1999. tb00407.x
35. Ko C.-H., Lin Z.-P., Tu J., Tsai C.-H., Liu C.-C., Chen H.-T., et al. Xylanase production by Paenibacillus campinasensis BL11 and its pretreatment of hardwood kraft pulp bleaching // International Biodeteriora-tion & Biodegradation. 2010. Vol. 64. Issue 1. Р. 1319. https://doi.org/10.1016/j.ibiod.2009.10.001
Критерии авторства
Ха Т.З., Канарский А.В., Канарская З.А., Щербаков А.В., Щербакова Е.Н., Пранович А.В. выполнили экспериментальную работу, на основании полученных результатов провели обобщение и написали рукопись. Авторы имеют на статью равные авторские права и несут равную ответственность за плагиат.
Конфликт интересов
Авторы заявляют об отсутствии конфликта интересов.
Все авторы прочитали и одобрили окончательный вариант рукописи.
СВЕДЕНИЯ ОБ АВТОРАХ
Ха Тхи Зунг,
аспирант кафедры пищевой биотехнологии, Казанский национальный исследовательский технологический университет, 420015, г. Казань, ул. Толстова, 8,
8, Tolstov St., Kazan, 420015, Russian Federation, e-mail: coldwind.91@mail.ru
Albert V. Kanarsky,
Dr. Sci. (Engineering), Professor, Department of Food Biotechnology, Kazan National Research Technological University,
8, Tolstov St., Kazan, 420015, Russian Federation, S e-mail: alb46@mail.ru
Zosia A. Kanarskaya,
Cand. Sci. (Engineering), Associate Professor Department of Food Biotechnology, Kazan National Research Technological University,
8, Tolstov St., Kazan, 420015,
Russian Federation,
e-mail: zosya_kanarskaya@mail.ru
Andrei V. Shcherbakov,
Cand. Sci. (Biology), Researcher, Laboratory of Microbial Technology, All-Russia Research Institute for Agricultural Microbiology,
3, Podbelsky Highway, St. Petersburg, 196608,
Russian Federation,
e-mail: microbe-club@inbox.ru
Elena N. Shcherbakova,
Cand. Sci. (Agriculture), Researcher, Laboratory of Microbial Technology, All-Russia Research Institute for Agricultural Microbiology,
3, Podbelsky Highway, St. Petersburg, 196608, e-mail: alonagonchar@mail.ru
Andrey V. Pranovich,
Cand. Sci. (Chemistry), Associate Professor, Department of Wood Chemistry, Laboratory of Wood and Paper Chemistry, Abo Academy,
3, Tuomiokirkontori, Turku/Abo, FI-20500, Finland,
e-mail: apranovi@abo.fi
Российская Федерация, e-mail: coldwind.91@mail.ru
Канарский Альберт Владимирович,
д.т.н., профессор кафедры пищевой биотехнологии,
Казанский национальный исследовательский технологический университет, 420015, г. Казань, ул. Толстова, 8, Российская Федерация, И e-mail: alb46@mail.ru
Канарская Зося Альбертовна,
к.т.н., доцент кафедры пищевой биотехнологии,
Казанский национальный исследовательский технологический университет, 420015, г. Казань, ул. Толстова, 8, e-mail: zosya_kanarskaya@mail.ru
Щербаков Андрей Владимирович,
к.б.н., научный сотрудник лаборатории технологии микробных препаратов, Всероссийский НИИ сельскохозяйственной метеорологии,
196608, С.-Петербург, шоссе Подбельского, 3, Российская Федерация, e-mail: microbe-club@inbox.ru
Щербакова Елена Николаевна,
к.с-х.н., младший научный сотрудник лаборатории технологии микробных препаратов,
Всероссийский НИИ сельскохозяйственной метеорологии,
196608, С.-Петербург, шоссе Подбельского, 3, Российская Федерация, e-mail: alonagonchar@mail.ru
Пранович Андрей Викторович,
к.х.н., доцент кафедры химии древесины и бумаги, Або Академия,
Туомиокирконтори 3, Турку/Або, FI-20500,
Финляндия,
e-mail: apranovi@abo.fi
