Based in the previous table, it can be concluded that other strains could function as model strains for investigating biodegradation of lignin-based aromatic monomers, instead of FMYD002. As an example, No 3.7 and No 3.19 isolates showed to grow in all of the tested cultured media and accumulated lipids as well. It could also be possible to obtain high degradation values by employing the isolates No 1.5, No 1.12, No 2.15, No 3.3, No 3.4, and No 3.11 due not only to their resistance to vanillin, but also to their excellent growing rate and ability to accumulate lipids.
5 Conclusions
The solvent system (mobile phase) used in the TLC:s was toluene: ethyl acetate: formic acid in a 4:5:1 (v/v/v) proportion. This system worked well for separating the spot believed to correspond to vanillyl alcohol from all other spots.
It is possible to subject vanillyl alcohol to a brief high temperature treatment, as it does not seem to affect its stability (as evidenced by TLC, Figures 10 and 12). However, TLC is not quantitative. The vanillin samples that undergone biodegradation by yeasts for 72 hours did not show spots of vanillyl alcohol. This could mean that transformation from vanillin to vanillyl alcohol is time-dependent, regardless of the treatment temperature. As for other biodegradation products, it is possible to see that there is a smear and also a new spot appearing below the other products on the TLC when the extracts were subjected to higher temperatures for longer periods of time (elongated spots near Rf = 0.76 - which is similar to that of vanillic acid), and an unidentified spot at Rf = 0.55, when samples were subjected to 24 hours of biodegradation.
Spots with similar Rf to that of the vanillic acid standard (Rf = 0.76) appeared in all of the samples, regardless of treatment temperature and time point of biodegradation. Their intensity, however, increased in samples that undergone 72 hours of bio degradation, as compared to those taken after 24 hours of biodegradation.
As regards the samples subjected to different pH regimes followed by pH adjustment, it can be concluded that all the samples exhibited similar spots, regardless of pH, i. e. at an Rf = 0.65 , similar to Rf of the for the vanillyl alcohol standard. All the samples at pH 2 were the only ones that showed spots consistently at Rf = 0.76, a value equivalent to the Rf of vanillic acid.
Under UV visualization of TLC plates, few spots with an Rf = 0.76 and Rf = 0.80 (vanillin standard) were observed for pH 7 and pH 9, but in the MBTH-stained version, there were none or only faint ones.
It has been shown that the best route for extraction of the compound presumed to be identical to vanillyl alcohol is found when the sample is adjusted to a pH 9. As seen in Figures 13 and 14, other spots aside that of vanillyl alcohol had disappeared or were faint at pH 9.
As regards the strains cultured in different media, it is not possible to say at this stage whether or not the analyzed strains accumulate lipids. This is because not all of the pressed filter papers showed a dark blue or violet cell color when subjected to Sudan B dye. Certain bias could have arisen due to the temperature used (60 oC), which perhaps did not allow the yeast contents to become exposed to the dye.
Only 26 out of 60 strains thrived in most of the different lignin-based culture media tested, regardless of the concentration of lignin. However, from this group of 26 strains, only 17 successfully grew in nitrogen reduced medium.
Based on the ability to grow in all of the tested media and the capacity of strains to accumulate lipids, it is advisable to use the strains with positions No 3.7 and No 3.19 as models for future studies of vanillin biodegradation.
Similarly, other strains such as those with positions: No 1.5, No 1.12, No 2.15, No 3.3, No 3.4 and No 3.11 demonstrated resistance to different concentrations of vanillin, whilst growing at high rate, even though not all of them grew in nitrogen-limited medium. They all demonstrated the capacity to accumulate lipids.
The previous findings show that certain strains have the ability to grow on media supplemented with lignosulfonate and lignin acid hydrolyzate, suggesting that they use these substrates as their primary carbon sources.
From here, it can be then hypothesized that these strains might be capable of degrading lignocellulose. If this was the case, their potential for use in industrial treatment of paper mill sludge is high.
The Evans staining technique employed the replica printing technique of multiple strains simultaneously onto plates containing different substrates, and it proved to be a valuable tool during the advancement of the investigation. However, it should be noted that having only one culture plate with original strains (a so-called Master plate) could cause cross contamination between the strains in the Master agar plate, rending the culture useless for further experiments. This peculiarity is due to the diverse textures of the various strains, which allow them to inadvertently become stuck or to “drop” off the replica printing tool, providing them with the opportunity to mix and cross-contaminate each other.
