Everything About Wood: 06/17/16

Friday, 17 June 2016

Exercise: Walking Vs. Running

Walking and running are effective cardiovascular exercises that can help you burn calories and improve your health. The Centers for Disease Control and Prevention recommends that adults up to age 65 have several sessions of moderate to vigorous physical activity per week such as aerobic exercise. Both walking and running can help you achieve the goal of including moderate to vigorous physical activity every week depending on the intensity you put into these exercises.

Although there has been much debate about the number of calories burned from walking versus running the same distance, a study in a 2012 "Journal of Strength and Conditioning Research" showed that participants who walked one mile burned 89 calories during the exercise and 110 calories over the next few hours after the workout, while participants who ran one mile burned 112 calories during the exercise and 159 calories as the day progressed.

The impact on your joints is generally less from walking than running because of the increased up-and-down movement of your body when running, which increases joint impact. You can work with an exercise science specialist or a physical therapist to improve your running gait and reduce your up-and-down motion. However, bouncing off the ground when you walk can increase the impact on your joints, so take care to walk evenly, with little bouncing.

One of the most important factors determining how effective your workout is from walking or running is the intensity of effort. For instance, running on a level path requires less effort than running up a hill, and walking at a brisk pace requires more effort than a leisurely walk does.

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Why Do You Stretch After Sleeping?

Every morning when you wake up, instinct takes over, and you feel compelled to stretch out your body. Why do we do this? Aside from feeling good, it actually does help to wake you up. Stretching works to increase flexibility and range of motion in the muscles and joints. It also improves circulation and relieves tension.

After a long, restful sleep you often find yourself waking up with stiff, tight muscles. This is usually caused by lying in the same position for an extended amount of time. Upon waking, you often stretch out the kinks in your neck, back and legs, maybe even subconsciously, leaving you feeling better. When your muscles are flexible, it makes getting out of bed easier, along with performing everyday tasks.

The main causes of morning stiffness are lack of daily physical activity, being overweight, poor diet, not sleeping properly, and a cold or damp environment, according to the Healthy Back Institute. Stretching in the morning helps improve the range of motion in the joints, as well as stiffness that often accompanies being inactive or overweight. Decreased flexibility in the joints may increase your likelihood of developing arthritis or having poor balance, which could result in injury.

Stretching in the morning dramatically improves blood circulation. A quick stretch upon waking can energize you to get out of bed. The heart rate is slowest just before rising, so when you stretch, your blood starts moving faster and circulating to the muscles in the extremities. Proper circulation helps you move and function throughout the day.

Stretching helps you relieve stress and tension. Sometimes it is hard to get out of bed in the morning, especially when you know that you have a stressful day ahead. Taking a few minutes first thing in the morning to stretch will help relieve some tension and allow you to face the day more positively. To get the most out of your stretches, hold each stretch for 30 seconds to give your muscles time to lengthen, then relax and breathe normally.

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Optimization of the Chitinase Production by Different Metarhizium anisopliae Strains under Solid-State Fermentation with Silkworm Chrysalis as Substrate Using CCRD

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DOI: 10.4236/abb.2012.35083

Author(s)

Azza Naik, Smita Lele

References

[1]	Jurenka, J. (2008) Therapeutic Applications of Pomegra nate (Punica granatum L.): A Review. Alternative medicine review, 13(2), 128-144.

[2]	Aghsaghali, A.M. (2011) Evaluating potential nutritive value of pomegranate processing by-products for ruminants using in vitro gas production technique. ARPN Journal of Agricultural and Biological Science, 6(6), 45-51.

[3]	EL-Nemr, S.E., Ismail, I.A., Raga, M. (1990) Chemical composition of juice and seeds of pomegranate fruit. Die Nahrung, 34(7), 601-606. doi:10.1002/food.19900340706

[4]	Xu, B., Wang, Q., Jia, X., Sung, C. (2005) Enhanced lovastatin production by solid substrate fermentation of Monascus rubber. Biotechnology and bioprocess engineering, 10, 78-84.

[5]	Manzoni, M., Bergomi, S., Rollini, M., Cavazzoni, V. (1999) Production of statins by filamentous fungi. Biotechnology letters, 21, 253-257. doi:10.1023/A:1005495714248

[6]	Samiee, S.M., Moazami, N., Haghighi, S., Mohseni, F.A., Mirdamadi, S., Bakhtiari, M.R. (2003) Screening of lovastatin production by filamentous fungi. Iranian biomedical journal, 7(1), 29-33.

[7]	Szakács, G., Morovján, G., Tengerdy, R.P. (1998) Production of lovastatin by a wild strain of Aspergillus terreus. Biotechnology Letters, 20(4), 411-415.

[8]	Jia, Z., Zhang, X., Zhao, Y., Cao, X. (2010) Enhancement of lovastatin production by supplementing polyketide antibiotics to the submerged culture of Aspergillus terreus. Appl Biochem Biotechnol, 160(7), 2014-25. doi:10.1007/s12010-009-8762-1

[9]	Kim, H., Moon, J.Y., Kim, H., Lee, D.S., Cho, M., Choi, H.K., Kim, Y.S., Mosaddik, A., Cho, S.K. (2010) Antioxidant and antiproliferative activities of mango (Mangifera Indica L.) flesh and peel. Food Chemistry, 121, 429-436. doi:10.1016/j.foodchem.2009.12.060

[10]	Pandey, A. (2003) Solid-state Fermentation. Biochemical Engineering Journal, 13, 81-84. doi:10.1016/S1369-703X(02)00121-3

[11]	Wei, P., Xu, Z., Cen, P. (2007) Lovastatin production by aspergillus terreus in solid state fermentation. Journal of Zhejiang, 8(9), 1521-1526. doi:10.1631/jzus.2007.A1521

[12]	Vilches Ferron, M.A., Casas Lopez, J.L., Sanchez Perez, J.A., Fernandez Sevilla, J.M., Chisti, Y. (2005) Rapid screening of aspergillus terreus mutants for overproduction of lovastatin. World journal of microbiology & biotechnology, 21, 123-125. doi:10.1007/s11274-004-3045-z

[13]	Valera, H.R., Gomes, J., Lakshmi, S., Gururaja, R., Suryanarayan, S., Kumar, D. (2005) Lovastatin production by solid state fermentation using aspergillus flavipes. Enzyme and microbial technology, 37, 521-526. doi:10.1016/j.enzmictec.2005.03.009

[14]	Chen, F., and Hu, X. (2005) Study on red fermented rice with high concentration of monacolin K and low concentration of citrinin. International Journal of food Microbiology,103(3),331-337. doi:10.1016/j.ijfoodmicro.2005.03.002

[15]	Pansuriya, R.C., and Singhal, R.S. (2010) Response surface methodology for optimisation of production of Lovastatin by Solid State Fermentation. Brazilian Journal of Microbiology, 41, 164-172. doi:10.1590/S1517-83822010000100024

[16]	Sayyad, S.A., Panda, B.P., Javed, S., Ali, M. (2007) Optimization of nutrient parameters for lovastatin production by Monascus purpureus MTCC 369 under submerged fermentation using response surface methodology. Applied. Microbio. Biotech,73, 1054-1058. doi:10.1007/s00253-006-0577-1

