RawData_BA_paper_Figure3

Selected Cell

Cell:

Value:

Figure_3_UPDATED N_assay_UPDATED PHA_data C_data

Experiment	Duplicate fed-batch fermentations
Exp info	Carry out P. putida CA3 fed-batch fermentation in Benzoate at a C/N ratio of 30:1 in the C&N feed. Batch phase was started with a C/N ratio of 7:1 [full N MSM with 1.96gC/l].Linear feed from T5 with feed rate, Q0 of 0.05 ml/min at m=0.0003
Fed batch feed	Linear feed
Duplicate	FB4	FB5
Exp date	2023-09-05T0:0:0+0000	31/5/2023
BB unit	BB2	BB2

Inoculum prep
Medium	Full N - MSM + BA
	FB4	FB5
10 mL	24 h old - 1.38 OD	23 h old - 1.42 OD
50 mL - 1	23 h old - 3.7	20 h old - 3.988
51 mL - 2	13 h old - 2.142 OD	19 h old - 2.81 OD
Reactor starting OD540 nm	0.1				FB5 [Rep 1 in manuscript]										FB4 [Rep 2]
					Time [h]	OD 540 nm	PHA [%]	PHA [g/L]	CDW [g/L]	N [mg/L]	BA [mM]	BA fed [mmol]	N fed [mg]		Time [h]	OD 540 nm	PHA [%]	PHA [g/L]	CDW [g/L]	N [mg/L]	BA-Na [mM]	BA fed [mmol]	N fed [mg]

					0	0.167				258.0755544271328	25.041476808038325	25.041476808038325	249.43927638869584		0	0.117				214.68448805043926	28.286720411263	25.041476808038325	249.43927638869584
					2	0.646				221.39988893488055	24.19359894068622	25.041476808038325	249.43927638869584		2	0.478				194.76982031209224	23.7774428476847	25.041476808038325	249.43927638869584
					4	2.065				158.14277781062898	19.505437551115786	25.041476808038325	249.43927638869584		4	1.513				157.4936507105785	20.463987225922033	25.041476808038325	249.43927638869584
				Feed start at T5	5	3.474				97.24098465376973	13.060940140982204	25.041476808038325	249.43927638869584	Feed start at T5	5	2.859				119.4393757603167	15.46370993496125	25.041476808038325	249.43927638869584
					7	11.546	0.29	0.0077575	2.675	0.6561242909164394	3.201177707676131	38.962510066686924	288.4574863886958		7	10.441				0.2528846551143493	6.510657787124665	38.962510066686924	288.4574863886958
					9	15.736	6.16	0.20422769230769233	3.3153846153846156	0.548452612407075	0.2635666160376991	53.72724231070818	329.8404363886959		9	13.161000000000001	6.53	0.28594526315789454	4.378947368421049	4.863159299277245E-4	0.30327218911866655	53.72724231070818	329.8404363886959
					25	27.611	17.97	1.15008	6.4	3.7012139487594053	21.95027456478561	206.37015084852177	757.6716863886959		25	21.901	19.25	1.6715416666666678	8.683333333333339	33.06953349228146	50.82804065895549	206.37015084852177	757.6716863886959
					27	29.434	17.25	1.216125	7.05	4.037687944101165	37.91542625696149				29	27.381	21.81	1.6721	7.666666666666667	105.23892778227423	81.53521049966896	254.127953941589	891.5284163886959
					29							254.127953941589	891.5284163886959		33	23.661	20.25	1.14668674698795	5.66265060240963	250.0397213951736	154.8450364139113	305.70460507371126	1036.088706388696
					31	29.036	22.25	1.7510749999999997	7.869999999999999	13.79543380901232	60.792740584959304				50	27.541	22.91	1.363145	5.95	751.4473737967613	165.768119328582	567.7619504514297	1770.5893963886958
					33							305.70460507371126	1036.088706388696
					50	25.786	18.24	1.10352	6.05	1144.011584161997	396.04268411418786	567.7619504514297	1770.5893963886958
				Mean		Productivity
						CDW [g/L/h]	PHA [mg/L/h]
			Timepoints	FB5	31 h	0.25387096774193546	56.486290322580636
				FB4	25 h	0.34733333333333355	66.86166666666671

