Lignocellulosic Conversion - Science topic

I don't think there is one standard protocol for this question. It will be dependent on the strain of S. cerevisiae that you are using and on the nature of the hydrolysate. Is your strain tolerant to inhibitors in the hydrolysate or is it a 'lab' yeast? Are you wanting papers that describe how to prepare the hydrolysate or just papers growing yeast in hydrolysates. Are you just looking for growth or will you have a product other than biomass? If a product, which? Growth conditions will be dependent on requirements for the product - e.g. to produce ethanol you want low oxygen or anaerobic, but to produce something like xylitol you want an aerobic culture. It is often a good idea to do a small-scale preliminary experiment to assess whether the hydrolysate is toxic and if so how toxic. (e.g. a set of low volume flasks with a series of dilutioins of they hydrolysate such as 100, 75, 50, 25% hydrolysate with a constant amount of added sugar, inoculate and assess whether all sugar is consumed in all concentrations). From this you can then define a proper experiment with replicates. If the hydrolysate is very toxic, you may also need to make a 'synthetic' medium in which you add each potential inhibitor in a separate flask so you can assess which ones are most toxic. And of course start with a good analysis of the hydrolysate itself, to know what it contains.

There are numerous reviews on hydrolysates which should provide links to the literature describing how to produce the hydrolysate. There are also reviews on further pre-treatments if they seem necessary. And abundant literature on growing S. cerevisiae in various hydrolysates. I recommend looking not at a single paper, but at several so you have an idea of the range of problems you may encounter. If the strain you are using has been published about before, focus on publications which have used it. If the material that is being hydrolysed is something that has been used before, than focus also on those publications.

Adarsh Shetty

PFA the file for reference, 0.5-2.0% H2O2 works for these types of biomass.

I would like to add to previous answers that salts containing sodium, potassium, magnesium and calcium are the major minerals found in biomass ash, these elements are part of the alkali and alkaline earth metals (AAEM). For example, during pyrolysis, these minerals, especially AAEMs, act as catalysts and possess a great potential to alter the resulting bio-oil composition. The feedstock could be pretreated prior to pyrolysis to reduce its ash content.

Hi! I have the same problem. I have check every detail (acid concentration, temperature, time, etc)... however, none of the expected monosaccharides appear in the HPLC chromatrograms.

I will to try highing the first hydrolysis step (72%) temperature.

You might find the following paper to be of some help.

 Thank you so much for your answers !

Yes, If you have access to anion exchange chromatography with amperometry detection xylan can be quantified.

Dear Shruti,

The term lignocellulosic waste can be used for apple pomace depending on its crude composition. If it contains peel, core, seed, calyx, stem, and soft tissue in it then the lignin content is considerable.  If seed, calyx and stem are not mixed with it then lignin content will be very less, in that case the term 'cellulosic' will be more appropriate to use. I hope got my point. You can find yourself more satisfactory answer if you go through these publications -  http://dx.doi.org/10.1080/07388550801913840  

hrcak.srce.hr/file/37780 agro.icm.edu.pl/agro/element/bwmeta1.element.agro.../praca_053_291-295.pdf

I hope these will be helpful to you.

Best wishes,

Shiladitya

Digestion of plant biomass in 72% H₂SO₄ at 30^oC for 1 hr and hydrolysis in 2 - 4% H₂SO₄ at 121^oC for 60 min is an optimum protocol for reproducible hydrolysis of cellulose and hemicellulose in plant. I carried a comparative analysis with 18, 36 and 72% H₂SO₄ using standard protocol and determined that biomass hydrolysis directly correlated with acid concentration. The poor hydrolysis of the biomass with the lower acid concentration was attributed to inefficient digestion of the biomass at this concentrations. The result indicated digestion step which denature and homogenized the cell wall components was critical for efficient acid hydrolysis of biomass. However, I believe that lower acid concentration could used for Klason lignin determination, if the biomass digestion time and probably the incubation time in the autoclave are increased to ensure efficient hydrolysis of the biomass.

Many thanks Prof.  Ewald for your kind help

Thank you All

Preparing a good compost of rice straw is not easier. You can add cattle dung to fasten the process of composting. You can easily determine the change in lignin, cellulose and sugar content over a period of time by following standard methods of AOAC or NREL. C and N content of any substrate can be determined and addition of cattle dung has nothing to do with it. But yes, addition of cattle dung does help in maintaining an appropriate C:N ratio which is essential for composting.  

