The Future of Research in
Russia: biosecurity projects
G. Yu. Laptev
PhD in Biology
ООО Biotrof
20 November 2012
T-RFLP analysis
Advantages:
1.
Convenient for study of livestock GIT microbial
community structure.
2.
Discovery of new GIT microorganisms and
identification of their role in the digestion of
feed
3.
Possible to follow the age, diet and disease
related changes in the bacterial population
4.
Detection of pathogenic microorganisms
Correlation between bacterial content in bull’s
rumen and ruminal digestion
Terminal restriction
fragment length, bp
рН
Volatile fatty
acids
Ammonia
Methanogenic archaea
Indicators
рН
Volatile
fatty
acids
Ammoni
a
Family Clostridiaceae
n.d.
-0.60
n.d.
Family Eubacteriaceae
n.d.
0.73
n.d..
Family Lachnospiraceae
n.d.
n.d.
-0.65
Family Alicyclobacillaceae
0.63
n.d.
-0.76
Family Lactobacillaceae
n.d.
0.90
0.63
Phylum Fusobacteria
n.d.
n.d.
-0.83
Bacteia
Indicators
68.83
-0.87
-0.99
0.99
74.11
-0.78
-0.97
0.98
91.95
-0.88
-0.99
0.99
102.69
-0.98
-0.96
0.96
248.26
0.84
0.99
-0.99
251.67
0.93
0.99
-0.99
292.09
0.84
0.99
-0.99
509.65
0.74
0.95
-0.96
Fungi
52.11
0.97
0.79
-0.77
53.81
0.98
0.82
-0.81
Nonculturable bacteria 87.34
0.34
-0.63
-0.72
56.17
0.98
0.8
-0.77
Nonculturable bacteria 90.98
0.58
n.d.
-0.55
73.68
0.97
0.8
-0.77
Nonculturable bacteria 199.4
-0.60
0.52
0.51
152.07
-0.99
-0.91
0.9
155.39
0.77
-0.8
n.d.
157.73
0.77
-0.8
n.d.
175.56
0.77
-0.8
n.d.
219.58
-0.91
-0.66
0.64
“Probiotic Cleaners”

Funded by Russian Ministry of Education
and Science, government contract
#16.512.11.2029 of 11 February 2011
Содержание аммиака в воздухе на
свинофермах (мг/м3).
Treatment with Agrotrof
Control
Experiment
Before treatment
9.04± 0.42
8.89± 0.36
1st treatment (7 days)
10.01± 0.36
5.92± 0.16
2nd treatment (14 days)
9.84± 0.44
4.6± 0.14
3rd treatment (21 days)
9.98± 0.39
4.7± 0.14
4th treatment (28 days)
10.6± 0.37
5.3± 0.19
5th treatment (42 days)
10.91± 0.51
6.0± 0.24
6th treatment (56 days)
10.82± 0.48
6.4± 0.24
Biological Product
Физико-химические свойства навозных
стоков до и после внесения биопрепарата.
before
Indicators
after
use of bioproduct
Suspended material, mg/L
80120
14608
Dry residue, mg/L
86600
21900
COD in mixed sample, mg/L
159744
63488
рН
6,74
7,90
Total nitrogen, mg/L
4405
3640
Ammonia nitrogen, mg/L
2375
3105
Total phosphorus in mixed sample
(Р2О5), mg/L
5077
911
Total phosphorus in filtered
sample (Р2О5), mg/L
35
59
Total potassium in mixed sample
(К2О), mg/L
3205
1956
Microbiological specifications of manure
effluents, before and after adding the biological
agent
Бактерии
Аммонифицирующие
Уробактерии
Численность микроорганизмов
до внесения препарата после внесения препарата
клеток/мл
7
7
2,5·10
2,5·10
5
4
2,0·10
2,0·10
Эффективность применения биопрепарата
для утилизации стоков
Indicators
Experiment
Control
Number of cattle heads at the
beginning of experiment
1800
2866
Average weight per head (kg) at the
end of experiment
101.2
102.7
Death loss
127
284
Death loss (%)
7.0
10.0
Average daily gain, g
670
616
Concentration of ammonia in the air at Noviy Svet pig
complex in Gatchina District (mg/m3). MAC – 20.0
mg/m3.
