Introduction #

Synthetic plas­tics are one of the great­est in­ven­tions of hu­mankind and have been de­vel­oped into a ma­jor in­dus­try and in­dis­pens­able com­mod­ity in hu­man life. The dura­bil­ity of these dis­posed plas­tics con­tributes to the en­vi­ron­men­tal prob­lems when they go into the waste stream. As the nat­ural en­vi­ron­ment is con­tin­u­ously pol­luted by these haz­ardous plas­tics, the de­vel­op­ment and pro­duc­tion of en­vi­ron­men­tal-con­served biodegrad­able plas­tics are rapidly ex­pand­ing to trim down our re­liance on syn­thetic plas­tics.

Straw is a soft­wood pro­duced as a byprod­uct of crop pro­duc­tion at har­vest. Straw is re­moved with the grains dur­ing har­vest and ends up be­ing piled or spread out in the field de­pend­ing on whether it was har­vested man­u­ally or us­ing ma­chines. According to International Rice Research Institute, Philippines, Globally, roughly 800 to 1,000 mil­lion tons per year of rice straw is pro­duced, with about 600 to 800 mil­lion tons per year pro­duced in Asia. This con­tin­ues to rapidly in­crease due to the shorter turn­around time re­quired for in­ten­si­fied rice crop­ping. As far as wheat straw is con­cerned, we pro­duce 529 mil­lion tons of wheat straw every year (Panwar et al., 2016).

As Wheat Straw con­tains 17.5 — 18 % Dry weight of Lignin and Rice Straw con­tains 15.6 — 17 % Dry weight of Lignin, they are the po­ten­tial source of Vanillin. However, Lignin is a com­plex mol­e­cule, and it is very hard to break the chain of Lignin.
However, us­ing Nitrobenzene Oxidation (NBO) Method, the Lignin (from soft­wood) can be bro­ken down into Vanillin.

Vanillin is the key com­po­nent re­spon­si­ble for form­ing Biodegradable plas­tics, com­pa­ra­ble to poly­eth­yl­ene tereph­tha­late (PET). Vanillin can be acety­lated us­ing Sodium Acetate and Acetic Anhydride, to form acetyl fer­ulic acid, which can be poly­mer­ized us­ing Zinc Acetate Zn(OAc)2 and processed fur­ther to form Bioplastics.

Materials and meth­ods #

Part 01: Extraction of Vanillin from Wheat Straw us­ing NBO Method #

Materials Required #

Wheat Straws were col­lected from lo­cal fields (Villages Bakaniya and Barkheda Salem) near IISER Bhopal. Various chem­i­cals used in the ex­per­i­ments in­clude Sodium Hydroxide, Nitrobenzene, Dichloromethane, Vanillin, Sodium Sulfate, Methanol, Hexane, Sulfuric Acid, Hydrochloric Acid and Distilled Water were ob­tained from Prof. Aasheesh Sir Lab (Lab No. 323) in IISER Bhopal.

Experimental Procedures #

Extracting Lignin out of Wheat Straw

1. We took 10 grams of washed Straw, and this time we ground them with the grinder such that the fi­nal straw size varies around 1 — 2 mm and trans­ferred them into Round Bottom Flask

Preparation of white liquor: We pre­pared the White Liquor un­der an ac­tive al­kali charge of 25% NaOH and Sulphidity of 30% Sodium Sulfide by weight in the 3:1 ra­tio of NaOH and Na2S.