					Mean	SD
				CDW [g/L]	8.276666666666669	0.5751135153650632
				PHA [g/L]	1.7113083333333337	0.056238559330369066
				CDW [g/L/h]	0.3006021505376345	0.06608787249541401
				PHA [mg/L/h]	61.67397849462367	7.33649897026575
				PHA [%CDW]	20.75	2.1213203435596424

Experiment	P. putida CA-3 duplicate batch fermentation																	Revision experiment
Date	May-jun, 2023							N=OD/0.297199787753071					N=OD/0.297199787753071					N=OD/0.411254667405275
								22/5/2023					12/6/2023 [Blank OD matches previous standard :)]					29/10/2024
								FB4					FB5					FB4 repeat
								Sample [h]	OD at 635 nm	N-concentration in well [mg/l]	Dilution	N [mg/l]	Sample	OD at 635 nm	N-concentration in well [mg/l]	Dilution	N [mg/l]	Sample [h]	OD at 635 nm	N-concentration in well [mg/l]	Dilution	N [mg/l]
								FB4 - [-1]	0.689333	2.3194262863092407	100	231.94262863092408	FB5 - [-1]	0.8	2.6917919627341105	100	269.17919627341104	FB4 - [-1]	0.8937999979019166	2.173349189058838	100	217.3349189058838
								FB4 - 0	0.741333	2.494392763886958	100	249.43927638869582	FB5 - 0	0.7669999999999999	2.580755544271328	100	258.0755544271328	FB4 - 0	0.8828999773025513	2.1468448805043927	100	214.68448805043926
								FB4 - 2	0.853333	2.8712436386697333	100	287.12436386697334	FB5 - 2	0.6579999999999999	2.2139988893488054	100	221.39988893488055	FB4 - 2	0.8009999767303467	1.9476982031209225	100	194.76982031209224
								FB4 - 4	1.016333	3.4196962510768083	100	341.9696251076808	FB5 - 4	0.47000000000000003	1.58142777810629	100	158.14277781062898	FB4 - 4	0.6476999894142151	1.574936507105785	100	157.4936507105785
								FB4 - 5	0.626333	2.1074476692439297	100	210.74476692439296	FB5 - 5	0.289	0.9724098465376974	100	97.24098465376973	FB4 - 5	0.4912000075340271	1.194393757603167	100	119.4393757603167
								FB4 - 25	0.161333	0.5428435909047278	100	54.284359090472776	FB5 - 7	0.195	0.6561242909164394	1	0.6561242909164394	FB4 - 7	0.1039999947309494	0.2528846551143493	1	0.2528846551143493
								FB4 -29	0.351333	1.182144182054079	100	118.2144182054079	FB5 - 9	0.163	0.548452612407075	1	0.548452612407075	FB4 - 9	1.9999969601631334E-4	4.863159299277245E-4	1	4.863159299277245E-4
								FB4 - 33	0.823333	2.7703014400672044	100	277.03014400672043	FB5 - 25	0.01100000000000001	0.037012139487594055	100	3.7012139487594053	FB4 - 25	0.13599999997615814	0.3306953349228146	100	33.06953349228146
								FB4 - 50	0.022333000000000006	0.07514473737967613	10000	751.4473737967613	FB5 -27	0.011999999999999997	0.04037687944101165	100	4.037687944101165	FB4 -29	0.4328000024318695	1.0523892778227424	100	105.23892778227423
								FB4 - 7	0.28433299999999995	0.9567066051750971	1	0.9567066051750971	FB5 - 31	0.04100000000000001	0.1379543380901232	100	13.79543380901232	FB4 - 33	1.0283000246047973	2.500397213951736	100	250.0397213951736
								FB4 - 9	0.132333	0.44526613225561634	1	0.44526613225561634	FB5 - 50	0.034	0.1144011584161997	10000	1144.011584161997
													FB5 - Exhaust	0.09	0.3028265958075874	10	3.028265958075874









