Richard Lab

Tom Richard[1]
Associate Professor, Department of Agricultural and Biological Engineering
Director, Biomass Energy Center, Penn State University

What we do: Engineer solid-state microbial bioconversion processes for renewable energy and sustainable agroecosystems

Penn State Richard website: http://www.abe.psu.edu/fac/Richard/Overview.htm
Penn State iGEM team: http://openwetware.org/wiki/IGEM:PennState
Penn State Biomass Energy Center: http://www.bioenergy.psu.edu

Richard Lab group

Post Doc

• Hala Chaoui email

Projects: Design and conduct experiments to (1) calibrate flux chamber for use with a photoacoustic sensor to meaaure greenhouse gas emissions; (2) evaluate greenhouse gas emissions from animal waste storage, and test the effect of biofilter types on theses emissions.

Future project: Test the feasibility of scaling down biodigesters.

Reviews: Biolfiters; biodigester exhaust gases remediation techniques, modeling of non-greenhouse gas emissions from animal facilities.

Graduate Students

• Deepti Tanjore

• Farzaneh Razaei

• Marlyse Williams

• Qin Chen

• Scott Shore

Undergraduate Students

• Allie Srebro

• Eric White

• Natalie Shewski

Our Methods

Corn stover harvest and storage

1. Call harvest center to make sure the machine availability;

2. Preparation: gloves (thinner and thick); working clothes; tapes; bags; buckets and car keys.

3. Measure the length of the field and set a sign for the end of harvesting;

4. Harvest ears by hands;

5. Place plastic bags into buckets;

6. Download the chopped stovers to buckets;

7. Label the buckets;

8. Weight the ears and stover and label them;

9. Weight the foil boxes and around 500 gram stovers with triplicates and record the weight and put them into oven at 105 C.

10. Weight the foil boxes and around 1200 gram corns with triplicates and record the weight and put them into oven at 105 C.

11. Shelling the cobs and shelling cobs.

12. Doing quick moisture measurement at the same time of processing cobs.

13. According to moisture content from 12 and do calculations, including water addition, enzyme addition, cobs addition.

14. Prepare water bottles, spray bottles, buckets, plastic bottles;

15. Make vacuum bags, 48 samples: 48 smaller bags and 48 bigger bags;

16. For no water addition samples:

1) Control:

a) Weight the cobs and stover for mixing;

b) Mix stover with cobs;

c) Weight the bags;

d) Seal stover into bags and record the weight;

e) Double bags;

2) Enzymes:

a) Weight two ½ required cobs;

b) Separate the stover into two buckets;

c) Mix half stover with half cobs individually;

d) Dilute the required enzyme with 20 ml D.I. water and spray them on these two buckets;

e) Mix the two buckets stover and cobs well;

f) Weight the bags;

g) Seal stover into bags and record the weight;

h) Double bags;

17. For water addition samples:

1) Control:

a) Prepare the required water and transfer to corresponding spray bottle;

b) Weight the cobs for mixing;

c) Weight the stover for mixing;

d) Mix stover with cobs;

e) Spray DI water and mix with stover and cobs;

f) Weight the bags;

g) Seal stover into bags and record the weight and label them;

h) Double bags;

2) Enzyme:

a) Prepare the required water and transfer to corresponding spray bottle;

b) Weight two ½ required cobs;

c) Weight two ½ required stovers;

d) Separate the stover into two buckets;

e) Mix half stover with half cobs individually;

f) Dilute the required enzyme with required D.I. water and spray them on these two buckets;

g) Mix the two buckets stover and cobs well;

h) Weight the bags;

i) Seal stover into bags and record the weight and label them;

j) Double bags;

The mixture ratio of cobs and stover can be calculated :

(from harvest corn and stover)
(from chopped cobs and shelled corn)

Moisture content measurement

1. Quick method

Weight around 200 gram stover and record the weight and put the stover into vortex, keep drying 40 mins, and then weight the dried stover and record the weight. To make sure the stover to be dried thoroughly, keep drying 10 to15 mins more and record the stover weight again, until there is no change of stover weight. Repeat the same steps for other replicates and cobs.

2. Traditional method

Weight around 500 gram stover and record the weight and put it into oven (105°C) 1 day and record the weight. Check the weight one day again and keep do it until there is no weight loss by continuously oven drying. The corn ears and cobs moisture content can be measured by the same way.

