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Effects of various microorganisms and pretreatments on microbial degradation of lignocellulosic materials.

INTRODUCTION

Increased concern for the negative impact of fossil fuels on the environment, particularly greenhouse gas emissions into the atmosphere, and rising prices of crude oil due to increasing fuel demands, has put pressure on society to find renewable alternative sources of bioenergy [1, 2]. Currently, research is being done to enhance the digestibility of lignocellulosic biomass mainly for the efficient conversion of indigestible materials such as lignin, hemicelluloses, and cellulose to reducing sugar, ethanol, methane, and hydrogen. Fortunately, biologically mediated processes seem promising for energy conversion, in particular for the conversion of lignocellulosic biomass into fuels [3].

Biological, enzymatic, or microbial hydrolysis commonly involves four biologically-mediated transformations: the production of saccharolytic enzymes (cellulases and hemicellulases); the hydrolysis of carbohydrate components present in pretreated biomass to sugars; the fermentation of hexose sugars (glucose mannose, and galactose); and the fermentation of pentose sugars, xylose and arabinose [3, 4]. However, it is unclear which characteristics of the lignocellulosic biomass are important to determine a successful pretreatment method [3].

Lignocellulosic biomass refers to plant biomass that is composed of cellulose and hemicellulose tightly bound to the lignin by hydrogen and covalent bonds, and is the most abundant organic material on earth, and is therefore a promising raw material for bioenergy production [5,6]. Cellulose is a polymer of glucose while hemicellulose ispredominantly composed of xylans which, after hydrolysis, yield the pentose sugar and xylose [7].

The four categories of biomass resources in the world include: (1) wood residues which are by far the largest current source of biomass for energy production, (2) municipal solid waste, the next largest, (3) agriculture residues and, (4) dedicated energy crops [8].

Generally, the use of celluloses and hemicelluloses (cellulosic biomass) is recommended instead of traditional feed stocks. Ideally, cellulosic biomass could be used as an inexpensive and abundantly available source of sugar for fermentation into the sustainable transportation fuel, ethanol [9, 10].

To initiate the production of industrially important products from cellulosic biomass, bioconversion of the cellulosic components into fermentable sugars is necessary [2]. The two main obstacles in the degradation of lignocellulosic materials are the resistance of lignin, and the crystal-like structure of cellulose fibrils [7]. It would therefore be of great benefit if microorganisms were developed that could utilize cellulose and other fermentable compounds available from pretreated biomass and produce desired product at high yield and titer [4].

In filamentous fungi, production of the cellulose- and hemicelluloses-degrading enzyme, cellulases and hemicellulases, are controlled at the transcriptional level by the available carbon source [11].

The filamentous fungus Trichoderma reesei is well known as an efficient producer of cellulases [12, 13]. This fungus is the main industrial source of cellulases and hemicellulases used to depolymerize biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol [14]. It has been proposed by other researchers [9, 15] that T. reesei produces a family of different cellulolytic enzymes, including endoglucanases, exocellobiohydrolases, and p-glucosidases; a view that is different than the one proposed by Kovacs and his colleagues [16] who proposed that p-glucosidases is practically not secreted by Trichoderma reesei. Still, others claim that the cellulolytic enzyme system of T. reesei can efficiently degrade crystalline cellulose to glucose [17]. Studying solid-state fermentation with Trichoderma reesei for cellulase production, Chahal [12] concluded that the cellulase potential of various mutants of T. reesei ranges between 160 and 250 IU/g of pure cellulose in liquid-state fermentation.

Higher fungi (white rot fungi) which cause white rot in wood are believed to be the most effective lignin-degrading microbes in nature [18, 19,] and are the most promising microorganisms used for biological pretreatment because of their abilities to selectively degrade. Pycnoporus cinnabarinus is the basidiomycete commonly known for its ability to efficiently degrade lignin by an unusual production of ligninolytic enzymes, ligninolytic phenol oxidases [20]. Pycnoporus cinnabarinus has a simple ligninolytic system. Neither lignin peroxidase nor manganese peroxidase activity has been detected, but laccase is produced [21. Pycnoporus cinnabarinus laccase appears to occur in only one single acidic form, a usual feature among fungal laccases [22]. Overall, P. cinnabarinus has proven to be an interesting model organism for studying new mechanisms of lignocelluloses degradation by white rot fungi [20, 22].

