Megan N. Marshall

Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

ABSTRACT Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.

Evaluation of PCR primers for denaturing gradient gel electrophoresis analysis of fungal communities in compost.

Aims: Three previously published fungal specific PCR primer sets, referred to as the NS, EF and NL primer sets, were evaluated for use in compost microbial community analysis by PCR and denaturing gradient gel electrophoresis (DGGE).

Effects of laccase on lignin depolymerization and enzymatic hydrolysis of ensiled corn stover

The aim of this study was to explore the synergies of laccase, a ligninolytic enzyme, with cellulose and hemicellulase amendments on ensiled corn stover. Molecular signals of lignin decomposition were observed by tetramethylammonium hydroxide thermochemolysis and gas chromatography-mass spectroscopy (TMAH-GC-MS) analysis. The significant findings suggest that ensilage might provide a platform for biological pretreatment. By partially hydrolyzing cellulose and hemicellulose into soluble sugars, ensilage facilitates laccase penetration into the lignocellulose complex to enhance lignin degradation. Downstream cellulose hydrolysis was improved 7% with increasing laccase loading rate. These results demonstrate the potential of enzymes, either directly amended or expressed by microbes during ensilage, to maximize utilization of corn stover for cellulosic biofuels and other downstream fermentations.

Managing compost stability and amendment to soil to enhance soil heating during soil solarization

Soil solarization is a method of soil heating used to eradicate plant pathogens and weeds that involves passive solar heating of moist soil mulched (covered) with clear plastic tarp. Various types of organic matter may be incorporated into soil prior to solarization to increase biocidal activity of the treatment process. Microbial activity associated with the decomposition of soil organic matter may increase temperatures during solarization, potentially enhancing solarization efficacy. However, the level of organic matter decomposition (stability) necessary for increasing soil temperature is not well characterized, nor is it known if various amendments render the soil phytotoxic to crops following solarization. Laboratory studies and a field trial were performed to determine heat generation in soil amended with compost during solarization. Respiration was measured in amended soil samples prior to and following solarization as a function of soil depth. Additionally, phytotoxicity was estimated through measurement of germination and early growth of lettuce seedlings in greenhouse assays. Amendment of soil with 10%(g/g) compost containing 16.9 mg CO2/gdry weight organic carbon resulted in soil temperatures that were 2-4 °C higher than soil alone. Approximately 85% of total organic carbon within the amended soil was exhausted during 22 days of solarization. There was no significant difference in residual respiration with soil depth down to 17.4 cm. Although freshly amended soil proved highly inhibitory to lettuce seed germination and seedling growth, phytotoxicity was not detected in solarized amended soil after 22 days of field solarization.

Implications of organic acids in wet storage and bioconversion of corn stover to ethanol

Narrow harvest windows and contamination concerns with field drying suggest that wet storage will likely be the preferred storage method for biofuel feedstocks in humid regions of the U.S. During wet storage for biofuel production, feedstock is stored under anaerobic and moist (= 25% wet basis) conditions, which result in the production of organic acids by natural acidogenic microorganisms. The organic acids have the potential to alter the feedstock structure and provide partial pretreatment, but can also inhibit subsequent biofuel fermentation. Pretreatment is necessary for lignocellulosic feedstocks since it allows plant cell wall degrading enzymes to have access to structural sugars (cellulose and hemicelluloses) and convert them to glucose and other simple sugars. Microbial inhibition results in reduced specific ethanol productivity (the amount of ethanol produced from the feedstock within a given time). In this study, wet storage of corn stover at seven different moisture contents (25-75%) was incubated under anaerobic conditions at two temperature levels (22oC and 37oC) for 0, 21, and 220 days. Dry matter loss ranged from less than 1% for 21-day storage to less than 4% for 220-day storage. The results also showed that the various conditions can be cluster into three groups based on organic acid profile using Ward’s hierarchical method. Fiber reactivity test was performed on unensiled stover and stover stored at 25% and 75% moisture without prior pretreatment to determine if organic acids produced during ensilage have any significant pretreatment effect on fiber structure. In general, the sugar yield from fiber reactivity was low but the results could be related to the three cluster groups and were indicative that ensilage does have a beneficial effect on fiber structure.