Dry matter (D.M.) content and moisture content (M.C.) can be calculated.

Calculate the amount of the enzyme addition

To explore the effects of enzyme addition on stover silage, an industrial enzyme (Multifect A40, Genencor, Palo Alto, CA) including cellulose and hemicelluose, will be added to the mixture of stover and cobs.

Enzyme activities were measured by the methods of Wood and Bhat (1988) and Bailey et al., (1992) by using carboxymethylcellulose (CMC) and birchwood 4-o-methyl glucoronoxylan (Roth 7500) as substrates of endo-1,4- -glucanase and xylanase.

The enzyme rate, 5 IU/ g DM, has been determined by former experiments of our lab. The lowest effective amount of enzyme was determined by the calculation of the ratio of the lowest effective enzyme rate to the sum of enzyme activities of xylan and CMC as (ml/g DM) Where EAxylan denoted xylan activity (IU/ml) and EACMC denoted CMC activity (IU/ml).

The addition of enzyme volume is ml/g DM.

Silage preparation

1. Silage stover without water addition Take 24 samples, half for control and half for enzymatic treatment, for example. According to the calculated ratio of cobs to stover, weight gram stover and gram of cobs. For control, mix chopped cobs, (gram) and stover, , very well. For enzyme treatment, separate the stover into three parts, and mixed each part of stover, , with one third of the required cobs, and enzymes well and then mixed the three boxes of stover with cobs and stover together for obtaining well mixed effects.

2. Silage stover with water addition Water will be added into stover to get desired moisture content, 60 %. The amount of water and stover addition can be calculated according to the following formula. For control, The stover weight: The cobs weight:

The water amount:

For enzyme treatment, The weight of the stover and cob is the same as control experiment, but water amount is different as following:

Weight the bag, around 20 cm 40 cm airtight bag made of polyethylene, and take around 500 g samples, including stover and cobs, and pack tightly, vacuumed and sealed by a food vacuum sealer. Record the weight of the bag and samples. Samples will be double bagged with each bag sealed to guarantee anaerobic conditions for stover fermentation.

The enzyme treated and control samples will be ensiled at 37 1 °C in incubator and destructively sampled on 0, 1, 7 and 21 days. Stover weight and pH value were measured immediately on each sampling date. The remaining stover was frozen (-15 ºC) for later chemical analysis, including as dry matter, fiber fractions, water soluble carbohydrates (WSC), monosaccharides, ethanol and organic acids.

Weight loss and pH measurement

On 0, 1, 7 and 21 days, the stover weight and pH will be measured. Stover weight was recorded before and after the ensilage process. Weight loss refers to the difference of the weight of stover with the inner bag before silage and that after silage.

After the sample bag was opened and the stover was mixed well, a 10 g composite sample of stover was subsampled from different parts of the bag. The 10 g sample will be put in a weighted cup, and then homogeneously mixed with 100 ml deionized water, and pH was measured by a pH electrode on the sample solution after equilibrating for 30 minutes.

Dry matter and Dry matter loss percentage

A100g composite sample of sover was subsampled from different parts of the bag and put into weighted food paper bags, dried at 60 ºC in a forced air oven for 2 days, and reweighed. The dry matter (D.M.) can be calculated as:

C.M.% = D.M.% = 1- C.M.% D.M. (g) = Dry mater loss percentage =

Fiber analysis

All the samples were ground with a hammer mill to pass through a 1 mm screen. Accurately 0.5 g powder from each sample was measured out for sequential analysis to determine the contents of NDF, ADF, ADL and ash by the ANKOM procedure (Ankom, 2005).

Note:

a. Measure the M.C. of the grinded powder by the following steps:

i. Weight the containers and record the weight for all the samples;

ii. Place around 1.5 to 2.0 gram power into the containers and record the weight;

iii. Place all the container into oven and dry around 2 days at 105 °C;

iv. Place the dried powder with container into desiccators for cooling;

v. Cool to room temperature and record the weight of stover with bag one by one.

vi. The M.C. can be calculated by the ratio of the weight difference to the net weight of initial power.

b. Label the filter bags with a solvent resistant maker;

c. Filter bag should be weighted and recorded before get stover powders;

d. Do not let powder fall near the open edge of bags;

e. Seal the bag within 0.4cm from the open edge by heat sealer.