Fusarium solani, a pathogenic plant fungus causes root rots, which results in considerably economical losses in many important crops [23], has a beneficial importance in degrading cellulosic materials in biomass. Among organisms isolated from the soil, F. solani was the most vigorous microbial degrader of a synthetic lignin, such as a dehydrogenation polymer of coniferyl alcohol. The findings of other researchers with different microbes strongly indicate that aeration of F. solani cultures with oxygen could dramatically increase the rate and quality of lignin degradation under most, if not all, otherwise suitable incubation conditions.

This study was therefore carried out to examine the effects of five microorganisms: Trichoderma reesei, Pycnoporus cinnabarinus, Fusarium solani, an unknown bacterial isolate, and an unknown fungal isolate on the degradation of cellulosic materials that had been subjected to pretreatments of heat and sulfuric acid, and incubated under aerobic and anaerobic conditions.

MATERIALS AND METHODS

Preparation of Lignocellulosic and Cellulose Materials

Samples of Southern pine sawdust (obtained from a sawmill in Taylorsville, Mississippi) were prepared according to treatments 1 thru 8, (Table 1). For Treatments 1 and 2, thirty mL of distilled water were added to 12 Erlenmeyer flasks that contained 10 g of the sawdust. The flasks were then covered and autoclaved (121[degrees]C, 15 psi) for three consecutive days for one hour each day. For treatments 3 and 4, ten g of sawdust were placed into 5 flasks each and 30 mL of 25% [H.sub.2]S[O.sub.4 were] added and mixed with the sawdust. The flasks were covered and incubated for three days at room temperature after which the sample was filtered four times using 20 mL of distilled water and saving the filtrate each time. Thirty mL of the filtrate were poured into each of 12 previously prepared flasks and covered. For treatments 5 and 6, ten g of original sawdust that had not been autoclaved or treated with 25% [H.sub.2]S[O.sub.4] was placed into 12 Erlenmeyer flasks. and covered. For treatments 7 and 8, five g of pure cellulose were added to 12 Erlenmeyer flasks and covered.

Liquid Media Preparation

Liquid medium was prepared according to Miller [24]. Briefly, in each 1000 mL of distilled water, the following reagents were added; 2 g potassium phosphate monobasic (K[H.sub.2]P[O.sub.4]), 1.4 g ammonium sulfate [(N[H.sub.4]).sub.2]S[O.sub.4], 0.3 g calcium chloride (Ca[CL.sub.2][H.sub.2]O), 0.3 g magnesium sulfate (Mg[SO.sub.4].7[H.sub.2]O), 0.6 g urea, 10 mg ferrous sulfate (FeS[O.sub.4].7[H.sub.2]O), 2.8 g zinc sulfate, ZnS[O.sub.4].7[H.sub.2]O), 3.2 g cobalt chloride (Co[Cl.sub.2].6[H.sub.2]O), 1.6 g manganese sulfate (MnS[O.sub.4].[H.sub.2]O), 0.1 % peptone, and 0.1% tween 80 (Polyoxyethylene (20) Sorbitan mono-oleate)/L, The resulting solution was then boiled with frequent agitations using a magnetic stirrer for ten minutes. One hundred fifty mL of the medium was added to each of the 48 previously prepared and described flasks containing a carbon source. The flasks were autoclaved at 121[degrees]C at 15 psi for 15 minutes.

Microorganisms

Fungal cultures including Trichoderma reesei (ATCC # 26921), Pycnoporus cinnabarinus (ATCC # 48748), and Fusarium solani (ATCC # 52176) were purchased from the American Type Culture Collection (ATCC) Manassas, VA. Unknown bacterial and fungal isolates from rotting wood samples were also used.