For further details log on website :
http://www.scirp.org/journal/PaperInformation.aspx?PaperID=22540

Production of iturin A through glass column reactor (GCR) from soybean curd residue (okara) by Bacillus subtilis RB14-CS under solid state fermentation (SSF)

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DOI: 10.4236/abb.2012.32021

Author(s)

Abdul Wahab Khan, Umme Salma Zohora, Mohammad Shahedur Rahman, Masahiro Okanami, Takashi Ano

References

[1]	Robinson, T., Singh, D. and Nigam, P. (2001) Solid-state fermentation: A promising microbial technology for secondary metabolite production. Applied Microbiology and Biotechnology, 55, 284-289. doi:10.1007/s002530000565

[2]	Mizumoto, S., Hirai, M. and Shoda, M. (2006) Production of lipopeptide antibiotic iturin A using soybean curd residue cultivated with Bacillus subtilis in solid-state fermentation. Applied Microbiology and Biotechnology, 72, 869-875. doi:10.1007/s00253-006-0389-3

[3]	Yang, S.Q., Yan, Q.J., Jiang, Z.Q., Li, L.T., Tian, H.M. and Wang, Y.Z. (2006) High-level of xylanase production by the thermophilic Paecilomyces themophila J18 on wheat straw in solid-state fermentation. Bioresource Technology, 97, 1794-1800. doi:10.1016/j.biortech.2005.09.007

[4]	Khare, S.K., Jha, K. and Gandhi A.P. (1995) Citric acid production from okara (soy-residue) by solid-state fermentation. Bioresource Technology, 54, 323-325. doi:10.1016/0960-8524(95)00155-7

[5]	Ohno, A., Ano, T. and Shoda, M. (1996) Use of soybean curd residue, okara, for the solid state substrate in the production of a lipopeptide antibiotic, iturin A, by Bacillus subtilis NB22. Process Biochemistry, 31, 801-806. doi:10.1016/S0032-9592(96)00034-9

[6]	Ohno, A., Ano, T. and Shoda, M. (1995) Effect of temperature on production of lipopeptide antibiotics, iturin A and surfactin by a dual producer, Bacillus subtilis RB14, in solid-state fermentation. Journal of Fermentation and Bioengineering, 80, 517-519. doi:10.1016/0922-338X(96)80930-5

[7]	Hiraoka, H., Ano, T. and Shoda, M. (1992) Characteristics of Bacillus subtilis RB14, copoducer of peptide antibiotics iturin A and surfactin. The Journal of General and Applied Microbiology, 38, 635-640. doi:10.2323/jgam.38.635

[8]	Phae, C. G., Shoda, M. and Kubota. H. (1990) Suppressive effect of Bacillus subtilis and its products on phytopathogenic microorganisms. Journal of Fermentation and Bioengineering, 69, 1-7. doi:10.1016/0922-338X(90)90155-P

[9]	Asaka, O. and Shoda, M. (1996) Biocontrol of Rhizoctonia solani damping off of tomato with Bacillus subtilis RB14. Applied and Environmental Microbiology, 62, 4081-4085.

[10]	Mizumoto, S., Hirai, M. and Shoda, M. (2007) Enhanced iturin A production by Bacillus subtilis and its effect on suppression of the plant pathogen Rhizoctonia solani. Applied Microbiology and Biotechnology, 75, 1267-1274. doi:10.1007/s00253-007-0973-1

[11]	Ohno, A., Ano, T. and Shoda, M. (1995) Production of a lipopeptide antibiotic, surfactin, by recombinant Bacillus subtilis in solid-state fermentation. Biotechnology and Bioengineering, 47, 209-214. doi:10.1002/bit.260470212

[12]	Phae, C.G. and Shoda, M. (1991) Investigation of optimal conditions for foam separation of iturin, an antifungal peptide produced by Bacillus subtilis. Journal of Fermentation and Bioengineering, 71, 118-121. doi:10.1016/0922-338X(91)90235-9

[13]	O’brien, J., Wilson, I., Orton, T. and Pognan, F. (2000) Investigation of the alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. European Journal of Biochemistry, 267, 5421-5426. doi:10.1046/j.1432-1327.2000.01606.x

[14]	Ohno, A., Ano, T. and Shoda, M. (1993) Effect of temperature change and aeration on the production of the antifungal peptide antibiotic Iturin by Bacillus subtilis NB22 in liquid cultivation. Journal of Fermentation and Bioengineering, 75, 463-465. doi:10.1016/0922-338X(93)90098-S

[15]	Nakano, M.M. and Zuber, P. (1998) Anaerobic growth of a “strict aerobe” (BACILLUS SUBTILIS). Annual Review of Microbiology, 52, 165-190. doi:10.1146/annurev.micro.52.1.165

For further details log on website :
http://www.scirp.org/journal/PaperInformation.aspx?PaperID=18454

Dinner for a Balanced Diet

A healthy dinner plan can help you avoid making unhealthy last-minute food choices. Organize your dinner plans by creating weekly menus and making a shopping list based on your meal plans. Consider trying new varieties of fruits, vegetables, grains and proteins, and choose whole foods over processed foods more often.

A balanced dinner includes two to three servings of fruits and vegetables, a serving of whole grains and a lean protein option. Include 1 cup of milk with your dinner if you need help getting the recommended amount. Adults need the equivalent of 3 cups of milk daily, according to the U.S. Department of Agriculture. The Centers for Disease Control and Prevention suggest using a small dinner plate, reserving the largest portion for vegetables, fruit and whole grains.

One cup of vegetables is the equivalent of two servings, with the exception of raw leafy greens which count cup for cup as a serving. One slice of whole wheat bread, 5 whole wheat crackers or 1/2 cup brown rice, whole grain pasta or bulgur constitute a serving of whole grains, according to the USDA. One serving of lean protein is approximately 3 oz. poultry, meat or fish, or 3/4 cup cooked dry beans.

Nutrients in vegetables vary by color and variety so include more than one color of vegetable on your dinner plate. Pair carrots with spinach, sweet potatoes with broccoli or a salad with tomatoes, for example. Consider serving less common grain varieties such as millet, quinoa or amaranth. Other options include a whole wheat roll or 6-inch whole wheat tortilla. The American Heart Association recommends serving fish at least twice a week for heart health. Other healthy protein options include chicken or turkey breast, lean bottom round or legumes such as black beans, chick peas or white beans.

A balanced diet helps you manage your calorie intake and get the nutrients you need for good health. Eating the recommended amounts of fruits and vegetables potentially reduces your risk for developing chronic diseases including stroke, cardiovascular disease and certain cancers, according to the CDC. The National Institutes of Health reports that increasing fiber, specifically fiber from grains, helps protect against diabetes, according to the National Institutes of Health. Eating two to three sources of protein a day provides enough for healthy cell function, according to Medline Plus.