2. As we took 10 grams of Straw, we had to mix white liquor and Straw in a 6:1 ra­tio. Assuming the den­sity of the Water to be 1 g/​ml, we had to make 60ml of the white liquor. We took 11.25g of NaOH pel­lets in 45ml of Distilled Water and 4.5g of Na2S in 15ml of Distilled Water and added the white liquor to the Round Bottom flask con­tain­ing the Straws.
3. We re­fluxed the above mix­ture at 160°C in an oil bath for 3 hours
4. After re­flux­ing, we ob­tained a dark brown coloured vis­cous so­lu­tion which we call Black liquor and the residue of un­treated Straw at the bot­tom.
5. We fil­tered the above so­lu­tion us­ing Vacuum fil­tra­tion to re­move the residue, and we got the black liquor as the fil­trate.
6. The pH of the Black Liquor was tested us­ing fil­ter pa­per. The pH was found to be around 13.
7. The Black liquor is neu­tral­ized us­ing 98% pure H2SO4 (for our ex­per­i­ment we took 7ml) and fur­ther acid­i­fied un­til the pH of the so­lu­tion reaches 2 — 3 (pH pa­per turned pink). pH 2 was nec­es­sary to in­crease the ef­fi­ciency of Lignin.
8. On neu­tral­iza­tion, the colour of the so­lu­tion was changed from Dark Brown to Peanut Brown Colour, and the pre­cip­i­ta­tion was ob­served.
9. We left the so­lu­tion overnight, and the next day, we fil­tered the so­lu­tion us­ing vac­uum fil­tra­tion and washed it with hot wa­ter 2 — 3 times to re­move ex­cess H2SO4.
10. The solid residue was taken out and dried for around 24 hours in a hot air oven at 65°C
11. After 24 Hours, a hard residue of Lignin is formed, which is woody brown, and then we grounded the Lignin us­ing Mortar and pes­tle till we got fine par­ti­cles and stored them in the Vial by weigh­ing them.
12. We tested the Lignin qual­i­ta­tively us­ing the Safranine dye and re­ported the re­sults.

Synthesis of Vanillin from Extracted Lignin us­ing the NBO method

1. We took 1.5 grams of dried Lignin in a round bot­tom flask and added 52.5ml of 2M NaOH (4.19 grams NaOH in 52.5ml of Distilled Water) and 3.75ml of Nitrobenzene. Nitrobenzene acts as a mild ox­i­diz­ing agent un­der al­ka­line con­di­tions and sup­ports side-chain ox­i­da­tion of Lignin.
2. We re­fluxed the above mix­ture in an oil bath for 3 hours at 160°C till a dark brown-coloured so­lu­tion was ob­tained.
3. After Refluxing, we trans­ferred the con­tents from the Round Bottom flask to the sep­a­rat­ing fun­nel and got two lay­ers of liq­uids.
4. We sep­a­rated the lower layer into a beaker and left the rest in the sep­a­rat­ing fun­nel for fur­ther test­ing.
5. We di­vided the so­lu­tion in the beaker into three parts and per­formed the fol­low­ing test­ing:
   • In Part 1, we ran the TLC against Laboratory–grade Vanillin us­ing 20% EtOAc/Hexane so­lu­tion as elu­ent, stained it with 2,4 DNP, and re­ported the re­sults.
   • In Part 2, we acid­i­fied the layer with 1M HCl and then ran the TLC of the pre­cip­i­tate against Laboratory–grade Vanillin us­ing 20% EtOAc/Hexane so­lu­tion as elu­ent, stained it with 2,4 DNP, and re­ported the re­sults.
   • In Part 3, we tried dis­solv­ing the so­lu­tion into cold di­ethyl ether and kept it overnight, cov­er­ing it to get the pre­cip­i­tate. The next day, we ran the TLC of the so­lu­tion against Laboratory–grade Vanillin us­ing 20% EtOAc/Hexane so­lu­tion as elu­ent, stained it with 2,4 DNP, and re­ported the re­sults.
   • From Part 3, we re­moved the di­ethyl ether by evap­o­rat­ing it and added 20 ml of Methanol and wa­ter mix­ture (1:1 ra­tio) to the re­main­ing so­lu­tion. Both the lay­ers were im­mis­ci­ble, and a cloudy so­lu­tion was ob­tained. We ran the TLC of the so­lu­tion against Laboratory–grade Vanillin us­ing 20% EtOAc/Hexane so­lu­tion as elu­ent, stained it with 2,4 DNP, and re­ported the re­sults.
6. We re­moved the up­per layer from the sep­a­rat­ing fun­nel in step 4 in the beaker and tested the pH of the so­lu­tion.
7. We neu­tral­ized the sep­a­rated up­per layer us­ing 98% pure HCl till the pH of the so­lu­tion reached around 4. We neu­tral­ized the so­lu­tion to con­vert the phe­no­lates to phe­nols.
8. After neu­tral­iza­tion, we passed the so­lu­tion through 90mm fil­ter pa­per us­ing Vacuum fil­tra­tion. Moreover, col­lected the fil­trate in a Buchner fun­nel.
9. We trans­ferred the fil­trate into the Separating Funnel, and as the layer was not sep­a­rated, so we added 20 ml of Dichloromethane (DCM). We then mixed both the so­lu­tions by shak­ing and let­ting the lay­ers sep­a­rate with time.
10. Once the lay­ers were sep­a­rated, we ex­tracted the lower layer of DCM into Round Bottom Flask and added more DCM into the sep­a­rat­ing fun­nel 2 more times to get the com­pound of our in­ter­est in the or­ganic layer and col­lected it in the Round Bottom Flask.
11. Using Rotavapor, we evap­o­rated the Organic Solvent (DCM) un­der re­duced pres­sure to get the crude prod­uct.
12. We stored the prod­uct in the 5ml Vial and ran the TLC against Laboratory–grade Vanillin us­ing four dif­fer­ent elu­ents, 10% EtOAc/Hexane so­lu­tion, 20% EtOAc/Hexane so­lu­tion, 30% EtOAc/Hexane so­lu­tion and 40% EtOAc/Hexane so­lu­tion, stained them with 2,4 DNP and re­ported the re­sults.