Isolation of Bacteria and Fungi from Rotting Wood

Ten grams of rotting-wood samples were dispensed into a 100 mL dilution bottle containing 90 mL of sterile distilled water. The bottle was vortexed for five minutes to ensure thorough mixing of the samples and the diluents. From the initial suspension ([10.sup.-1]), serial dilutions were made using sterile distilled water as the diluent [25]. Aliquots (0.1 mL) of the chosen dilutions (i.e., [10.sup.-3] to [10.sup.-7]) were dispensed onto nutrient agar for bacteria, and potato dextrose agar for fungi. Solidified petri dishes and spread plates were prepared and incubated at 24[degrees]C for five days. Colonies that developed on the plates were subcultured for subsequent use in this experiment.

Media and Culture Conditions

Potato dextrose agar (PDA) was used to grow Trichoderma reesei culture at 24[degrees]C. A known culture of Pycnoporus cinnabarinus was grown in ATCC medium 200 Yeast Mold Agar (YM agar, BD 271210) on petri dishes and slants, and then incubated at a room temperature (24[degrees]C). A culture of Fusarium solani was grown on potato dextrose agar (PDA) plates and slants at 24[degrees]C. All cultures were grown for 72 hours.

A bacterial isolate from a rotting wood sample was inoculated into nutrient agar plates and incubated for three days. Fungal isolate with the highest growth was selected and inoculated into potato dextrose agar plates and incubated for two days. After the designated growth period, both cultures of bacterium and fungi were harvested and maintained at 4[degrees]C for use in this experiment.

Microbial Inoculation of Liquid Media

Ten mL of each culture were inoculated into each of the twenty 250 mL Erlenmeyer flasks containing 150 mL of liquid medium plus a carbon source as specified in Table 1. Four flasks containing a carbon source were not inoculated with microorganisms and represented the control. Foam plugs were inserted into each of the flasks and the flasks were left undisturbed on the laboratory table at room temperature. This was considered as an anaerobic condition. For aerobic treatments, another set of 24 flasks were prepared as described above, covered with parafilm, and placed into a shaker and shaken at a speed of 60 rpm. Thus, a total of forty-eight flasks were used in this experiment.

Determination of Reducing Sugar (Glucose)

At specified time intervals (day 0, 6, 9, 13, 16, 22, and 28), samples were analyzed to determine the concentration of glucose present in each using the dinitrosalicylic acid (DNS) method of Miller [24]. Five mL of each sample were pipetted into centrifuge tubes. The samples were then centrifuged for two minutes, and one mL of the supernatant from each sample was pipetted into 25 mL test tubes and replicated 3 times. One mL of DNS was added to each test tube and the test tubes were allowed to boil for ten minutes. The color of the solution changed to red. The tubes were then transferred into a cold water bath. After cooling, the solution was diluted to 25 mL with distilled water. The absorbance readings of the samples were determined with a Cole Palmer SQ-2800 UV/Visible Spectrophotometer at 520 nm.

Glucose Standard Preparation

A stock solution of 1.0 mg/mL of glucose was first prepared in a test tube. Using this stock solution, the following standards were then prepared: 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mg/mL. One mL of dinitrosalicylic (DNS) acid reagent was added to each tube containing the glucose standard solutions. The test tubes were then boiled for ten minutes (the color of the solution changed to red) and were transferred into a water bath with a temperature of 4[degrees]C. After cooling, the solution in each tube was diluted to 25 mL with distilled water and the absorbance reading was taken at a wavelength of 520 nm using a SQ-2800 UV/Visible Spectrophotometer (Cole Palmer).

Statistical Analysis

Statistical analyses were performed using SAS Computer Software Program. Comparisons of data between incubation period, and among pretreatment methods were made by the analysis of variance (ANOVA) and Tukey's Honestly Significant Difference (HSD) test. Pretreatments were defined as: heat (autoclaved), acid (sulfuric acid), no pretreatment (untreated sawdust) and pure cellulose. Statistical differences were assessed at p < 0.05 (95% confidence).

RESULTS AND DISCUSSION

Our study further revealed that samples that were treated with Trichoderma reesei produced higher amounts of suga under the conditions of heat and acid, especially Overall, results of this study suggest that pretreatment of Southern pine sawdust with heat and sulfuric acid increased the degradability of lignin, hemicelluloses and a cellulose to produce reducing sugars under both aerobic (p = 0.0047) and anaerobic (p = 0.0101) conditions, respectively (Table 2).