A healthy and balanced diet consists of at least three meals a day. To reap the benefits of a balanced dinner, include a healthy breakfast and lunch in your eating plan. Your total calorie intake is also an important factor in your diet. Consult the My Pyramid Food Intake Patterns to identify how many servings you need from each food group daily to meet your calorie goal.

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An Average Heart Rate After Drinking a Monster Energy Drink

Many consumers are drawn to energy drinks to feel effects such as energy boosts, enhanced concentration and increased stamina. There are many brands of energy drinks, including Monster, that provide these desired outcomes. The concern for many health professionals is the impact these drinks are having on the body, particularly the heart. There is a significant increase in heart rate each time these drinks are consumed.

Monster comes in different many varieties, such as Lo Carb, XXL and assault. Each of the types have slight differences but the main ingredients remain the same. To provide the desired outcomes for energy, Monster energy drinks are filled with caffeine, taurine and sugar. In an average 8-oz. serving of Monster, there are 27 grams of sugar, 1,000 milligrams of taurine and 80 milligrams of caffeine. There are between two and three servings per container.

The heart's electrical system is the controller of your heart rate. In a normal rhythm, each beat starts at the top of the heart at the sinoatrial node, passes through the atrioventrical node and then down into the lower ventricles. This provides blood to the rest of the body. When the body is provided a stimulant, the heart cannot always keep up with the demand, and it may cause an abnormal heartbeat. Consistent stress on the heart makes the heart more susceptible to arrhythmias.

Caffeine and taurine have been shown to have effects on the heart's function. Although each person reacts to energy drinks differently, these ingredients can negatively affect the heart over time. In a study from "Circulation," the average heart rate increases between five and seven beats per minute after consuming energy drinks. The average resting heart rate is between 60 and 100 beats per minute. If your heart rate is consistently above 100 while drinking Monster energy drinks you should discontinue use.

Consuming large amounts of caffeine can lead to health conditions such as anxiety, insomnia, digestive issues and high blood pressure. Although there are benefits to Monster energy drinks, they should be consumed in moderation. If you feel any palpitations, heart racing or uncontrollable restlessness, seek medical attention. Never use alcohol while consuming Monster energy drinks.

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Negative Effects on the Heart From Energy Drinks

Caffeine remains the most widely consumed drug. Present in chocolate and coffee, manufacturers also add the stimulant to sports and energy drinks. These beverages have become increasingly popular, especially with college students. According to 2011 review in "Kardiologia Polska," ingesting large amounts of caffeine increases the damage caused by heart disease. Energy drinks have other adverse effects on the heart as well. Speak with a doctor before regularly consuming caffeinated beverages.

Cardiac arrest, or heart failure, remains a leading cause of death throughout the world. According to a 2004 review in the "Internist," people need to exercise more and eat less. Avoiding toxic chemicals, or at least minimizing your exposure to them, remains equally important. A case described in the 2009 volume of the "Medical Journal of Australia" illustrates the consequences of overindulgence. A healthy 28-year-old male athlete spent an entire day performing at a motocross competition. He consumed large amounts of energy drinks throughout that day. During the competition, the man experienced mild chest pains. At the event's end, he collapsed of heart failure. Paramedics were able to revive him, and he eventually returned to an active lifestyle

Having bradycardia, an unusual slowing of the heart, places you at risk for heart attack. Arrhythmias remain difficult to treat despite increased medical knowledge and awareness. Nutritional supplements were thought to provide a treatment option. However, according to a 2005 report in the "Journal of the American Medical Association," supplements such as fish oil can worsen arrhythmias. An experiment presented in the 2006 edition of "Amino Acids" looked at the cardiac effects of substances commonly added to energy drinks. Healthy subjects received either caffeine and taurine or an inert treatment during a single testing session. The simulated energy drink significantly decreased heart rate, a symptom of bradycardia.

Atrial fibrillation is another common type of heart arrhythmia. The atrium, or the upper two chambers of the heart, flutters instead of clearly contracting if you have this type of heart pathology. According to a 1997 article in "Acta Cardiologica," caffeine can trigger atrial fluttering in animal subjects. Studies done with human patients have shown equivocal results. Yet, two cases presented in the 2011 edition of the "Journal of Medical Case Reports" indicate that ingesting caffeinated energy drinks can trigger atrial fibrillation. Two adolescent boys reported to the hospital with chest pains after drinking energy drinks. Each patient had no history of heart problems, but each boy showed atrial fluttering. Medication alleviated the flutter of one boy, and hydration resolved it in the other.

In postural tachycardia, your heart races when you quickly move into the standing position. Other health problems become associated with postural tachycardia. According to a study reviewed in the 2011 volume of the "Journal of Clinical Sleep Medicine," people with postural tachycardia also experience a reduction in life quality and have difficulty sleeping. The mechanism underlying this syndrome remains unknown. A 2008 report in "Clinical Autonomic Research" describes the case of an adolescent volleyball player who regularly ingested excessive amounts of energy drinks. The hospital admitted the girl due to repeated fainting spells. Upon close inspection of her routine, the doctors determined that the athlete was inadvertently overdosing on the energy drinks. This toxicity triggered postural tachycardia that remitted when the girl stopped drinking them.

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Natural Foods for Dry Mouth

Dry mouth, also called xerostomia, can cause serious problems that affect the enjoyment of food and the condition of the mouth. Without saliva, food and bacteria linger in the mouth, accelerating tooth decay. In addition, lack of saliva alters the taste of food and prevents the start of digestion. There are numerous causes of a dry mount including medication, cancer therapy, and Jorgen’s Syndrome, according to academic papers published on the University of Chicago at Illinois website In addition to medication, treatment also includes altering the diet with natural foods.

The American Cancer Society recommends drinking 8 to 10 cups of liquids a day when suffering from a dry mouth. It is especially important to drink liquids during meal times. The chosen liquids should be sugar free, given the propensity for tooth decay with dry mouth. Water is the preferred beverage, according to the University of Illinois at Chicago, but sugar free juices, caffeine free diet soda, sports drinks, club soda, and decaffeinated hot tea with lemon are also acceptable. Folks with a dry mouth may have less of a desire to eat due to the taste changes, and the American Cancer Society suggests nutritional supplements or milkshakes to help meet needs while keeping the mouth moist.

The American Cancer Society says to eat soft foods with xerostomia. It also suggests that foods be eaten at room temperature. Examples of soft natural foods for people with a dry mouth include tender meats, chicken, and fish, smooth peanut butter, cream soups, strained soups, cottage cheese, yogurt, canned fruits, soft cooked/blended vegetables, mashed potatoes, soft cooked pasta, cooked cereals, ice cream, pudding, popsicles, smoothies and slushies. The American Cancer Society also suggests using gravies, sauces, and broth to add moisture to foods to ease swallowing.

According to Sjorgen’s Syndrome Foundation, sugar free candies, sugar free gum, diabetic candies, fruit pits of the cherry or olive and lemon rinds can help stimulate saliva. They recommend foods sweetened with xylitol, which has been shown to help prevent tooth decay. The American Cancer Society also suggests lemon drops to stimulate saliva.