Experiment scenarios
Treatment
Distance above the
floor
1.5 m
No treatment
1.0 m
1.5 m
1.0 m
Ventilation is ON. Measurements for three replications.
1
12.8±3.2
13.6±3.4
15.8±4.0
16.2±4.1
2
12.6±3.2
13.8±3.5
15.6±3.9
16.2±4.1
3
12.6±3.2
13.6±3.4
15.8±4.0
16.4±4.1
Ventilation is OFF. Measurements for three replications.
1
22.2±5.6
21.4±5.4
28.1±7.0
28.4±7.0
2
22.6±5.7
21.8±5.5
28.4±7.1
28.6±7.2
3
22.2±5.7
21.8±5.5
28.4±7.1
28.6±7.2
Physical and chemical characteristics of manure effluents
sampled from left and right sides of section 32 before and after
the triple treatment with Agrotrof product
Right side
Left side
Section 32
Indicators
before
after
before
after
Treatment with Agrotrof
24.06.08
16.07.08
24.06.08
16.07.08
Suspended matter, mg/L
6660
34400
10640
114900
Dry residue, mg/L
12220
52310
15790
123990
рН
7.65
6.99
7.16
7.23
COD (organic compounds) in mixed
sample, mg/L
20000
71000
16667
144000
COD in filtered sample, mg/L
10300
32000
8300
27000
Total nitrogen in mixed sample, mg/L
2023
4506
1948
6746
1687
2612
1423
2911
1409
1850
1007
1757
301
2090
521
2993
218
1082
422
1061
1289
2082
1202
2016
1274
1815
1174
1902
Total nitrogen in filtered sample, mg/L
Ammonia nitrogen N-NH4, mg/L
Total phosphorus in mixed sample, mg/L
Total phosphorus in filtered sample, mg/L
Total potassium in mixed sample, mg/L
Total potassium in filtered sample, mg/L
Experimental Design
Options
Control
Number of
cattle heads
Weight Agrotrof treatment plan for animal stalls
before
experimen
t
2866
24.5
Without treatment
Experiment 1800
24.5
1st month: once in 7 days
2nd month: once in 10 days
3rd month: once in 15 days
Cost Efficiency
Indicators
Experiment
Number of cattle heads at the beginning of 1800
Control
2866
experiment
Implementation, number of heads
Initial weight (kg)
1650
2582
24.5
24.5
Average weight per head (kg) at the end of 101.6
102.7
experiment
Death loss
127
284
Death loss (%)
7.0
10.0
Average daily weight gain, (g).
670
616
• Contract number: 16.515.11.5063
• Topic: Development of technological basis for processing
beet pulp into feed
• Priority Area: 1.5. – Environmental Management
• Critical Technology: Environment state monitoring and forecasting, pollution
prevention and response.
• Duration: 1 January 2012 – 20 June 2012
• Planned Funding: 17.5 mln rub.
• Budget allocation
- 14.0 mln rub
• Extra funds - 3.5 mln rub
• Contractor: OOO „BIOTROF‟
• Director: G.Yu. Laptev, PhD
13
Beet pulp as a by-product of sugar production
Sugar beet, 1000 kg
Preparation for processing
Extraction of juice
Extraction
Wet pulp, 540 kg
Sugar crystallization
Water extraction
Boiling and centrifuge
Granulated sugar, 150 kg
Pressed pulp, 290 kg
Drying
Dry pulp, 60 kg
Beet pulp accounts for the major part (85-90%) of
beet factory waste (appr. 22-24 mln t/yr). Fresh beet
pulp (including pressed pulp) quickly turns sour
and cannot be stored. Beet pulp decomposes
quickly, releasing toxic decay products (ammonia,
skatole, indole, mycotoxins, etc.). This raises the
environmental hazard profile of beet pulp from
class V to class IV and increases the
environmental impact.