Our study further revealed that samples that were treated with Trichoderma reesei produced higher amounts of sugar under the conditions of heat and acid, especially on day 13 under aerobic (Figure 1) and anaerobic (Figure 2) conditions. Trichoderma reesei is well known as an efficient producer of cellulases [12, 13] and is the main industrial source of cellulases and hemicellulases used to depolymerize biomass to simple sugars that are converted to chemical intermediates and biofuels such as ethanol [14]. Sugar production in sawdust with no pre-treatment and treatments of pure cellulose were the lowest (Figures 1 and 2). Both conditions, aerobic (Figure 1) and anaerobic (Figure 2) showed variation in reducing sugar production with peaks on days 13 and 16 followed by a gradual decline on day 22.

Samples that were pretreated with acid and P. cinnabarinus under aerobic conditions (Figure 3) produced almost the same amount of reducing sugar (0.837 mg/L) as samples under anaerobic conditions (Figure 4) (0.810 mg/L) on days 9 and 22, respectively.

Fusarium solani showed a different pattern of variation in reducing sugar production under aerobic (Figure 5) and anaerobic (Figure 6) conditions. Maximum amount of reducing sugar was produced on day 22 under anaerobic condition (1.159 mg/L) with acid pretreated samples followed by heat pretreated sample under aerobic (0.732 mg/L) condition. The mean amount of reducing sugar produced by acid pretreated samples (0.49 mg/L) was lower compared to those of heat-pretreated samples (0.51 mg/L) under aerobic conditions (Table 2). Under anaerobic conditions acid pretreated samples produced more sugar (0.75 mg/L) as compared to heat-pretreated samples (0.61 mg/L). In all cases, untreated samples produced the least amount of sugars under both aerobic and anaerobic conditions (0.15 mg/L and 0.15 mg/L, respectively) (Table 2). Generally, F. solani is one of the most vigorous microbial degraders of synthetic lignin, and it has been indicated that aeration of F. solani cultures with oxygen could dramatically increase the rate of lignin degradation under most incubation conditions [23]. The difference between our findings and the findings of other investigators are unexplained at this time.

In nature, cellulosic materials are degraded with the cooperation of many microorganisms. It has been reported that the utilization of microbial community composed of one cellulolytic bacterium and another non-cellulolytic bacterium or comprising of aerobic and anaerobic bacteria could degrade cellulosic materials effectively [27]. In this study, a bacterial isolate was incubated under aerobic (Figure 7) and anaerobic conditions (Figure 8). The highest sugar production was observed on day 6 under aerobic conditions and day 13 under anaerobic conditions, respectively. However, less sugar was produced by the unknown bacterial isolate as compared to T. reesei, F. solani, and P cinnabarinus (Table 2). Overall, samples that were pretreated with acid or heat showed significant differences in the amount of sugar production when compared to untreated samples (p = 0.0069).

Fungal isolates produced the highest amount of reducing sugar on day 16 where we observed 0.48 mg/L and 0.40 mg/L under aerobic (Figure 9) and anaerobic conditions (Figure 10), respectively. There were high amounts of sugar produced after 16 and 22 days of incubation (Table 3). Generally, under both aerobic and anaerobic conditions samples that were not pretreated either by heat or sulfuric acid produced less sugar compared to pretreated samples. Our results support the position that pre-treatment: (1) improve the formation of sugars or the ability to subsequently form sugars; (2) avoid the degradation or loss of carbohydrate; and (3) avoid the formation of by-products inhibitory to the subsequent hydrolysis and fermentation processes [28].

CONCLUSIONS

There is a significant amount of low-value or waste lignocellulosic materials that are currently burned or wasted. These materials are particularly well suited for reducing sugar, ethanol and for energy applications because of their large scale availability, low cost and environmentally benign production [4].

Combined pretreatment methods can be recommended to enhance the effectiveness of conversions of lignocellulosic materials from Southern pine sawdust. The study demonstrated that the combined pretreatments of acid plus microorganisms and heat plus microorganisms, were effective for sugar production. However, in order to realize the full potential of these methods, microorganisms must be developed that utilize cellulose and other fermentable compounds available from pretreated biomass with high rate and high conversion, and which produce a desired product at yield and titer [4].