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Fate of Carbohydrates and Lignin during Composting and Mycelium Growth of Agaricus bisporus on Wheat Straw Based Compost

Author

Published: October 5, 2015
http://dx.doi.org/10.1371/journal.pone.0138909

Abstract

In wheat straw based composting, enabling growth of Agaricus bisporus mushrooms, it is unknown to which extent the carbohydrate-lignin matrix changes and how much is metabolized. In this paper we report yields and remaining structures of the major components. During the Phase II of composting 50% of both xylan and cellulose were metabolized by microbial activity, while lignin structures were unaltered. During A. bisporus’ mycelium growth (Phase III) carbohydrates were only slightly consumed and xylan was found to be partially degraded. At the same time, lignin was metabolized for 45% based on pyrolysis GC/MS. Remaining lignin was found to be modified by an increase in the ratio of syringyl (S) to guaiacyl (G) units from 0.5 to 0.7 during mycelium growth, while fewer decorations on the phenolic skeleton of both S and G units remained.

Figures

Citation: Jurak E, Punt AM, Arts W, Kabel MA, Gruppen H (2015) Fate of Carbohydrates and Lignin during Composting and Mycelium Growth of Agaricus bisporus on Wheat Straw Based Compost. PLoS ONE 10(10): e0138909. doi:10.1371/journal.pone.0138909

Editor: Kristiina Hildén, University Of Helsinki, FINLAND

Received: May 8, 2015; Accepted: September 6, 2015; Published: October 5, 2015

Copyright: © 2015 Jurak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: The work described in this manuscript was performed at the Laboratory of Food Chemistry, Wageningen University, The Netherlands. EJ, WA, MK and GH were financially supported by the Technology Foundation STW (grant number 11108), applied science division of NWO and the technology programme of the Ministry of Economic Affairs (The Netherlands) and by Walkro International B.V., Sylvan Inc. and DSM Food Specialities. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Wim Arts is employed by C4C Grondstoffen B.V. C4C Grondstoffen B.V. provided support in the form of salary for author WA, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the ‘author contributions’ section.

Competing interests: EJ, WA, MK and GH were partly supported financially by CNC Grondstoffen B.V., Walkro International B.V., Sylvan Inc. and DSM Food Specialities. Wim Arts is employed by C4C Grondstoffen B.V. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Highlights:

50% of xylan and cellulose are metabolized in composting.
During A. bisporus’ mycelium growth 45% of lignin was metabolized.
S:G ratio of remaining lignin increases from 0.5 to 0.7 during mycelium growth.
Part of the guaiacyl units of lignin become water soluble during mycelium growth.

Introduction

In the conventional European process, compost for mushroom growth is produced from a basic mixture (BM) of straw bedded horse manure, wheat straw, poultry manure and gypsum [1]. The BM composition and duration of composting phases can differ in different parts of the world, but compost always serves as carbon and nitrogen source for Agaricus bisporus’ mushroom growth.

The main ingredient in the European compost, wheat straw, contains about 57% (w/w) of carbohydrates, mostly cellulose (44 mol%) and xylan (46 mol%), and 27% (w/w) of lignin [2]. Cellulose is a non-branched polymer of β-1,4-linked glucosyl units. Xylan in grasses, like wheat straw, is composed of a 1,4-linked β-D-xylopyranosyl-backbone with arabinosyl, O-acetyl and (4-O-methyl-) glucuronic acid side chains [3]. However, the exact amounts and distribution of all substituents on wheat straw xylan is not reported. Lignin is composed of three main monolignols: p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) phenylpropanoid units. In wheat straw lignin all three units are present (H:G:S ratio of 6:64:30; [4]). Cellulose, xylan and lignin form together a complex, hard to degrade, network. In grasses, like wheat straw, xylan can adsorb to cellulose, but also be oxidatively cross-linked with other xylan molecules and with lignin via hydroxycinnamic acid residues [5]. Composting aims at opening up such a complex to facilitate release of monosaccharides, which serve as carbon source during A. bisporus’mushroom growth [2]. As such, composting has a similar goal as many other pre-treatments of lignocellulosic plant biomass aiming at an improved enzymatic release of fermentable monosaccharides to produce biofuels and chemicals from. Therefore, insights in the reactions occurring could be of use for other pre-treated plant materials.

The industrial production of compost is carried out in closed tunnels and involves three phases, described in detail elsewhere [2]. In brief, meso- and thermophilic microbiota decompose BM (Phase I (PI)), causing a rise in temperature to 80°C and release of ammonia. In the next phase (PII), microorganisms, in particular actinomycetes and fungi, consume at a maximum of 60°C about 40% of the ammonia present, while the other part disappears in the air [6]. As a result of two composting phases, compost has become accessible and specific for A. bisporus mycelium growth in the third phase at temperatures around 24°C for 16 days (PIII–16). In PIII, the A. bisporus mycelium is known to consume (part of) the microbiota present [7]. For optimal growth the A. bisporus mycelium needs also to degrade and consume the carbohydrates and, possibly, lignin present [1, 2]. PIII–16 compost is considered mature and by adding a casing layer on top of this compost the fruiting body formation starts [6].

Composting is an accelerated version of natural decomposition of lignocellulose by the microorganisms present [8]. The activity of these microorganisms and growth of A. bisporuschemically alters the compost [1, 2]. Quantification of remaining components, like xylan, cellulose, lignin and protein, however, has not been reported. So far, mainly qualitative changes in compost composition have been reported, with a focus on a decrease in carbohydrate and protein as based on total dry matter [1, 2, 9]. Lignin degradation has been mentioned, but only indirect evidence was shown, either by investigating whether A. bisporus can grow on radioactive labelled 14C lignin or by determining the presence of laccase-activity and manganese peroxidase. The latter is hypothesized to be linked to lignin degradation during mycelium growth [10, 11]. Although important, these results lack the possibility to determine absolute quantities of carbohydrates, lignin and protein metabolized.

In our research, in a tunnel-experiment at industrial scale, a mass balance was conducted for dry matter as well as for proteins, cellulose, xylan, lignin and ash. In addition, the structural changes of xylan and lignin were studied. Mapping the amounts and structures of the main components available for mushroom growth is essential for improving the process. Generally, our study contributes to the understanding how wheat straw compost is degraded.

Materials and Methods

Composting process

At the composting company CNC-C4C (Milsbeek, The Netherlands) basic mixture (BM) was obtained by mixing on a wet basis, 63% w/w of fresh horse manure, 2% w/w of gypsum, 1% w/w of ammonium sulphate solution (20% w/v (NH4)2SO4 in water), 17% w/w of filtered percolate water, 11% w/w of chicken manure, 4% w/w straw and 2% w/w of water. Fresh horse manure and wheat straw were collected in October 2013 and the experiment was carried out in October and November 2013. For this experiment, one tunnel was assigned for compost production from which all samples were taken from. The composting phases are described elsewhere [2]. In addition to the previously described information, it should be mentioned that the PI phase lasted for 5 days, and reached 80°C under formation of ammonia, after which PI compost was obtained. To PI compost 10 g kg−1 of PII compost was added to introduce viable microflora necessary for the conditioning phase. The duration of PII was also 5 days. To PII compost 4.5 g kg−1 of rye-based spawn was added and inoculated for 16 days after which PIII–16 mycelium grown compost was obtained.