15
Chemical composition of beet pulp from different
processing factories, g in kg of natural feed
Prot
ein
732
Pho
Calci
spho
um
rus
Fat
Fibe
r
Sug
ar
Ash
20.2
0.8
40.7
34.8
12.4
0.2
2.4
858
12.6
1.0
25.4
11.7
9.0
0.1
1.2
Liski-Sakhar
863
12.5
1.0
22.9
18.2
7.3
0.1
Sadovskiy Sugar
Factory
936
10.0
0.5
19.5
2.4
6.2
0.1
Factory
Moistur
e
Voronezh-Sakhar
Yelan-Kolenovskiy
Sugar Factory
1.1
1.1
16
Bacteriological and mycological tests of beet
pulp
Factory
Liski-Sakhar: fresh
Microorganisms
patogenic
nonpatogenic
C. freundii,
titre 103
Cryptococcus
OGCh-11000
D/g
after week’s
storage in a pit
E. coli,
titre 105
-
-
after 2 week’s
storage in a pit
E. coli,
titre 105
-
ОGCh-16000
D/g
enterobacteria,
titre105
enterobacteria,
titre105
ОGCh-5340
D/g
OGCh-7330
D/g
-
OGCh-14000
D/g
Voronezh-Sakhar
-
Yelan-Kolenovskiy
Sugar Factory
-
Sadovskiy Sugar
Factory
E. coli,
titre 105
17
Organoleptic assessment of silage made of pulp (after
one month of storage)
Silage
Color
Beet pulp without additives
- control
Light-grey
Beet pulp + preservation
agent
light
Smell
Distinctly sour
Slightly sour
Presence of mold
small mold growth under
film
no
18
Chemical composition of siloed pulp,
% of dry solids
Forage
Protein
Fat
Fiber
Nitrogen-free
extractive
substance
Metabolic
energy, MJ
Pulp – initial
substance
75.1
6.2
144.1
724.1
9.55
74.8
19.7
200.6
646.7
9.12
75.3
17.5
173.9
679.5
9.37
Beet
pulp
without
additives
–
control
Beet pulp +
preservation
agent
19
Study design for milk cows
Group
Analogues
Number of heads
Feeding specifics
1-control
cows
10
Basic diet (BD)
10
BD1, including beet
pulp prepared with an
added agent
10
BD1 + probiotic agent to
optimize
digestion
(Cellobakterin)
2-experimental
3-experimental
Cows
Cows
20
Lactation curve for newly calved
cows
21
Study design for fattening bulls
b
Analogue
Number of heads
Feeding specifics
1-control
Bulls
10
Basic diet (BD)
2-experimental
Bulls
10
BD1, including beet
pulp prepared with a
preservation agent
3-experimental
bulls
10
BD1 + prebiotic agent to
optimize digestion
22
Bull body weight increase during the
experiment
(on average per one head in 100 days, n=10)
Indicator
Group
1-control
2-experimental
3-experimental
324.7±2.08
322.9±1.23
325.9±1.38
415.0±2.57
419.9±3.17
427.1±3.06
gross, kg
90.3±1.89
97.0±2.75
101.2±2.58
daily average, g
903±18.86
970±27.53
1012±25.81
in % to control
100
107.4
112.1
Body weight, kg:
at the beginning of
experiment
at the end of
experiment
Body weight gain:
23
Results of T-RFLP-analysis
Microbial count, %
Microorganisms,
Control
1 experimental
2 experimental
systematics
(BD)
(BD1)
(BD1+probiotic)
11.15 ±
Phylum Bacteroidetes
2.88
13.98 ± 3.95
8.99 ± 1.2
Uncultured Bacteroidetes
0.54 ± 0.24
1.19 ± 0.89
1.22 ± 0.58
Family Bacteroidaceae