Part 02: Formation of Acetyl fer­ulic acid from Vanillin and fur­ther poly­mer­iz­ing it us­ing Zinc Acetate #

Materials Required #

The ex­per­i­ments’ Apparatus and var­i­ous chem­i­cals, in­clud­ing Vanillin, Pyridine, Acetic Anhydride, Sodium Acetate, Zinc Acetate and Distilled Water, were ob­tained from Prof. Aasheesh Sir Lab (Lab No. 323) in IISER Bhopal.

Experimental Procedure: #

1. We took 1.00 grams of Vanillin in a 25 ml round bot­tom flask and added 1.08 grams of Sodium Acetate, .7 ml of Acetic Anhydride
2. We re­fluxed the above mix­ture in an oil bath at 40°C for 24 hours till the brown-coloured so­lu­tion as ob­tained.
3. After 24 hours, We poured the so­lu­tion over 50 rams of crushed ice con­tained in a 100 ml beaker nd stirred for 45 min­utes till a brown sticky ub­stance was ob­tained.
4. After stir­ring, we kept the mix­ture overnight in he freezer main­tained at 4°C.
5. Next day, we re­moved the wa­ter, and dis­solved the olu­tion in di­ethyl — ether and re­moved the di­ethyl ether by evap­o­ra­tion.
6. After evap­o­rat­ing di­ethyl — ether, layer of cetylfer­ulic acid is formed in­di­cated by Beige el­low coloura­tion which is then dried in oven for 4 hours.
7. After 24 hours, we poly­mer­ized it us­ing Zinc Acetate in 1 mol% of Acetyl Ferulic Acid.
8. We set the re­ac­tion mix­ture con­taing Acetylferulic acid and Zinc Acetate as a cat­a­lyst and heat the re­ac­tion to ap­prox. 200 de­grees Celsius in oil bath for 2 hours.
9. Ater poly­mer­iza­tion, we vac­u­umed the Round Bottom flask to re­move any mois­ture if pre­sent.