Samples

All samples were taken from the same original BM (same timeline). The first tunnel (35 x 4 x 4 meter) was filled with 200 tons of BM. Of this BM 100 kg was kept apart and divided into 3 batches (A, B, C) of about 33 kg. Each batch was handled separately and placed in onion mesh bags in the same tunnel (Fig 1). Per batch two bags (biological duplicates) were prepared and weighed (min 15 kg), labelled (e.g. A–1 and A–2) and placed over the length of the tunnel, about 30 cm below the surface of BM compost. After phase PI, both bags from the corresponding batch were weighed and afterwards thoroughly mixed, and one sample (min 1 kg) was taken from each batch (A, B and C). After sampling, the material was again mixed and divided over two bags and placed in the tunnel for phase PII (35 x 4 x 3.62 meter). Total compost in PII tunnel was 200 tons. The same procedure was followed for phase PIII (tunnel 35 x 4 x 3.62 meter, total compost 144 tons). So, for each sampling step (end of each phase) three samples (biological triplicates) were obtained (min 1 kg). Throughout the complete composting process, the batches (A, B and C) were weighed at the end of each phase and all the changes (addition of water, spawn) were noted. Weight of each sample (batch) was determined after sampling and after collecting, samples were immediately frozen at -18°C. First, the dry matter content was determined for each sample (100 g, 105°C overnight). From the fresh weight of each batch the dry matter yield was determined for PI, PII and PIII–16. The dry matter yield was 91.9% of PI, of PII 77.1% and of PIII–16 69.4% (average of three batches, STDEV 1.2, 1.9 and 1.9, respectively). Dried samples were milled (<1 mm) using an MM 2000 mill (Retsch, Haan, Germany) prior to further analysis. Samples were analyzed for their protein, carbohydrate, ash and lignin contents for each sample (batch). Contents of all analyzed components was summed up and compared to the dry matter content of corresponding sample and the recovery was found to be >95%. In addition, the carbohydrate and lignin composition was analyzed.

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Fig 1. Schematic representation of sampling.

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Preparation of water un-extractable solids (WUS)

For batch A, freeze dried, milled samples (5 g) of BM, PI, PII and PIII–16 was suspended in water (175 mL) and boiled at 100°C for 5 min. Next, the suspension was stirred for 16 h at 21°C. The supernatant was removed after centrifugation (10 000 x g, 30 min, 20°C) and the residue was washed twice with water (60 mL and 75 mL). The final residues were freeze dried and collected as water un-extractable solids (e.g. PI-WUS). Samples were analyzed for yield, protein and dry matter contents.

Analytical techniques and methods

Carbohydrate content and composition.

The neutral carbohydrate and uronic acid content and composition was determined in duplicate, as described by Jurak et al. [2].

Nitrogen and protein content.

Samples (7–10 mg) were analyzed for nitrogen content in duplicate using the combustion (DUMAS) method on a Flash EA 1112 Nitrogen Analyzer (Thermo Scientific, Sunnyvale, CA, USA). Methionine (Acros Organics, Geel, Belgium) was used as a standard. Nitrogen content in the water soluble extract was calculated by difference. Nitrogen to protein conversion factor of 6.25 was used [12]. For PIII–16, due to the presence of A. bisporus mycelium in compost, protein was not specified (n.s. Table 1).

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Table 1. Mass balance of dry matter and organic matter and structural components (carbohydrates, nitrogen, lignin and ash) in the compost during composting and mycelium growth.

BM: basic mixture; PI: compost after Phase I; PII: compost after Phase II; PIII–16: compost after 16 days of mycelium growth for batch A.

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Ash content.

Freeze dried samples (1 g) or lignin residues (200–400 mg; see 2.4.4.) were dried in the oven overnight (105°C) and weighed, then put at 575°C for 5 h. Next, samples were weighed and difference between the mass at 105°C and 575°C was taken as ash content. Additionally, samples were burned at 575°C for 16 h more and afterwards weighed. No difference in mass was observed between residue after 5 h and 21 h.

Klason lignin residue and acid soluble lignin (ASL).

To each sample of 1 g (dry matter) 10 mL of 72% w/w H2SO4 was added and samples were hydrolyzed for 1h at 30°C. Next, 100 mL of distilled water was added to each sample and samples were put in a boiling water bath for 3h and shaken every half hour. Next, the suspensions were filtered over G4 glass filters. The filtrate was measured for acid soluble lignin (ASL) spectrophotometrically at 205 nm. ASL was calculated according to the formula: ASL = (A * B * C)/(D * E), with A = absorption relative to 1M H2SO4, B = dilution factor, C = filtrate volume, D = extinction coefficient for lignin (110 g L−1 cm−1), and E = weight of substrate (g). The residual part was washed until it was free of acid (determined by using pH paper) and dried overnight at 105°C. The final residues were corrected for ash and considered as a measure for the acid insoluble lignin (Klason) content after ash-correction. To this end, acid insoluble lignin was burned for ash. Total lignin was defined as a sum of Klason lignin residue, corrected for ash, and acid soluble lignin. For wheat straw, Klason lignin content corrected for ash was 27% (w/w) and acid soluble lignin content was 1.9% (w/w) based on dry matter.

Lignin analysis by analytical pyrolysis-GC-MS (Py-GC/MS).

Pyrolysis was performed with a 2020 microfurnace pyrolyzer (Frontier Laboratories, New Ulm, MN, USA) equipped with an AS-1020E Autoshot. Components were identified by GC-MS using a Trace GC equipped with a DB–1701 fused-silica capillary column (30 m x 0.25 mm i.d. 0.25 μm film thickness) coupled to a DSQ-II (EI at 70 eV) (both Thermo Scientific, Waltham, MA, USA). The pyrolysis was performed at 500°C for 1 min. Helium was the carrier gas (1 mL min−1). Samples (60–70 μg) were pyrolyzed and each measurement was performed at least in triplicate. Initial oven temperature was 70°C (2 min hold) and it increased to 230°C with a rate of 5°C min−1, to 240°C by 2.5°C min−1 and finally to 270°C min−1 by 2.5°C min−1. Pure compounds were used as standards (Sigma Aldrich, St. Louis, MO, USA; Brunshwig Chemie B.V., Amsterdam, The Netherlands and Fisher Scientific, Landsmeer, The Netherlands) and peak molar area was calculated as defined by del Rio [13]. For wheat straw a cut-off of 1% molar area for single S (syringyl-like lignin structures) and G (guaiacyl-like lignin structures) compounds was applied and only the fate of remaining compounds (>1% molar area) was analyzed for compost samples. Compounds with a molar area >1% in wheat straw are specified in Fig 2. For WUS, the fate of the same S and G compounds as in original compost was compared. Remaining S and G compounds were annotated as Rest S* and Rest G*. The same cut-off level was applied for phenolic furanose/pyranose (F/P) and unknown compounds based on total area of these compounds. F/ P compounds with a molar area >1% are annotated in S1 Table. The remaining compounds are specified in S2 Table. Amdis software (version 2.71, NIST, USA) was used for identification and deconvolution of peaks. For deconvolution the following parameters were set: adjacent peak subtraction = one, resolution = medium, sensitivity = high and shape requirements = low. For identification a target compound library (based on referents standards) was built. Referents standards were measured in order to obtain retention time (RT) information and mass spectra (Fig 2, S1 Table and S2 Table). Compounds identified based on referents standards were, first, selected based on RT (± 1.0 min; or ± 0.1 min for isomers). If RT was within the selected window an annotation was given if reversed search (RS) value was higher than 80%. Finally, for all WS compounds, also the ones identified based on Ralph and Hatfield [14], spectra were checked manually. Total annotated area of S- and G- lignin units in wheat straw was ±80%.