Ball Milling is the crucial step in successful lignin extraction. As long as you can crush the biomass matrix, it will be okay.  I have no experience with bead beating and therefore cannot say for sure if it is strong enough. The paper I referred to in the first comment (the no.1) shows the picture of how the solution should look like if the biomass has been subjected to sufficient ball milling. If you use bead beating, you should continue doing so until you obtain the same clarity of solution. (If it appears cloudy, then it's not sufficient, you either have to do it longer or at higher frequency)

In term of heat generation, this is also an issue for ball-milling and hence you need to stop ball milling (or bead beating, if it creates heat, which it should, otherwise i'm sure it's not strong enough) once in a while to let the temperature cools down. In our lab we use liquid nitrogen to freeze the whole metallic container so that we ensure no heat damage to lignin.

Additionally, if you could turn off sonication it would be better. Sonication has been reported to induce minor structural change. Altho should not be a serious issue if you cannot. (Ref:  A. Yoshioka, T. Seino,M. Tabata and M. Takai,Holzforschung, 2000,

54, 357–364.)

I have also came across the same problem while working with fungi, and the probable reason might be degradation of Cellulose content which apparently increase insoluble lignin content. For example initial Lignin and Cellulose were 20% and 80%, If cellulose will be degraded, then Lignin will be increased. 

I am totally agreeing with Dr Babak Salamatinia. pH sensitivity is the vital cause.

After pre-treatment of the substrate with alkali you should have to neutralize that substrate again by washing the substrates with distilled water till it be near to 7.0 pH.

Or else it will affect the growth of fungi as we know fungi love to grow as acidic to neutral not much in alkaline conditions.

All the best.

-AKM 

when acetic acid role as acid,  acid hydrolysis of xylan is faster than other polysacchrides. Also part of lignin are degrade by acetic acid.

if your enzyme hydrolysis environment are gentle, it may can be happended that some bacterials, which have the ability consumping the reducing suger at the enzyme hydrolysis time, multiply rapidly in the liver on your sugar. maybe you should put a little ethyl acetate in this system at the first? trying again.

all the best!!!

@ Dr Henrik Romar, in case of further works and a possible column blocking, which alternative mobile phase would you recommend ?

Theoretical yield of bioethanol from any lignocellulosic residue (kg ethanol/kg lignocellulosic residue) = 0.58 * (Pentosans content per kg of lignocellulosic residue) + 0.57 *  (Hexosans content per kg of lignocellulosic residue).

The above formula may provide you an approximate yield of ethanol.

From the stoichiometry, 1 kg of pentosan yields 0.58 kg of ethanol. 0.57 kg of ethanol is yielded per kg of hexosan.

I want to duly notify that I have stopped working on this project

It strikes me, sometimes when using errosive strains /Trametes versicolor, Phanerochaete chrysosporium/ of white-rot fungi, a small amount of D-glucose to water prior to biomass sterillization can be added. The aim is to supress the fungi production of cellulolytic enzymes.

When smaller dimensions biomass particles are used for biopulping, the suspension of an inoculum may be sprayed /sprinkled/ directly onto them. After homogenzation the inoculated material is transported into the bioreactor.    

At 175 C the vapor volume of a 1 ul injection of water is around 1000 ul which is more than many injection liners can tolerate, so you will get flashback into the carrier gas lines to the injector. This can cause a lot of problems, including carryover and imprecision. As Kae Kwon said, you need a higher oven temperature, but also reduce the injection volume. A lot of analysts are afraid of injecting water, but most modern bonded phase columns can tolerate water without degradation. However, you do not mention what type of capillary column you are using. It should be a polar phase to give good retention and peak shape, such as a bonded Carbowax. It could also be done on a PLOT column such as Rt-Q-BOND. Also, the ethanol must elute well separated from the water. If they co-elute then the disturbance of the FID will yield very poor precision. Check the literature of your column supplier for the application.

Hi Florian. I am not sure about Folin Cioceltau method, but I use method reported by National Renewable Energy Laboratory (NREL) for determination of lignin content. You could access more information from their website. See link below:

Hope this help. Good luck!

Thank you again Buenaflor for your response.

We already have the fungi growing on lignocellulose media but my goal now is to make a model of the environment from which we isolated the fungi; hence, my question regarding the specific conditions for a pretreatment analog.