1.48 ± 0.99
1.21 ± 0.39
0.87 ± 0.17
Family Flavobacteriaceae
5.74 ± 2.41
6.47 ± 3.0
4.24 ± 0.54
Family Flexibacteraceae
2.98 ± 0.73
4.83 ± 1.2
2.53 ± 0.42
Family Prevotellaceae
0.42 ± 0.56
0.29 ± 0.16
0.14 ± 0.1
Thermoanaerobacteriaceae
1 ± 0.06
0.67 ± 0.47
10.79 ± 3.28
Family Clostridiaceae
9.99 ± 1.35
8.49 ± 0.88
7.15 ± 2.02
Family
24
Family Eubacteriaceae
5.79 ± 1.7
5.35 ± 1.35
7.83 ± 2.2
Family Lachnospiraceae
25.93 ± 1.41
21.33 ± 1.17
21.27 ± 5.44
Family Peptostreptococcaceae
0.06 ± 0.08
0.27 ± 0.18
0.77 ± 0.05
Family Ruminococcaceae
4.83 ± 0.74
4.45 ± 0.67
2.89 ± 0.26
Family Veillonellaceae
5.54 ± 0.62
4.4 ± 0.76
5.05 ± 0.34
Order Bacillales
1.7 ± 0.33
4.79 ± 1.41
3.48 ± 0.97
Family Bacillaceae
1.46 ± 0.41
3.7 ± 0.81
2.8 ± 0.3
Family Alicyclobacillaceae
0.05 ± 0.02
0.43 ± 0.27
0.45 ± 0.52
Family Paenibacillaceae
0.2 ± 0.1
0.67 ± 0.45
0.24 ± 0.17
Order Lactobacillales
0.32 ± 0.15
0.65 ± 0.25
0.42 ± 0.21
Family Lactobacillaceae
0.32 ± 0.15
0.63 ± 0.25
0.42 ± 0.21
Family Enterococcaceae
0
003 ± 0.03
0
Family Bifidobacteriaceae
1.37 ± 0.29
0.81 ± 0.22
0.4 ± 0.14
Family Pseudomonadaceae
1.52 ± 0.45
2.84 ± 0.28
2.99 ± 1.72
Family Burkholderiaceae
0.63 ± 0.41
0.72 ± 0.56
1.17 ± 0.05
Family Enterobacteriaceae
1.93 ± 0.86
1.49 ± 0.5
2.63 ± 0.14
Phylum Actinobacteria
9.27 ± 1.95
6.31 ± 0.38
8.08 ± 0.72
Family Staphylococcus
0.04 ± 0.04
0.21 ± 0.18
0.06 ± 0.07
Family Helicobacteraceae
0.28 ± 0.37
0.07 ± 0.05
0
Phylum Fusobacteria
1.53 ± 0.24
1.73 ± 0.38
0.81 ± 0.25
Phylum-level unclassified bacteria
16.74 ± 1.77
20.55 ± 2.09
15.21 ± 0.49
25
Business Analitica Company Data
• Russian beer market is the 5th largest in
the world
• Growth in 2007 amounted to 17%
• Amount of waste grows every year,
brewer‟s draff is the major part of beer
industry waste.
• 2,876,000 t of brewer‟s draff was produced
in 2007.
Brewer’s draff fungal community
Aspergillus sp. Link ex Fries
Aspergillus fumigatus Fresenius
Aspergillus niger van Tieghem
Aspergillus versicolor Tiraboshi
Fusarium oxysporum E.F.
Sm.Swingle
Penicillium chryzogenium Thom
Penicillium notatum Westl.
Penicillium purpurogenum Stoll
Penicillium sp. Link ex Fries
Penicillium tomii Maire
Saccaromyces cereviseae Meyen
Dynamics of mycotoxins concentration (mg/kg)
Determination by enzyme immunoassay
24 hours
Aflatoxin В1
Т-2 toxin
Ochratoxin
(MPL = 0.05 mg/kg)
0
2
5
7
24 hours
(MPC=0.1 mg/kg)
0.0069
mpl
mpl
0.0063
mpl
mpl
0.0065
mpl
mpl
mpl
mpl
0.083
Desoxynivalenol
ZEA
Fumonisin
0.27
0.027
0.38
mpl
0.1
0.17
0.34
mpl
0.025
n/d
0.036
n/d
(MPC=1.0 mg/kg)
0
2
5
7
Brewer‟s draff from
factory
Preparation of
working solution
(1 L per 8 t of
brewer‟s draff)
Silage trench
Sealed to create
anaerobic
conditions
Dosing pump
Brewer‟s Draff Preservation Schematic