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Fig 2. Identities of lignin-derived phenolic S (syringyl-like) and G (guaiacyl-like) compounds identified with Py-GC/MS and relative molar area higher than 1% in wheat straw (out of total S+G molar area).

aInterpretation based on pure compounds. bInterpretation based on Ralph and Hatfield (1991), reverse search of compound in compost or WUS versus compound in wheat straw: 34S>99%, 42S>97%, 49S>99%. RS = reverse search

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Estimation of lignin quantities with analytical Py-GC/MS.

To estimate absolute amounts of lignin in the samples, the areas of Py-GC/MS pyrograms were assumed to indicate amounts of lignin units present. As a base, the total lignin content (sum of Klason lignin (26.5% w/w) and ASL (1.9% w/w)) of wheat straw was correlated with the area under the Py-GC/MS pyrograms of wheat straw. Molar areas of S- and G-units annotated in Fig 2 and S2 Table were summed up as total molar area. For wheat straw and compost samples about 85% of dry matter was pyrolyzed in the Py-GC/MS, based on gravimetric analysis prior and after the pyrolysis. As the same amount of sample was weighed and pyrolyzed for wheat straw and compost samples it was assumed that correlation between wheat straw and compost lignin could be made. Also, in compost samples, lignin originates only from wheat straw. The correlation between total molar area of S and G with the w/w % of total lignin in wheat straw, was used to calculate the w/w % of lignin in compost samples based on the molar Py-GC/MS areas of the compost samples analysed. Lastly, obtained values for w/w % of lignin based on dry matter in compost samples was used to calculate the mass balance of lignin in PI, PII and PIII–16.

Results and Discussion

Dry matter, organic matter, carbohydrate and protein mass balance during composting and mycelium growth phases

The contents based on dry matter of carbohydrates, ash, Klason lignin residue (- ash) and nitrogen were analyzed for all three batches (A, B and C) are presented on a dry matter basis in Table 2. Previously, the composition of compost was reported for compost samples from BM, PI, PII and PIII–16 [2]. In that research, samples were obtained by mixing multiple compost samples from different tunnels with the aim to study the remaining carbohydrate structures, and a mass balance could not be performed. However, in order to fully understand the changes in the compost, a mass balance for dry matter as well as for proteins, cellulose, xylan, lignin and ash and, therefore, samples from the same timeline is needed. From Table 2, batch C was found to be an outlier with respect to carbohydrate content and dry matter content of Phase I (PI). Namely, in 1000 kg of basic mixture (BM), based on the carbohydrate content, 424 kg of carbohydrates were present compared to 440 kg in PI, for batch C. While 439 kg in BM and 412 kg in PI and 449 kg in BM and 420 kg in PI of carbohydrates, were calculated for batch A and B, respectively. So, only for batch C this would, impossibly, indicate a gain in carbohydrates in PI. Its carbohydrate content was analyzed at least 3 times indicating that this outlier was not due to an analytical error. Considering the correct values of PII and PIII–16, the error appeared to have occurred in the sampling after PI. Hence, after carbohydrate and ash analysis, batch C was excluded from further analysis. Nevertheless, to our opinion the values obtained for batch A and B give representative data for the mass balance, also, because the numbers obtained are very close to the yearly average mass balance values of CNC-C4C (personal communication with CNC-C4C).

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Table 2. Carbohydrate, ash, nitrogen, Klason lignin and dry matter content (based on dry matter) for compost after PI, PII and PIII–16. BM: basic mixture; PI: compost after Phase I; PII: compost after Phase II; PIII–16: compost after 16 days of mycelium growth; A, B, C different batches.

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The carbohydrate contents (based on dry matter) of batch A and B was found to be, on average, 44% w/w for BM, 46% w/w for PI, 26% w/w for Phase II (PII) and 26% for PIII–16. Ash content was found to be 21%, 22%, 29% and 30% for BM, PI, PII and PIII–16, respectively (w/w based on dry matter). Total nitrogen content was 1.3%, 1.4%, 2.1% and 2.2% for BM, PI, PII and PIII–16, respectively and water insoluble nitrogen content was found to be 0.8%, 0.8%, 1.6% and 2.1% for BM, PI, PII and PIII–16, respectively (w/w based on dry matter). Lastly, Klason lignin contents, corrected for ash, were 21%, 22%, 23% and 21% for BM, PI, PII and PIII–16, respectively (w/w based on dry matter).

Next, for batch A the mass balance concerning ash, protein, carbohydrates and lignin during composting and mycelium growth is presented (Table 1) based on a starting amount of 1000 kg dry matter BM. The totals of all analyzed components covered 95% w/w or more of the total amount of dry matter, indicating the completeness of the analyses performed. Compared to BM, a decrease of 8% w/w of dry matter was analyzed for PI, 23% w/w for PII and 31% w/w for PIII–16.

Overall, some variations in the absolute amounts of ash was observed (Table 1). Previously, variations in the amount of inorganic materials during composting have been reported [1]. Ash present mainly originated from sand and stones found in the commercial compost solids. Possibly, these are introduced together with recycled process-water, and therefore present in various amounts in the different samples (personal communication CNC-C4C). Such ash-recycles may also contribute to the higher decrease in organic matter (OM) compared with DM (Table 1).

Total nitrogen remained rather similar in the compost during composting and mycelium growth. Given the low nitrogen values (% w/w, Table 2), comparison of absolute nitrogen amounts should be performed with caution. Given this, a tendency in increase of water insoluble nitrogen might be observed in PII compost compared to PI compost. During PI rise in temperature to 80°C [2] and formation of ammonia was observed (personal communication CNC-C4C) indicating microbial growth of meso- and thermophilic microbiota [6], however, no big differences in carbohydrate and lignin content were observed (Table 2). In contrast to this, a tendency in increase in the amount of total nitrogen and protein in PII (mass balance, Table 1) could be interpreted. During PII a decrease in the organic matter (18% w/w) and carbohydrates (±50% w/w for both xylan and cellulose present (Fig 3)) was observed. During this phase up to 40% of ammonia is reported to be removed by microbiota, actinomycetes and fungi, present [6] with temperatures around 50°C [2] and higher humidity compared to PI (Table 1). With caution it could be proposed that one of the possible explanations for this observation is the growth of nitrogen-fixating and other viable microbiota introduced into compost at the beginning of PII [15, 16].