The biodegradability of lignocellulosic biomass is limited by several factors like crystallinity of cellulose, available surface area, and lignin content. Pretreatment methods have an effect on one or more of the operating conditions. Acid-pretreated samples and heat-pretreated samples produced differently with different microorganisms and under aerobic and anaerobic conditions.

ACKNOWLEDGEMENT

This research was made possible through support provided by NASA through the University of Mississippi to Jackson State University under the terms of Agreement No. 300112306A/NNG05GJ72H and 13-08-003/NNX10AJ79H. The opinions expressed herein are those of the authors and do not necessarily reflect the views of NASA or the University of Mississippi.

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Rose Kishinhi, Yasmin Partee, Terry Wilborn, LeoAlexander Harris, Natalie Anderson, Gloria Miller, Maria Begonia, and Gregorio Begonia

Department of Biology, P.O. Box 18540, College of Science, Engineering and Technology, Jackson State University, Jackson Mississippi 39217 USA

Corresponding Author: Maria Begonia maria.f.begonia@jsums.edu

Table 1. Organisms used in this study were: Trichoderma
reesei, Pycnoporus cinabarinus, Fusarium solani, an
Unknown Bacterial Isolate and an Unknown Fungal
Isolate. Pretreatments are: Heat (Autoclaved), Acid
(Sulfuric Acid), No Pretreatment (Untreated Sawdust)
and Pure Cellulose.

     Pretreatments                        Aerobic   Anaerobic

1.   Autoclaved (Sawdust + Water             1          1
     + Liquid Medium (Control)
2.   Autoclaved (Sawdust +                   5          5
     Water) + Liquid Medium + 1
     of 5 Organism per flask
3.   Sawdust + [H.sub.2]S[O.sub.4]           1          1
     + Liquid Medium (Control)
4.   Sawdust + [H.sub.2]S[O.sub.4]+          5          5
     Liquid Medium + 1 of 5 Organism
     per flask
5.   Sawdust original + Liquid               1          1
     Medium (Control)
6.   Sawdust original + Liquid Medium +      5          5
     1 of 5 Organism per flask
7.   Pure cellulose + Liquid                 1          1
     Medium (Control)
8.   Pure cellulose + Liquid Medium          5          5
     + 1 of 5 Organism per flask

Table 2. Microbial effects of pretreated pine sawdust on
reducing sugar production (mg/L). Data are means [+ or -] of
standard deviations of three replications.

Pretreatment                   Aerobic             Anaerobic

Trichoderma reesei

Heat                      0.52 [+ or -] 0.17   0.54 [+ or -] 0.24
Acid                      0.58 [+ or -] 0.20   0.59 [+ or -] 0.23
No pretreatment           0.49 [+ or -] 0.19   0.41 [+ or -] 0.22
Pure cellulose            0.39 [+ or -] 0.13   0.40 [+ or -] 0.17

Pycnoporus cinnabarinus                        0.59 [+ or -] 0.06

Heat                      0.52 [+ or -] 0.11
Acid                      0.63 [+ or -] 0.13   0.67 [+ or -] 0.10
No pretreatment           0.43 [+ or -] 0.08   0.41 [+ or -] 0.11
Pure cellulose            0.50 [+ or -] 0.06   0.55 [+ or -] 0.07

Fusarium solani                                0.61 [+ or -] 0.21

Heat                      0.51 [+ or -] 0.17
Acid                      0.49 [+ or -] 0.11   0.75 [+ or -] 0.29
No pretreatment           0.15 [+ or -] 0.04   0.15 [+ or -] 0.05
Pure cellulose            0.37 [+ or -] 0.14   0.33 [+ or -] 0.05

Bacterial Isolate

Heat                      0.27 [+ or -] 0.13   0.29 [+ or -] 0.17
Acid                      0.27 [+ or -] 0.15   0.30 [+ or -] 0.20
No pretreatment           0.11 [+ or -] 0.02   0.13 [+ or -] 0.03
Pure cellulose            0.20 [+ or -] 0.11   0.28 [+ or -] 0.09