As seen in Table 5, Fungal strains with serial numbers No 2.7, No 2.4 and No 2.15 grew at a concentration of 5 mM of vanillin, but did not successfully grow in a concentration of 1 mM of vanillin. This can be explained by the difficulty of dissolving vanillin in LiBa medium, which could result in precipitation problems when pouring plates, leaving the odd spots in the growth medium with higher concentration of vanillin than the expected 1
6 Future work
This investigation came to the conclusion that most of the spots that appeared on the TLC plates appeared to correspond to those of the standards of vanillyl alcohol and vanillic acid. However, it is not possible to determine only by means of TLC and GC techniques that the degraded products actually are identical to the previously named monomers. It is necessary to grow a large culture and extract the resulting supernatant (minimum of 250 ml) at a 34-hour-culture. Such an extract would be used primarily to get hold of a large quantity of the metabolite presumed to be vanillyl alcohol, which ultimately should be sent for NMR and mass spectrometry analysis.
Prior to the NMR analysis, other techniques, such as preparative TLC, high performance liquid chromatography (HPLC) and GC would help in getting a grip on as to whether or not the biodegradation product could be vanillyl alcohol.
During the experiment of subjecting the biodegradation products of vanillin to different pH regimes, the time point at which extraction was carried out after 31 hours of vanillin biodegradation. Experiments done by Nehvonen (2017) showed that the peak biodegradation occurred after 34 hours of inoculation with strain FMYD002. That work showed that it is advisable to harvest the samples at that point in time to obtain the maximum output of the metabolite presumed to be identical to vanillyl alcohol.
A time course experiment could be a valuable investigation in order to see how other compounds, e.g. vanillyl alcohol and perhaps vanillic acid (which both presumably would be produced at earlier time points than 72 hours), react in the solvent systems. This could also help in establishing the point time at which the biodegradation product presumed to be vanillyl alcohol would start to appear and the time it would have completely been biodegraded and thus would be absent in the culture media.
So far, the isolated fungal strain FYMD002 has been employed for investigating biodegradation of lignin-based monomers with acceptable results. The present investigation has come to the conclusion that other isolates, such as those labeled No 1.5, No 1.12, No 2.15, No 3.3, No 3.4, No 3.7, No 3.11 and No 3.19 could survive the poisonous medium which would result as a consequence of adding vanillin, and at the same time might be accumulating lipids. Future studies will ascertain if they can grow in different vanillin concentrations, and their biodegradation capabilities could be monitored by using the TLC technique.
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Appendix A
TABLE 6.LIST OF THE FIRST 20 STRAINS USED IN THE INVESTIGATION AND THEIR ABILITY TO GROW IN DIFFERENT MEDIA (+) OR NOT VISIBLE INDICATION OF GROWING (-).FOLLOWING THE SAME NOMENCLATURE, LIPID ACCUMULATION IS SHOWN AS POSSIBLE (+) OR NOT VISIBLE (-). LAH, LIGNIN ACID HYDROLYZATE MEDIUM;LS,
LIGNOSULFONATE MEDIUM;VA, VANILLIN MEDIUM;N-REDUCED, NITROGEN REDUCED MEDIUM AND LIBA, LILLY &BARNETT MEDIUM.THIS LIST IS REFERRED AS GROUP 1.
N0 Other media Growing ability in the presence of a lignin-based media
Lipid
N-
reduced* LiBa 1mM VA 5mM
VA LAH 2.5% LAH
5.0% LAH 10% LAH
20%
LS 2.5g/l
LS 5.0g/l
LS 10g/l
LS 20g/l
1.1 + + + + + + ++ ++ + + ++ + -
1.2 - - - - - - - - - - - - -
1.3 - - - - - - - - - - - - -
1.4 - - - - - - - - - - - - -
1.5 ++ + ++ - ++ ++ + - + + ++ ++ +
1.6 ++ + ++ - ++ ++ + - + + ++ ++ -
1.7 - + - - - - - - - - - - -
1.8 - - - - - - - - - - - - -
1.9 + + ++ - ++ ++ + - + + + ++ +
1.10 + - - - - - - - - - - - -
1.11 - - - - - - - - - - - - -
1.12 + + ++ - + + + + + + ++ ++ +
1.13 - - - - - - - - - - - - -
1.14 - - - - - - - - - - - - -
1.15 ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ -
1.16 - + + - - - - - + + - - -
1.17 - - - - - - - - - - - - -
1.18 + + + - ++ ++ ++ ++ + + ++ ++ -
1.19 - - - - - - - - - - - - -
1.20 - - - - - - - - - - + + -
* Plates were 1 year old
TABLE 7LIST OF THE SECOND 20 STRAINS USED IN THE INVESTIGATION AND THEIR ABILITY TO GROW IN DIFFERENT MEDIA (+) OR NOT VISIBLE INDICATION OF GROWING (-).FOLLOWING THE SAME NOMENCLATURE,
LIPID ACCUMULATION IS SHOWN AS POSSIBLE (+) OR NOT VISIBLE (-). LAH, LIGNIN ACID HYDROLYZATE MEDIUM;LS, LIGNOSULFONATE MEDIUM;VA, VANILLIN MEDIUM;N-REDUCED, NITROGEN REDUCED MEDIUM
AND LIBA,LILLY &BARNETT MEDIUM.THIS LIST IS REFERRED AS GROUP 2.