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Fig 3. Mass balance of constituent monosaccharides during composting and mycelium growth for batch A and B (BM, PI, PII, PIII–16). BM = basic mixture, PI = compost after Phase I, PII = compost after Phase II, PIII–16 = compost after 16 days of mycelium growth; Rha = ramnosyl, Ara = arabinosyl, Xyl = xylosyl, Man = mannosyl, Gal = galactosyl, Glc = glucosyl, UA = uronyl.

aCalculation was performed based on 1000 kg of DM using values presented in Tables 1and 4.

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In PIII–16 (Table 1) temperatures are maintained around 25°C [2] and humidity is higher (±8%) compared to PII. The amount of protein in PIII–16 increased further, mainly seen in the increase in water insoluble nitrogen, and the amount of carbohydrates decreased slightly both in xylan and cellulose (Fig 3), most likely as a result of the observed mycelium growth in this phase [17, 18]. It should be noted that due to the formation of mycelium dry matter, partly built from glucan, the decrease in compost-glucan (cellulose; Fig 3) derived from the starting material is underestimated. Namely, in our analysis, total glucan was analyzed, regardless whether it originated from plant or microbial origin. Finally, mannitol was analyzed to be present in PIII–16 compost, which is a known soluble carbohydrate in the mycelium of A. bisporus [19].

Overall, the molar composition of the compost carbohydrates in BM, PI, PII and PIII–16 (Table 3) remained rather similar and is in line with previously reported data [2]. However, a decrease in xylosyl residues could be observed in PIII–16 compared to PII. In all phases, the main carbohydrate constituents were xylosyl (28–35 mol%) and glucosyl (52–56 mol%) residues, which is in agreement with previously published data [2]. Recently, it was shown that during PIII compost xylan is partly degraded, thereby, making it more water soluble [2] which is expected to provide more easily accessible carbohydrates during fruiting of A. bisporus. In the present study, no division between water soluble and water insoluble glucans and xylan was performed.

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Table 3. Carbohydrate composition (mol%) and degree of substitution of xylan in different compost phases, based on dry matter.

BM: basic mixture; PI: compost after Phase I; PII: compost after Phase II; PIII–16: compost after 16 days of mycelium growth.

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Lignin mass balance during composting and mycelium growth phases

First, the Klason lignin analysis was applied to a lab-cultivated A. bisporus mycelium sample, which allowed us to observe the fate of mycelium in this analysis. It was shown that more than 50% of the mycelium dry matter, was collected as ‘Klason lignin’. This indicated that the Klason lignin analysis in samples containing substantial amounts of mycelium, like in PIII–16, would give an overestimation of the lignin present as mushroom mycelium would be analyzed as “lignin” by this method. Also, denatured proteins are known to remain in the Klason lignin residues [1]. Nevertheless, Klason lignin residues were analyzed for batch A (Table 1), allowing comparison with the scarce previously reported data on compost composition [1]. For PI and PII values obtained for Klason lignin corrected for ash were in line with values reported by Iiyama et al. [1].

Lignin structure and content was also analyzed as single monolignol units by analytical Py-GC/MS. Based on the correlation between the wheat straw Klason lignin content, and the area of annotated S- and G-units in the wheat straw pyrogram obtained, from the pyrogram-areas of the compost samples (in triplicate) the lignin yield in these samples was calculated, based on 1000 kg BM dry matter (Table 4). In general, the amount of pyrogram based lignin remained rather similar during composting (PI and PII). In contrast, a decrease of 45% w/w in the amount of lignin, based on pyrolysis, was observed after 16 days of mycelium growth. The overall difference in kg between BM and PIII–16 is more pronounced by the GC/MS analysis than by the classical Klason lignin analysis. This also accounts for the decrease in dry matter during the PIII phase. In our opinion, the Py-GC/MS data are more representative for the lignin amounts present than the Klason lignin residue analysis, because with the former technique only lignin derived pyrolysis units were taken into account in the quantification in mycelium grown compost samples. The GC/MS analysis leads to the total lignin yield based on constituent units present after pyrolysis. Hence, this technique also provided valuable data on compositional changes during the different phases.

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Table 4. Relative pyrogram area (%) and Py-GC/MS pyrogram based lignin for wheat straw and different compost phases for batch A.

BM: basic mixture; PI: compost after Phase I; PII: compost after Phase II; PIII–16: compost after 16 days of mycelium growth.

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Structural changes of lignin during composting and mycelium growth phase

The Py-GC/MS lignin-fingerprints of the BM, PI, PII and PIII–16 composts were annotated based on the fully annotated pyrogram of untreated wheat straw (Fig 4A). The pattern and main annotated peaks of lignin compounds for wheat straw were in line with previously reported data [4]. Due to better baseline separation and additional spectra measured from standard lignin compounds, some peaks (e.g. trans-isoeugenol, 4-methylsyringol, vanilin) were differently annotated than previously reported (Fig 2, S1 Table and S2 Table) [4].

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Fig 4. Py-GC/MS pyrograms of wheat straw, basic compost mix (BM) and compost after 16 days of mycelium growth (PIII–16) (A) and water un-extractable solids (WUS) of Phase I and PIII–16 (B) for batch A.

The identities and structures of main syringyl and guaiacyl (and p-hydroxyphenyl) compounds are listed in Fig 2, S1 Table and S2 Table. PI: compost after Phase I: PII: compost after Phase II.

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For BM the pattern and the ratios between peaks is quite similar to those of wheat straw, which was expected as lignin in BM-compost originates from wheat straw. Also, the pyrograms of PI and PII composts were majorly similar as the ones of BM and wheat straw. In Phase III, however, the ratios (in molar area) between some monolignol-units, mainly vinyl-guaiacol, guaiacol, vinyl-syringol and syringol, were very different between the BM and PIII–16 pyrograms (Fig 4).

In order to understand the differences observed during mycelium growth, first, the various monolignol-units present in the pyrogram of wheat straw are discussed. As previously stated, wheat straw lignin is mainly composed of S- (syringyl-like) and G- (guaiacyl-like) lignin units, and to a minor extent of H (p-hydroxyphenyl) units. Therefore, we focused on S and G lignin units.

The S:G ratios of wheat straw and different compost samples was calculated and is shown in Fig 5A. The S:G ratio in wheat straw was 0.49 (Fig 5A), which is in line with the value reported by del Rio [4], where vinyl-syringol and vinyl-guaiacol were excluded from the S:G ratio. The S:G ratio in PI and PII remained 0.51. After 16 days of mycelium growth (PIII–16), the S:G ratio changed to 0.68 (Fig 5A), indicating a modification in lignin by A. bisporus mycelium. The pyrograms obtained for compost samples as well as S:G values are found to be in line with values reported by Chen et al. [20] however, annotated peaks were not specified and the samples used were collected from different stages in the process. Therefore, these results are difficult to compare.

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Fig 5. S:G ratio (A) and distribution of syringyl (S) and guaiacyl (G) units (B), based on molar area, of wheat straw (WS) and total compost samples during composting BM, PI, PII and mycelium growth PIII–16 and in water insoluble compost PIwus, PIII-16wus.