		NanoPlateReader

		22/5/2023			BLANK	No colour	OD 0.061
		N-std
		Concentration range	MSM N-full 2x dilutions starting at 26.1850 mg/l
			N-concentration in well [mg/l]	OD at 635nm
			2.6185	0.802333
			1.30925	0.41933299999999996
			0.654625	0.21933300000000003
			0.3273125	0.119333
			0.16365625	0.068333
			0.081828125	0.046333											0.29719978775307115	0.025541466787355555	slope	intercept
			0.0409140625	0.044333											0.0028497276392292427	0.002724740167410772	se(slope)	se(intercept)
			0.02045703125	0.043332999999999997											0.9992650117645763	0.0072119984500542125	r2	se(y)
			0.010228515625	0.032333											10876.527961713358	8	F	df
			0.0051142578125	0.015333											0.5657199966268512	4.161033731486749E-4	ss reg	ss resid
			0.00255712890625	0.0043330000000000035
			0.001278564453125	0.0033330000000000026











		Tecan plate reader

		29/10/2024			BLANK	No colour	OD 0.0559
		N-std
		Concentration range	MSM N-full 2x dilutions starting at 26.1850 mg/l
			N-concentration in well [mg/l]	OD at 635nm
			26.185000000000002	over
			13.092500000000001	over
			6.546250000000001	2.6820000186920168
			3.2731250000000003	1.3668999448776245
			1.6365625000000001	0.6788999811172486
			0.8182812500000001	0.3385000005722046											0.41125466740527516	3.9063613246881346E-4	slope	intercept
			0.40914062500000004	0.14899999651908874											0.0017230462860985977	0.004118686081971158	se(slope)	se(intercept)
			0.20457031250000002	0.08479999754428863											0.9998595889744866	0.01090157439403204	r2	se(y)
			0.10228515625000001	0.042099997437000276											56967.582727535395	8	F	df
			0.051142578125000004	0.02290000025033951											6.770273874470369	9.507545941489203E-4	ss reg	ss resid
			0.025571289062500002	0.011900000309944155
			0.012785644531250001	0.006000001019239427

Experiment	Duplicate linear fed-batches
Date	2023-05-01T0:0:0+0000
Folder	2305_MW_KB
Retention times [min]
Internal standard	10.17
C4	5.47
C6	9.5
C8	16.1
C10	23.13
C12	31.3




			GC - peak area data										Mass of PHA monomer										PHA monomer distribution
Sample name	Sample amount [mg]	Methanolysis conversion efficiency, X	Internal standard peak	C4	C5	C6	C7	C8	C9	C10	C11	C12	C4	C5	C6	C7	C8	C9	C10	C11	C12	Total PHA [mg]	C4	C5	C6	C7	C8	C9	C10	C11	C12	Total %PHA
FB4-9	12.3	0.85	199.8	0	0	12.6	0	116.4	0	552.1	0	51.5	0	0	0.013473916461916462	0	0.1136609297297297	0	0.5094171812865497	0	0.045712284893589236	0.6822643123717851	0	0	1.9748821999902795	0	16.659369055169435	0	74.66566432525843	0	6.700084419581853	6.5257227390892885
FB4-25	7.7	0.85	240.1	0	0	35.1	0	335.1	0	1174.9	0	73.2	0	0	0.03123444155844156	0	0.27229268571428566	0	0.9021111879699248	0	0.0540679751552795	1.2597062903979315	0	0	2.479501912193742	0	21.61556926323441	0	71.61281918223611	0	4.292109642335743	19.24684935672928
FB4-29	11.5	0.85	218.4	8.4	0	53.4	0	504.7	0	1800.9	0	122	0.00974723076923077	0	0.05224051048951049	0	0.45085239230769225	0	1.5201564595141699	0	0.09906685618729098	2.1320634492678945	0.4571735786089182	0	2.4502324500449917	0	21.14629339302756	0	71.29977581277703	0	4.646524765541496	21.811390785349303
FB4-33	12.6	0.85	218.4	9.3	0	55.3	0	516.8	0	1827.1	0	123.5	0.010791576923076923	0	0.05409925524475524	0	0.4616614153846153	0	1.542272123481781	0	0.10028489130434783	2.1691092623385764	0.49751191009355733	0	2.494077001286203	0	21.28345599736573	0	71.10163375629199	0	4.623321334962533	20.25312103023881
FB4-50	11.9	0.85	218.4	10.5	0	58.3	0	544	0	1957.7	0	135.4	0.01218403846153846	0	0.057034115384615384	0	0.4859593846153846	0	1.6525127995951414	0	0.10994796989966554	2.3176383079563454	0.5257092282135317	0	2.4608721381942944	0	20.967869876292106	0	71.30158290541459	0	4.743965851885483	22.91288490317692
FB5-7	10.7	0.85	226	0	0	0	0	0	0	31.8	0	0	0	0	0	0	0	0	0.025940002049371212	0	0	0.025940002049371212	0	0	0	0	0	0	100	0	0	0.2852116772883036
FB5-9	11.8	0.85	237.7	0	0	18	0	139.5	0	628.5	0	0	0	0	0.016179388840019886	0	0.11449821960454352	0	0.4874470265482807	0	0	0.6181246349928441	0	0	2.6174962012648453	0	18.523484281753152	0	78.85901951698202	0	0	6.162758075701337
FB5-25	12.7	0.85	249.9	9.8	0	76.6	0	597.6	0	1895.5	0	0	0.00993835294117647	0	0.06549095187165775	0	0.4665498352941176	0	1.3983273684210527	0	0	1.9403065085280045	0.512205308671366	0	3.3752889857253456	0	24.045161588828627	0	72.06734411677466	0	0	17.97412235783237
FB5-27	14.2	0.85	249.9	10.6	0	81.8	0	640.2	0	1917.9	0	122	0.010749647058823529	0	0.0699368128342246	0	0.4998079058823529	0	1.4148520495356036	0	0.08657943734015344	2.081925852651158	0.5163318878592508	0	3.359236485063381	0	24.006998387857543	0	67.95881072007958	0	4.158622519140236	17.248764313597004
FB5-31	9.6	0.85	249.9	8.7	0	68.4	0	520.5	0	1819	0	0	0.008822823529411765	0	0.05848017112299467	0	0.40635741176470586	0	1.3418926315789468	0	0	1.8155530379960592	0.48595790620086066	0	3.2210665234843776	0	22.38201822036707	0	73.91095734994768	0	0	22.249424485245825
FB5-50	13.3	0.85	249.9	0	0	83	0	628.5	0	2033.5	0	0	0	0	0.07096278074866309	0	0.4906736470588235	0	1.5001312074303403	0	0	2.061767635237827	0	0	3.4418418223194904	0	23.798688012784904	0	72.7594701648956	0	0	18.237661523554415