N0 Other media Growing ability in the presence of a lignin-based media
Lipid
N-
reduced* LiBa 1mM VA
5mM VA
LAH 2.5%
LAH 5.0%
LAH 10%
LAH 20%
LS 2.5g/l
LS 5.0g/l
LS 10g/l
LS 20g/l
2.1 - - + - - - - - - - - - -
2.2 - - - - - - - - - - - - -
2.3 - - + - - - - - - - - - -
2.4 + + + + ++ ++ ++ ++ ++ ++ ++ ++ -
2.5 - + - - ++ + - - + + ++ + -
2.6 - - + - - - - - - - - - -
2.7 - ++ - + ++ ++ ++ - ++ + ++ ++ -
2.8 - - - - - - - - - - - - -
2.9 - - ++ - - - - - - - - - -
2.10 + + - - ++ + ++ - ++ ++ ++ ++ -
2.11 - - - - - - - - - - - - -
2.12 - - - - - - - - - - - - -
2.13 - - - - - - - - - - - - -
2.14 - + ++ - ++ + + - + + + + -
2.15 - ++ - + ++ ++ ++ + ++ ++ ++ ++ +
2.16 - - ++ - - - - + - - - - -
2.17 - ++ - ++ ++ + ++ - ++ ++ ++ ++ -
2.18 - - + - - - - - - - - - -
2.19 - - - - - - - - + ++ ++ ++ -
2.20 - ++ + + ++ ++ + - ++ ++ ++ ++ -
* Plates were 1 year old
TABLE 8LIST OF THE THIRD 20 STRAINS USED IN THE INVESTIGATION AND THEIR ABILITY TO GROW IN DIFFERENT MEDIA (+) OR NOT VISIBLE INDICATION OF GROWING (-).FOLLOWING THE SAME NOMENCLATURE, LIPID ACCUMULATION IS SHOWN AS POSSIBLE (+) OR NOT VISIBLE (-). LAH, LIGNIN ACID HYDROLYZATE MEDIUM;LS,
LIGNOSULFONATE MEDIUM;VA, VANILLIN MEDIUM;N-REDUCED, NITROGEN REDUCED MEDIUM AND LIBA, LILLY &BARNETT MEDIUM.THIS LIST IS REFERRED AS GROUP 3.
N0 Other media Growing ability in the presence of a lignin-based media
Lipid
N-
reduced* LiBa 1mM VA
5mM VA
LAH 2.5%
LAH 5.0%
LAH 10%
LAH 20%
LS 2.5g/l
LS 5.0g/l
LS 10g/l
LS 20g/l
3.1 - - - - - - - - - - - - -
3.2 - + + - + + + + ++ ++ ++ ++ -
3.3 - + ++ + ++ ++ ++ - ++ ++ ++ ++ +
3.4 - +++ ++ ++ ++ ++ ++ + ++ ++ ++ ++ +
3.5 - + - - - - - - - - - - -
3.6 + + + - ++ ++ ++ + + + + ++ -
3.7 + + + + + ++ ++ ++ ++ + + ++ +
3.8 - - - - - - - - - - - - -
3.9 - - - - - - - - - - - - -
3.10 - - - - - - - - - - - - -
3.11 + ++ ++ - ++ + + - ++ ++ ++ + +
3.12 - - - - - - - - - - - - -
3.13 + + + + ++ ++ ++ ++ ++ ++ ++ ++ -
3.14 - + ++ + ++ ++ + - ++ + ++ + -
3.15 + + ++ + ++ ++ + + ++ ++ ++ + -
3.16 + + ++ + ++ + + + ++ ++ ++ + -
3.17 - - - - - - - - - - - - -
3.18 + + ++ + ++ ++ ++ ++ ++ ++ ++ ++ -
3.19 + + ++ + ++ ++ ++ ++ ++ ++ ++ ++ +
3.20 - - - - - - - - - - - - -
* Plates were 1 year old
TABLE 9RELATIONSHIP OF ISOLATES COLOR AND ABILITY TO GROW ON DIFFERENT MEDIA
Category
Inhibition by the medium
Number of isolates
Corresponding isolate color and designation
yellow white black pink
1 none 9 1.1, 1.4 1.15, 3.7,
3.13, 3.15, 3.18, 3.19
3.16 -
2 5 mM VA 3 - 3.6 - 1.12,
1.18
3 20 % LAH 1 - - - 2.7
4 5 mM VA
and 20 % LAH
3 - - 1.6
1.5, 3.11,
1.9
5 All but
1mM VA 6 2.1, 2.3, 2.6, 2.9,
2.16, 2.18 - - -
6 All but
LiBa 1 1.7 - - -
7 All but
N-reduced
1 1.10 - - -
8 All the
substrates 21
1.2, 1.3, 1.4, 1.8, 1.11, 1.13, 1.14, 1.17, 1.19, 2,2, 2.8,
2.11, 2.12, 2.13, 3.1, 3.8, 3.9, 3.10,
3.12, 3.17, 3.20
- - -
Appendix B
Sequences employed during the development of growing characterization in different media.