UPSCALING

1. We took 7.00 grams of Vanillin in a 100 ml round bot­tom flask and added 7.56 grams of Sodium Acetate, 3 ml of Acetic Anhydride
2. We re­fluxed the above mix­ture in an oil bath at 40°C for 24 hours till the brown-coloured so­lu­tion as ob­tained.
3. After 24 hours, We poured the so­lu­tion over 150 rams of crushed ice con­tained in a 200 ml beaker nd stirred for 45 min­utes till a beige yel­low ticky sub­stance was ob­tained.
4. After stir­ring, we kept the mix­ture overnight in he freezer main­tained at 4°C.
5. Next day, we re­moved the wa­ter, and dis­solved the olu­tion in di­ethyl — ether and re­moved the di­ethyl ether by evap­o­ra­tion.
6. After evap­o­rat­ing di­ethyl — ether, layer of cetylfer­ulic acid is formed in­di­cated by Beige el­low coloura­tion which is then dried in oven for 4 hours.
7. After 24 hours, we poly­mer­ized it us­ing Zinc cetate in 1 mol% of Acetyl Ferulic Acid.
8. We set the re­ac­tion mix­ture con­taing cetylfer­ulic acid and Zinc Acetate as a cat­a­lyst nd heat the re­ac­tion to ap­prox. 200 de­grees Celsius n oil bath for 2 hours.
9. Ater poly­mer­iza­tion, we vac­u­umed the Round Bottom lask to re­move any mois­ture if pre­sent.

Part 03: Formation of Bioplastic us­ing poly­mer­ized Acetyl fer­ulic acid and test­ing it. #

1. Once the poly­mer­ized com­pound is dried, we took the com­pound and grind it to get yel­low colour pow­der.
2. We mixed the pow­der with 0.9 ml glyc­erol which act as a plas­ti­cizer and delig­ni­fied straws (utilization of straw fi­bres) around 1 gram and heat it in a mi­crowave for 45 sec­onds.
3. After heat­ing it in the mi­crowave, we moulded the plas­tic in the re­quired shape and dried it in hot air oven at 45 de­gree Celsius for 3 days.

Observations and Analysis #

Part 01: Extraction of Vanillin from Wheat Straw us­ing NBO Method #

Observations: #

Following are the ob­ser­va­tional re­sults:

Part 01: Extraction of Lignin from Wheat Straw:

Part 02: Synthesis of Vanillin from ex­tracted Lignin:

Analysis #

Pros:

1. Vanillin is formed as the ma­jor prod­uct of this Trial.
2. The ap­pear­ance of Vanilla like smell on smelling the Vial af­ter 3 days.
3. As side prod­ucts are formed, they will not in­ter­fere with fur­ther re­ac­tions as Vanillin is the ma­jor prod­uct.

Cons:

1. Many side prod­ucts are formed when viewed un­der UV or stained with 2,4 DNP, as shown in Figure 9(a)
2. Due to the pres­ence of these side prod­ucts, the ex­trac­tion of pure Vanillin makes it time-con­sum­ing and ex­pen­sive!

Part 02: Formation of Acetyl fer­ulic acid from Vanillin and fur­ther poly­mer­iz­ing it us­ing Zinc Acetate #

Observations: #

Following are the ob­ser­va­tional re­sults:

Analysis #

Pros:

The Polymer of Acetyl fer­ulic acid is suc­cess­fully formed as shown in the Figure 11(h).

Cons:

The sig­nif­i­cant quan­tity of acetylfer­ulic acid is lost due to its sticky na­ture. So, we have low yield of acetylfer­ulic acid.

Areas of Improvement:

Acetyl fer­ulic acid is a sticky com­pound so, it must be dried by dis­solv­ing it in or­ganic sol­vent and then fil­ter­ing it off us­ing Vacuum fil­ter­ation and dry­ing the residue in hot air oven overnight.

Part 03: Formation of Bioplastic us­ing poly­mer­ized Acetyl fer­ulic acid and test­ing it. #

Observations: Trial 01 #

Following are the ob­ser­va­tional re­sults for Trial 1:

Analysis — Trial 01: #

Pros:

The plas­tic was formed.

Cons:

1. Plastic was formed but it was wet even af­ter heat­ing it in oven at 54°C for 3 days.
2. We added straws, and put the mix­ture in the mi­crowave, so the mix­ture got burnt and gave the black­ish ap­pear­ance

Areas of Improvement:

The straws should be mixed af­ter melt­ing the glyc­erol and poly­mer mix­ture in oven for 30 sec­onds with the in­ter­vals of 10 sec­onds each.

