have description about how to produce and purify biochemical products

1. Acceptable storability and aerobic stability of
by-products ensiled as a total mixed ration
Ajmal Wali*, JianJian Hou*, Takeshi Tsuruta*, Naoki Nishino*
Graduate School of Environmental and Life Science, Okayama University
13-Jun- 2023

2. Introduction
ORANGE JUICE PULP ORNAGE PEEL SOYBEAN CURD
SUGAR BEET PULP POTATO PEEL WET BREWERS GRAIN

3. • Wet by-products soon become spoilage
• How we can prevent these valuable by-product from deterioration?
• We used wet by-products as mixture in TMR silage to inhibit spoilage
Wet by-products spoilage
Introduction

4. Silage is high moisture crop material produced by controlled
(anaerobic) fermentation.
Forage crop silage
Prepared from one crop
TMR (total mixed ration) Silage
Prepared from different wet and dry
by products and forage
1
2
Introduction

5. Total mixed ration
(TMR) silage made by
mixing the wet
by‐products with
roughage is in practice
at dairy farms in Japan
Introduction
Mixed storage is one of the important
process for efficient utilization of wet by-
products

6. 1. TMR silage has
higher aerobic stability.
Objectives
To find the potential of aerobic stability in TMR silage with the
mixture of wet by-products
Wang et al., 2018
Figure 1

7. 2. The ingredients of TMR silage are locally available in each
country
Objectives
To improve silage fermentation and aerobic stability stored at
low and high temperature.
How to improve TMR silage quality and aerobic stability in cold
and tropical countries from lab scale silo to practice?

8. Material and methods
TMR composition
• Wet by-products
• Hay
Aerobic Stability Test: Visual looking, silage temperature
record, change in pH and fermentation products were
the indicators for AST evaluation
• brewer’s grains
• soybean curd
residue
• Orange juice pulp
• Sugar beet pulp
• Cracked maize
• Rapeseed meal
• Sudan grass hay
• Wheat bran
Main ingredients
14d 60d
Sampling time
AST

9. Understanding the quality of TMR silage
Microorganisms
Reducing sugars Lactic acid
Kern et al., 2020
Fermentation Products

10. Figure 2. Fermentation products of TMR silage stored at various
temperatures for 14 and 60 days
0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
10 C 25 C RT C 40 C 10 C 25 C RT C 40 C 10 C 25 C RT C 40 C
14 days 60 days Aerobic exposure
Ethanol
1, 2 Propanediol
Propionic acid
Acetic acid
Lactic acid
2 Way ANOVA
P
**
T
**
p x T
**
** ** NS
** ** **
* ** **
** ** **
Results
Lactic acid is the major product of silage fermentation
which is important for silage preservation
Acetic acid is second major product of silage
fermentation which is important aerobic stability
o Small amount of acid was produced at low temperature during short time
ensiling while silage fermentation was improved in long time
o Excellent fermentation was achieved with all temperatures, surprisingly
high content of LA was existed after aerobic exposure

11. 0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
10 25 R 40 10 25 R 40 10 25 R 40
PEM-
A
14 days 60 days AST (67 days)
unidentified Stenotrophomonas
Pseudoxanthomonas Pseudomonas
Acinetobacter Gulbenkiania
Sphingomonas Acetobacter
Rhodobacter Paracoccus
Agrobacterium Methylobacterium
Ochrobactrum Bradyrhizobium
Megasphaera Ruminococcus
Butyrivibrio Clostridium
Streptococcus Weissella
Pediococcus Lactobacillus
unclassified LB unclassified LB
Tetragenococcus Enterococcus
Staphylococcus Lysinibacillus
Aeribacillus Thermobacillus
Paenibacillus Brevibacillus
Aneurinibacillus Bacillus
Chitinophaga Sphingobacterium
Cloacibacterium Prevotella
Figure 3. Genus-level bacterial microbiota of TMR silage
Results
o Lactobacillus
o Acinetobacter
o Bacillus
o Un Lactobacillus
o Bacillus family was significantly enhanced by
high temperature, and the silage
fermentation was supported by Bacillus

12. -0.6 -0.4 -0.2 0 0.2 0.4
CAP1
-0.6
-0.4
-0.2
0
0.2
0.4
CAP2
Similarity
70
SFG1
PE-A
14d-10
14d-25
14d-RT
14d-40
60d-10
60d-25
60d-RT
60d-40
AST-10
AST-25
AST-RT
AST-40
Chitinophaga
Bacillus
Aneurinibacillus
Brevibacillus
Enterococcus
Tetragenococcus
Lactobacillus
Streptococcus
Acinetobacter
Pseudoxanthomonas
Figure 4. Conical analysis of principal coordinates (CAP) characterizing
bacterial microbiota of TMR
Results
The silage of high temperature was grouped
separated by Bacillus, however the silage quality
was not changed. This result indicate that TMR
silage is good practice at tropical regions

13. 0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
10 25 R 40 10 25 R 40 10 25 R 40
PEM-
A
14 days 60 days AST (67 days)
Rhizopus Wallemia
Trichosporon Cutaneotrichosporon
Papiliotrema Vishniacozyma
Dioszegia Filobasidium
Cystofilobasidium Sporobolomyces
Rhodotorula Malassezia
Xylodon Suillus
Pleurotus Gibberella
Wickerhamiella Diutina
Candida Zygosaccharomyces
Torulaspora Saccharomyces
Naumovozyma Nakaseomyces
Kluyveromyces Kazachstania
Issatchenkia Wickerhamomyces
Kodamaea Clavispora
Schwanniomyces Millerozyma
Lodderomyces Hyphopichia
Blumeria Aspergillus
Alternaria Epicoccum
Coniothyrium Cladosporium
unidentified
Figure 5. Genus-level fungal microbiota of TMR silage
Results
o Aspergillus
o Kazachstania
o Diutina
o Although the abundance of Kazachstania enhanced
at low and moderate temperature, the silage was
aerobically stable.
o The growth of Aspergillus was suppressed by cold
temperature and was active at high temperature.

14. -0.4 -0.2 0 0.2 0.4 0.6
CAP1
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
CAP2
Similarity
70
SFG1
PE-A
14d-10
14d-25
14d-RT
14d-40
60d-10
60d-25
60d-RT
60d-40
AST-10
AST-25
AST-RT
AST-40
Aspergillus
Clavispora
Kazachstania
Kluyveromyces
Candida
Diutina
Trichosporon
Wallemia
Figure 6. Conical analysis of principal coordinates (CAP) characterizing fungal
microbiota of TMR
Results

15. Conclusion
• We found that high amount of acetic acid is responsible for
aerobic stability at low and high temperatures
• According to excellent fermentation at high temperature,
TMR silage might be good practice at tropical countries
• Agro Industrial by-products (raw material) provided
appropriate environment for microbial fermentation in
TMR silage
• TMR silage is better process for proper utilization of wet
by-products