Sequence
1 Group 1 Group 2 Group 3
LiBa
2.5%
LAH
5%
LAH
10%
LAH
20%
LAH
20 g/l LS
Sequence
2 Group 1 Group 2 Group 3
N- reduced
1mM VA
5mM VA
2.5 g/l LS
5 g/l LS
10 g/l LS
Appendix C
LiBa (Lilly & Barnett) medium
Solution A, B and C are needed as well as MilliQ water in the following percentage volumes:
A= 9.75 %; B= 10 %; C= 0.2 %; MilliQ= 80%
The recipe for preparing LiBa medium in stock volumes for solution A, B and C can be seen in Table 10.
TABLE 10.SOLUTIONS A,B AND C THAT COMPOUND LIBA MEDIUM
A: 200 ml stock
20 g D+Glucose 2 g KH2PO4
1 g Mg SO4 200 ml MilliQ water
Autoclave the solution
B: 200 ml stock
4 g asparagine 200 ml MilliQ Filter sterilize the solution
C: 500 ml stock
0.001 g FeSO4 x 7H2O;
0.87 g ZnSO4 x 7H2O;
0.3 g MnSO4 x H2O;
0.01 g biotin;
0.01 g thiamine Dissolved in 500 ml MilliQ and filter
sterilized. Bottle covered with aluminum foil and stored at 4 °C
Stock solution of 0.1M vanillin
In order to make 0.1M stock of vanillin, it is necessary to start from the molar solution concentration equation:
𝑀 = 𝑚 𝑉 1
𝑚𝑤 E
QUATION 5
Where M is the molarity of the concentration of the solute in mol l-1. m is the mass of solute in grams. V is the volume of solution in liters and mv is the molecular weight is g mol-1.
Solving for the mass one obtains:
𝑚 = 𝑀 ∗ 𝑉 ∗ mw EQUATION 6
The molecular weight of vanillin is 152.15 g mol-1 and we need 20 ml of solution.
Solving for Equation 6 one obtains:
𝑚 = 0.1 𝑚𝑜𝑙 𝑙−1 ∗ (0.02 𝑙) ∗ (152.15 𝑔 𝑚𝑜𝑙−1) EQUATION 7
𝑚 = 0.304 𝑔 𝑣𝑎𝑛𝑖𝑙𝑙𝑖𝑛
The result shows that we need to add 0.304 g of vanillin to 20 ml LiBa in order to have a stock solution of vanillin at 0.1 M.
Obtaining 1mM solution for casting of the plates
In order to obtain 1mM solution to cast the plates with agar, it is necessary to know the volume from the stock solution needed. Starting with the equation for unknown concentration, where C2 and V2 are the final concentration and volumes required:
𝐶1 ∗ 𝑉1 = 𝐶2 ∗ 𝑉2 EQUATION 8 Solving for V2:
𝑉1 =𝐶2𝑉2 𝐶1
EQUATION 9
Knowing that 0.1M vanillin is equal to 100 mM and that we might need 500 ml of LiBa solution from which 250 ml will be needed for casting around 5 agar plates, we obtain:
𝑉1 = 1𝑚𝑀 (500 𝑚𝑙) 100𝑚𝑀
EQUATION 10
𝑉1 = 5 𝑚𝑙
This is the volume need from the stock solution to be added to 500 ml of LiBa (with 2%
w/v agar) in order for it to contain 1mM vanillin.