f. Double seal the bag to prevent from powder loosing;

g. Flicking the bag to avoid clumping;

h. Each tray can be used for 3 bags, and all of them should be stack trays on center with each one rotated 120 degree with a little bit of overlap to each other for keeping them on the trays in the running process;

i. The maximum number of bags is  ;

j. The weight should be placed on the top of the empty tray to make the suspender can be submerged into detergent.

k. Put the suspender into apparatus, close the valve, then add NDF solution and 20 g sodium sulfite and 4 ml heat stable α-amylase.

l. Place the suspender into solution and make sure that it is submerged into it.

m. Turn Agitate and heat ON, close the vessel lid and then set 75 minutes.

n. Heat about 5 times of 2000ml for rinsing;

o. Exhaust hot solution into strain with tag water before open the lid;

p. Close the valve and use about 2000ml preheated water with 4.0 ml α-amylase and agitate 5 mins, then exhaust water and repeat rinse 4 times (total 5 times);

q. Drain the water and remove suspender.

r. Rise vessel with cold tap water for next run.

s. Remove filter bags and press out water gently.

t. Place all the bags into beaker and then cover them with acetone around 5 to 10 mins;

u. Remove filter bags and press out acetone gently.

v. Transfer acetone into recycle bottles for recycle.

w. Place all the bags into hood and let them dry around 1 to 2 hours.

x. Place filter bags in oven and dry around 3 hours at 105 °C;

y. Remove 5 to 8 bags into MoistureStop weigh punch and remove air by flattening the bag.

z. Put the bag into desiccators around 20 mins;

aa. Place another MoistureStop weight punch with 5 to 8 bags into desiccator before weight the cooled filter bags;

bb. Cool to room temperature and weight and record the weight of stover with bag one by one. Here the weight is the sample with bag weight after NDF.

cc. Transfer the filter bag from MoistureStop weight punch by a tweezer to scale.

dd. After get the samples with bag after NDF, spread sample uniformly by flicking the bag again;

ee. Put the filter bags into vessel and use the same way to place them on the trays and use 1900-2000ml ADF solution;

ff. Agitate and heat the samples about 70 mins;

gg. Heat about 3 times of 2000ml for rinsing;

hh. Use the boiling water to rinse three times by the same way as that of NDF, but without α-amylase.

ii. Repeat steps from q. to cc..

jj. The weight of the sample with bag after this process is called sample and bag weight after ADF.

kk. Place the weighted sample with bags into MoistureStop for ADL experiment;

ll. Make 72 % by weight sulfuric acid by adding 1200 H2SO4 to 2000ml beaker with 440 ml H2O ; (Use rubber gloves and face shield)

mm. After cooling, transfer it into volumetric flask for later use; (Use rubber gloves and face shield)

nn. After get the samples with bag after ADF, spread sample uniformly by flicking the bag again;

oo. Put 24 samples into one glass jar, add 500ml 72% H2SO4 to cover bags;

pp. Place jars into DaisyII.

qq. Turn rotate on for 3 hours without heating.

rr. Heat about 6 times of 2000ml for rinsing;

ss. Pour off H2SO4 into recycle bottle and rinse the filter bags with boiling water;

tt. Repeat 6 or more times until the pH is neutral.

uu. Place the bags of the same bottle into 250 ml acetone for 3 mins for removing water;

vv. Put all the bags into hood and let them dry around 1 to 2 hours;

ww. Place all the bags into oven and dry for 4 hours.

xx. Repeat y to cc and record the weight, which is the sample and bag.weight after ADL. yy.

Note: The bag correction factor for each step is calculated by dividing the weight of the empty bag in the run by it's original weight. NDF, ADF, ADL, and Lig calculations will have different bag correction factors.

After ADL determination the sample bags were folded and put into individually weighed porcelain crucibles, which were combusted thoroughly in a muffle furnace at 550 ºC for 5 hours, and the crucibles with ash were then cooled in desiccators and reweighed. Ash was determined by the weight difference between the crucible with sample before combustion and after complete combustion.

Reference: www.ankom.com

Also, thanks Trish for sharing her experience.

Note: All the formula from word can not be pasted here.

Managing the lab

Notices

Lab meetings: Fall 2006, Friday afternoons

1:30 - 2:30 Lignicellulosic hydrolysis/biofuels/microbial fuel cells
2:30 - 3:30 Everyone, special topics each week
3:30 - 4:30 Greenhouse gas emissions/biogas/compost
4:30 - ???? TGIF