Rest S* and Rest G* S2 Table, BM: basic mixture; PI: compost after Phase I: PII: compost after Phase II; PIII–16: compost after 16 days of mycelium growth, A and B are biological duplicates and each sample measurement was performed in quadruplicates.

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Changes in distribution of S and G lignin units were determined for S and G structures with molar area larger than 1% of total S+G molar area (Fig 2). Remaining annotated compounds are presented in S1 Table and S2 Table (see materials and methods), but not taken into account further.

Compared to PII compost, a lower proportion of substituted vinyl-syringyol and vinyl-guaiacol lignin compounds in PIII–16 compost was present in favor of the less substituted guaiacol and syringol (Figs 2 and 4). This may point at cleavage of substituents on the phenolic skeleton during PIII (Fig 5B). The observed modification of substituents is mainly observed in “vinyl- groups” leading to a relative decrease in vinyl-guaiacol and vinyl-syringol during PIII. Lignin structures analyzed by NMR in wheat straw [4] indicate that such vinyl-decorations are mainly responsible for inter-lignin linkages. Therefore, our findings suggest that A. bisporus is capable of cleaving larger lignin structures into smaller ones, and further remove the decorations leaving mainly the basic S and G phenolic skeletons of the lignin structures.

In previous research [4], vinyl-guaiacol and vinyl-syringol were excluded from the S:G ratio as during pyrolysis p-hydroxycinnamates are known to result in the same compounds as those derived from lignin. If these compounds were only part of xylan, it could lead to overestimation of lignin. However, in BM, due to high pH, no free and ester bound FA (ferulic acid) and very low amounts of pCA (p- coumaric acid) (<0.1% w/w based on dry matter) were found [2]. This indicated that in BM compost less than 0.3% (w/w based on dry matter) of ester bound FA and pCA were present. Also, the amount of vinyl-guaiacol that could be formed from FA and pCA after pyrolysis was less than 4% of the total of vinyl-guaiacol analyzed in the wheat straw pyrogram. The remaining ether-bound FA and pCA are expected to account for less than 0.5% w/w based on dry matter [21].

Composition and S:G ratio of water insoluble lignin in compost samples is presented in Fig 5and corresponding pyrograms are presented in Fig 4. As no major compositional changes in the relative distribution of S and G lignin compounds were observed between BM and PII (Fig 5B), only for PI (PIwus) and PIII–16 (PIII-16wus) water insoluble lignin was analyzed in detail in particular for batch A. For PI the distribution of S and G compounds of PIwus-A (Fig 5A) was found to be rather similar as that of the total sample (PI-A). On the contrary, in PIII-16wus a relatively lower S:G ratio was found compared to the total sample of PIII–16 indicating that part of lignin in PIII–16 compost became more water soluble (Fig 5B).

Lignin modification in the compost by A. bisporus mycelium was previously indicated based on the degradation on 14C-labelled lignin [22] and A. bisporus was shown to produce the with lignin degradation correlated activities of manganese peroxidase and laccase in liquid cultures [23]. Recently, annotation of the A. bisporus genome indeed showed that A. bisporus contains genes encoding manganese peroxidase (MnP) and laccases [24, 25]. Regulation and expression of laccases and MnP was investigated and two genes encoding laccases and the predicted MnP gene were found to be highly expressed during mycelium growth in the compost and lower expression in the later stages of mushroom growth. In addition, the secretion of corresponding proteins indicated that laccases are secreted to a higher extent compared to MnP [24, 26]. These findings support the data presented in our research showing that A. bisporus, during the vegetative growth, was able to modify lignin structures. It is proposed that observed lignin degradation and modification increase the bioavailability of the carbohydrates in the wheat based compost.

To our knowledge, this is the first time that degradation and metabolization of lignin by A. mycelium was shown directly on the lignin structure in mycelium grown wheat straw based compost. Overall, our research provides more insights in how A. bisporus mycelium degraded lignocellulosic biomass for mushroom growth, and in general, give new insights in lignocellulosic plant biomass degradation.

Conclusion

During PI of composting, no changes in carbohydrates and lignin were observed in the compost. In PII, 50% of carbohydrates, both cellulose and xylan were metabolized, while lignin structure was not. During 16 days of mycelium growth (PIII–16) 45% of lignin was metabolized and the remaining lignin was modified resulting in an increased S:G ratio (0.51 to 0.68). Furthermore, from both S and G phenylpropanoid units the decorations, mainly vinyl-groups, were removed from the phenolic skeleton.

Supporting Information

S1 Table. Identities of lignin-derived phenolic F/P and unknown compounds identified with Py-GC/MS with relative molar area higher than 1% in wheat straw (out of total F/P and unknown compounds molar area)

doi:10.1371/journal.pone.0138909.s001

(DOCX)

S2 Table. Identities of lignin-derived phenolic S, G, F/P and unknown compounds below 1% of relative molar area in wheat straw (for S and G out of total S+G molar area, and for F/P and unknown out of total F/P + unknown molar area) identified with Py-GC/MS.

doi:10.1371/journal.pone.0138909.s002

(DOCX)

Author Contributions

Conceived and designed the experiments: EJ WA MK HG. Performed the experiments: EJ. Analyzed the data: EJ AP. Contributed reagents/materials/analysis tools: EJ AP WA MK HG. Wrote the paper: EJ AP WA MK HG.

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Friday, 17 June 2016

Calories Burned

Joint Impact

It's All About Intensity

Muscle Flexibility

Joint Flexibility

Improved Circulation

Stress Relief

Identification

Components

Types

Effects

Considerations

Monster Energy Drink

Heart Rate

Monster and the Heart

Considerations

Cardiac Arrest

Bradycardia

Atrial Fibrillation

Postural Tachycardia

Overview

Liquids

Soft Foods, Moist Foods

Foods That Stimulate Saliva

Figures

Highlights:

Introduction

Materials and Methods

Composting process

Samples

Preparation of water un-extractable solids (WUS)

Analytical techniques and methods

Carbohydrate content and composition.

Nitrogen and protein content.

Ash content.

Klason lignin residue and acid soluble lignin (ASL).

Lignin analysis by analytical pyrolysis-GC-MS (Py-GC/MS).

Estimation of lignin quantities with analytical Py-GC/MS.

Results and Discussion

Dry matter, organic matter, carbohydrate and protein mass balance during composting and mycelium growth phases

Lignin mass balance during composting and mycelium growth phases

Structural changes of lignin during composting and mycelium growth phase

Conclusion

Supporting Information

S1 Table. Identities of lignin-derived phenolic F/P and unknown compounds identified with Py-GC/MS with relative molar area higher than 1% in wheat straw (out of total F/P and unknown compounds molar area)

S2 Table. Identities of lignin-derived phenolic S, G, F/P and unknown compounds below 1% of relative molar area in wheat straw (for S and G out of total S+G molar area, and for F/P and unknown out of total F/P + unknown molar area) identified with Py-GC/MS.

Author Contributions

References