note:
Actual internal standard peak area for samples, FB4-33, FB4-50 and FB5-,FB5- were higher due to interference from accumulated BA-Na in the culture. So, tnternal standard peak areaa of previous samples were used.

		Actual Internal standard peak area
		FB4-33	928.6
		FB4-50	434.4
		FB5-27	286.8
		FB5-31	467.3
		FB5-50	964.8

Experiment	P. putida CA-3 duplicate linear batch fermentations FB4 and FB5
Date	May-jun, 2023


Run Date	Agilent infinity 2

	BA-na standard
	RT = 5.60
	Concentration [mM]	Peak area
	100	38930.897
	50	26488.097				641.9249867451587	172.10082958946623
	25	15972.812				10.006308583743312	102.12568016315453
	12.5	8583.559				0.9985442132180414	228.89247830466408
	6.25	4404.592				4115.482674768923	6
	3.125	2238.151				2.1561740784346524E8	314350.59974670666
	1.5625	1133.563
	0.78125	574.736
	0.390625	291.076
	0.1953125	149.191







		Experiment	Timepoint[h]	Peak area	BA-Na [mM]
		FB4	T-[-1]	17398.246	27.103237917202165
			T-0	18157.953	28.286720411263
			T-2	15263.335	23.7774428476847
			T-4	13136.345	20.463987225922033
			T-5	9926.542	15.46370993496125
			T-7	4179.354	6.510657787124665
			T-9	194.678	0.30327218911866655
		(10x diluted)	T-25	32627.79	50.82804065895549
			T-29	52339.49	81.53521049966896
		100x diluted	T-31	99398.9	154.8450364139113
			T-50	106410.7	165.768119328582
		FB5	T-[-1]	15898.199	24.766443120302217
			T-0	16074.75	25.041476808038325
			T-2	15530.476	24.19359894068622
			T-4	12521.028	19.505437551115786
			T-5	8384.144	13.060940140982204
			T-7	2054.916	3.201177707676131
			T-9	169.19	0.2635666160376991
		10x diluted	T-25	14090.43	21.95027456478561
			T-27	24338.86	37.91542625696149
			T-31	39024.38	60.792740584959304
		100x diluted	T-50	254229.7	396.04268411418786
			T-Exhaust	0	0