Obtaining 5mM solution for casting of the plates
Once the previously plates are casted, the remaining 250 ml of LiBa (with 2% w/v agar) will be balanced as follows:
𝑉1 = 5𝑚𝑀 (250 𝑚𝑙) 100𝑚𝑀
EQUATION 11
𝑉1 = 12.5 𝑚𝑙
It is important to notice that the solution already contained 5 ml of vanillin at 1 mM.
That is why, the real value to be added to the remaining 250 ml is half the volume added to the initial 500 ml; that is 2.5 ml:
𝑉1 = 12.5 𝑚𝑙 − 2.5 𝑚𝑙 𝑉1 = 10 𝑚𝑙
This is the exact volume from the stock vanillin solution to be added to the remaining 250 ml of LiBa + agar in order for the new solution to be at a concentration of 5 mM vanillin.
Lignin acid hydrolyzate (LAH): 2.5 %
Autoclave MilliQ water along with LAH and agar needed for the casting solution prior to add the solutions A, B and C.
Add the following solutions in order to get 150 ml LiBa + agar for casting 5 to 6 agar plates:
TABLE 11A,B AND C CHEMICAL SOLUTIONS FOR CASTING 5 TO 6 PLATES OF 2.5%LAH WITH 150 ML LIBA AND AGAR
Solution amount A 14.3 ml B 14.6 ml C 0.29 ml MilliQ 117 ml LAH 3.75 ml Agar 3 g
Lignin acid hydrolyzate (LAH): 5 %
Autoclave MilliQ water along with LAH and agar needed for the casting solution prior to add the solutions A, B and C.
Add the following solutions in order to get 150 ml LiBa + agar for casting 5 to 6 agar plates:
TABLE 12A,B AND C CHEMICAL SOLUTIONS FOR CASTING 5 TO 6 PLATES OF 5%LAH WITH 150 ML LIBA AND AGAR
Solution amount A 13.9 ml B 14.25 ml C 0.28 ml MilliQ 114 ml LAH 7.5 ml Agar 3 g
Lignin acid hydrolyzate (LAH): 10 %
Autoclave MilliQ water along with LAH and agar needed for the casting solution prior to add the solutions A, B and C.
Add the following solutions in order to get 360 ml LiBa + agar for casting 10 to 12 agar plates:
TABLE 13A,B AND C CHEMICAL SOLUTIONS FOR CASTING 5 TO 6 PLATES OF 10%LAH WITH 150 ML LIBA AND AGAR
Solution amount A 31.75 ml B 32.4 ml C 0.648 ml MilliQ 259 ml
LAH 36 ml Agar 7.2 g
Lignin acid hydrolyzate (LAH): 20 %
Autoclave MilliQ water along with LAH and agar needed for the casting solution prior to add the solutions A, B and C.
Add the following solutions in order to get 360 ml LiBa + agar for casting 10 to 12 agar plates:
TABLE 14A,B AND C CHEMICAL SOLUTIONS FOR CASTING 5 TO 6 PLATES OF 20%LAH WITH 150 ML LIBA AND AGAR
Solution amount A 28.2 ml B 28.8 ml C 0.576 ml MilliQ 230 ml
LAH 72 ml Agar 7.2 g
Lignosulfonate (LS): 2.5 g l-1
Autoclave MilliQ water along with lignosulfonate and agar needed for the casting solution prior to add the solutions A, B and C.
Add the following solutions in order to get 150 ml LiBa + agar for casting 5 to 6 agar plates:
TABLE 15A,B AND C CHEMICAL SOLUTIONS FOR CASTING 5 TO 6 PLATES OF 2.5 G L-1LAH WITH 150 ML LIBA AND AGAR
Solution amount A 14.7 ml
B 15 ml
C 0.3 ml MilliQ 150 ml LS 0.375 g Agar 3 g
Lignosulfonate (LS): 5 g l-1
Autoclave MilliQ water along with lignosulfonate and agar needed for the casting solution prior to add the solutions A, B and C.
Add the following solutions in order to get 150 ml LiBa + agar for casting 5 to 6 agar plates:
TABLE 16A,B AND C CHEMICAL SOLUTIONS FOR CASTING 5 TO 6 PLATES OF 5 G L-1LAH WITH 150 ML LIBA AND AGAR
Solution amount A 14.7 ml
B 15 ml
C 0.3 ml MilliQ 150 ml LS 0.75 g Agar 3 g