Upscaling Observations and Analysis #

Analysis #

Pros:

1. Although the orig­i­nal colour of plas­tics is pale yel­low, Plastics can be dyed in dif­fer­ent colours, as shown in fig­ure 12 (g).

Cons:

1. Texture was rough, a lit­tle more time in oven will re­solve this is­sue.

Results #

Part 01: Extraction of Vanillin from Wheat Straw us­ing NBO Method #

Experimental Results from Trial 03:

1. Mass of Lignin Extracted from 10 grams of wheat Straw: 3.152 grams (31.52 %)
2. Safranine Dye changes colour from Deep Red to Pink
3. Determination of Vanillin (from 1.5 grams of lignin):
   • Mass of Empty Vial = 3.300 grams
   • Mass of Vial with Compound (Containing Side Products) = 3.401 grams
   • Mass of Compound (Including Side Products): 3.401 — 3.300 = 0.101 grams

Testing the com­pound on TLC against Laboratory — grade Vanillin us­ing 10% EtOAc/Hexane so­lu­tion as elu­ent.

(P — Pure Vanillin, CoS — Co-Spotting, C - Compound)

4. Testing the com­pound on TLC against Laboratory — grade Vanillin us­ing 20% EtOAc/Hexane so­lu­tion as elu­ent.

(P — Pure Vanillin, CoS — Co-Spotting, C - Compound)

5. Testing the com­pound on TLC against Laboratory — grade Vanillin us­ing 30% EtOAc/Hexane so­lu­tion as elu­ent.

(P — Pure Vanillin, CoS — Co-Spotting, C - Compound)

6. Testing the com­pound on TLC against Laboratory — grade Vanillin us­ing 40% EtOAc/Hexane so­lu­tion as elu­ent.

(P — Pure Vanillin, CoS — Co-Spotting, C - Compound)

Part 02: Formation of Acetyl fer­ulic acid from Vanillin and fur­ther poly­mer­iz­ing it us­ing Zinc Acetate #

Experimental Results from Trial 02:

1. Mass of Acetylferulic Acid:
   • Mass of Butter Paper = 0.249 g
   • Mass of Paper + Compound = 0.921 g
   • Mass of Compound = 0.921 — 0.249 = 0.672 g
2. Mass of Polymer from 0.672g of Acetylferulic Acid:
   • Mass of Empty Vial = 1.11 g
   • Mass of Vial con­tain­ing the poly­mer = 1.46 g
   • Mass of Polymer = 0.350 g

Experimental Results from Upscaling Step:

1. Mass of Acetyl Ferulic Acid Formed:
2. Mass of Acetyl fer­ulic acid taken for fur­ther con­sid­er­a­tion: 4.705 g
3. Mass of Polymer formed from 4.705 g of Acetylferulic Acid: 4.055 g
4. Mass of Bioplastic formed from 4.055 grams of poly­mer: 5.304 g

Part 03: Formation of Bioplastic us­ing poly­mer­ized Acetyl fer­ulic acid and test­ing it — Upscaling Step. #

1. Weight of plas­tic formed from 4.705 grams of acetyl fer­ulic acid:

NOTE: Separate sec­tion is de­voted to mea­sure the prop­er­ties of Bioplastics

Cost Analysis #

References #

1. M.Kapoor, D.Panwar, G.S.Kaira, 2016, Bioprocesses for Enzyme Production Using Agro-Industrial Wastes: Technical Challenges and Commercialization Potential, Agro-Industrial Wastes as Feedstock for Enzyme Production, 61 — 93, https://​doi.org/​10.1016/​B978-0-12-802392-1.00003-4
2. L. Mialon, Stephen A. Miller, 2013, Poly(dihydroferulic acid) a biore­new­able poly­eth­yl­ene tereph­tha­late mimic de­rived from Lignin and acetic acid and copoly­mers thereof, University of Florida Research Foundation, https://​patents.google.com/​patent/​US20130137847A1/​en
3. Shah, Manzoor & Schmid, Markus & Aggarwal, Ankit & Wani, Ali Abas. (2017). Testing and Quality Assurance of Bioplastics: Testing & Quality Assurance. https://​doi.org/​10.1201/​9781315374390-10.