Fungal isolate

Heat                      0.35 [+ or -] 0.09   0.35 [+ or -] 0.09
Acid                      0.37 [+ or -] 0.11   0.39 [+ or -] 0.13
No pretreatment           0.13 [+ or -] 0.02   0.15 [+ or -] 0.04
Pure cellulose            0.44 [+ or -] 0.20   0.32 [+ or -] 0.08

Table 3. Amount of reducing sugar (mg/L) produced at various
incubation periods. Data are means [+ or -] standard deviation of three
replications.

Organism       Condition   Day 0                Day 6

Trichoderma    Aerobic     0.36 [+ or -] 0.05   0.42 [+ or -] 0.07
reesei
               Anaerobic   0.34 [+ or -] 0.03   0.43 [+ or -] 0.07
Pycnoporus     Aerobic     0.54 [+ or -] 0.49   0.44 [+ or -] 0.57
cinnabarinus   Anaerobic   0.61 [+ or -] 0.58   0.44 [+ or -] 0.53

Fusarium       Aerobic     0.22 [+ or -] 0.08   0.41 [+ or -] 0.26
solani
               Anaerobic   0.21 [+ or -] 0.08   0.49 [+ or -] 0.34
Bacterial      Aerobic     0.21 [+ or -] 0.07   0.35 [+ or -] 0.16
isolate        Anaerobic   0.26 [+ or -] 0.12   0.24 [+ or -] 0.07

Fungal         Aerobic     0.23 [+ or -] 0.07   0.27 [+ or -] 0.10
isolate        Anaerobic   0.26 [+ or -] 0.03   0.29 [+ or -] 0.11

Organism       Day 9                Day 16

Trichoderma    0.39 [+ or -] 0.07   0.77 [+ or -] 0.14
reesei
               0.30 [+ or -] 0.06   0.87 [+ or -] 0.12
Pycnoporus     0.55 [+ or -] 0.60   0.45 [+ or -] 0.47
cinnabarinus   0.55 [+ or -] 0.62   0.54 [+ or -] 0.53

Fusarium       0.36 [+ or -] 0.17   0.38 [+ or -] 0.19
solani
               0.40 [+ or -] 0.21   0.51 [+ or -] 0.31
Bacterial      0.20 [+ or -] 0.06   0.25 [+ or -] 0.09
isolate        0.19 [+ or -] 0.06   0.43 [+ or -] .021

Fungal         0.27 [+ or -] 0.17   0.39 [+ or -] 0.17
isolate        0.26 [+ or -] 01.0   0.32 [+ or -] 0.13

Organism       Day 22               Day 22

Trichoderma    0.65 [+ or -] 0.11   0.38 [+ or -] 0.08
reesei
               0.55 [+ or -] 0.19   0.40 [+ or -] 0.16
Pycnoporus     0.42 [+ or -] 0.46   0.59 [+ or -] 0.59
cinnabarinus   0.53 [+ or -] 0.51   0.65 [+ or -] 0.67

Fusarium       0.40 [+ or -] 0.21   0.50 [+ or -] 0.18
solani
               0.44 [+ or -] 0.21   0.66 [+ or -] .046
Bacterial      0.08 [+ or -] 0.04
isolate        0.13 [+ or -] 0.06

Fungal         0.48 [+ or -] 0.26   0.31 [+ or -] .020
isolate        0.40 [+ or -] 0.16   0.29 [+ or -] 0.18

Organism       Day 28

Trichoderma
reesei

Pycnoporus     0.53 [+ or -] .046
cinnabarinus   0.54 [+ or -] 0.45

Fusarium       0.40 [+ or -] 0.20
solani
               0.51 [+ or -] 0.35
Bacterial
isolate

Fungal
isolate
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Author:Kishinhi, Rose; Partee, Yasmin; Wilborn, Terry; Harris, LeoAlexander; Anderson, Natalie; Miller, Glo
Publication:Journal of the Mississippi Academy of Sciences
Article Type:Report
Date:Oct 1, 2014
Words:4605
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