Wet corn stover storage: correlating fiber reactivity with storage acids over a wide moisture range

Wet storage is synonymous with ensilage, a simple biotechnology that has been used to preserve forage for livestock feed for millennia. In this natural process, organic acids are produced by anaerobic microbial degradation of a small fraction of the biomass, and these acids reduce the pH to levels that minimize further microbial activity and can preserve the biomass for years as long as anaerobic conditions are maintained. These organic acids also result in mild pretreatment with potential to enhance downstream conversion processes, making this an effective storage strategy. However, the degree and significance of this natural pretreatment capability of ensiled storage on downstream processes has not previously been quantified across a range of storage conditions. In this study, the degree of pretreatment was investigated by measuring the reactivity of corn stover fiber to cellulolytic enzymes. Although the results indicated significant improvement in hydrolytic outcomes after wet storage, by a factor of up to 2.4, saccharification of cellulose to sugar monomers was still limited. The results also show that dominance of lactic acid in the ensilage process is key to wet storage [pretreatment] effectiveness as in the livestock feed industry. Lactic acid pKa value is lower than the pKa of other silage acids and lower than typical silage pH. This gives lactic acid the advantage of being in the more dissociated form, with more protons available to facilitate pretreatment hydrolysis. However, unlike the livestock feed industry, where quality feedstock is attainable within very narrow storage moisture range, for biofuel purposes, a wider range of 35 ―65% is appropriate in achieving a similar quality outcome. This is true both for the immediate fiber response to enzymes and with subsequent pretreatment. This wider moisture range implies more flexibility in harvest schedule without sacrificing feedstock quality, thus alleviating concerns over feedstock quality that biomass suppliers or biorefineries may have.

Effects of Pretreatment Conditions on Downstream Bioconversion of Poplar Wood (Populus sp.) into Biofuels

One of the biggest challenges for using lignocellulosic biomass to generate biofuels is the pretreatment process, which involves the breakdown of the plant cell wall structure to release cellulose and hemicellulose from lignin. The structural mechanisms of lignocellulose breakdown during pretreatment are not yet understood. Poplar, especially hybrid varieties, has a lot of potential for biomass conversion because of its fast growing nature and the ability to regenerate from roots and trunks. The main goal of this research is to compare the effects of pretreating poplar wood using two different devices: 1) Accelerated Solvent Extraction (ASE) system (Micro-bench scale); and 2) Parr reactor (Bench scale) at various solid loading rates. Results of pretreatment experiments indicated that sugar yields did not change significantly when the solid loading concentration was increased, using a solvent saver mode with the ASE. It was also observed that sugars released during pretreatment increased as the pretreatment temperatures were increased from 170°C to 200°C. Fermentation and enzymatic degradation experiments carried out with these pretreated materials will be used to determine the pretreatment conditions that lead to maximum alcohol yield. Pretreatment conditions at laboratory-scale that mimic pilot-scale conditions to give similar yields will be identified. These results will be useful in developing process models for the pretreatment of poplar wood, in order to obtain high sugar and subsequently high biofuel yields. Results of nanoscale imaging, transport property studies and compositional analyses of the plant cell wall can be correlated with results of pretreatment and degradation experiments to better understand the breakdown mechanisms during pretreatment.

From Fields to Fuels: A Student Workshop on Conversion of Biomass to Ethanol

Many biomass materials can be converted into ethanol, with the conversion process varying based on the biological and structural complexity of the material. This process provides multiple opportunities to highlight important principles in biological engineering, with topical, compelling, and relevant exercises for science, technology, engineering and math (STEM) education. In 2009 we piloted a four-day workshop designed to provide hands-on lab experiences with ethanol production from biomass. The workshop was offered as part of a summer camp for high school girls, sponsored by the Penn State Women in the Sciences and Engineering (WISE) Institute. The participants were divided into three groups, each group performing their own experiments with five different materials - sugarcane, paper, corn stover, poplar, and switchgrass. Workshop participants performed the following procedures: (1) hot water pretreatment to alter biomass structure and increase enzyme accessibility, (2) enzymatic hydrolysis to release sugars from cellulose, (3) fermentation to convert sugars to ethanol, and (4) distillation to recover ethanol from the fermentation broth. Ethanol concentrations in the fermentation broth ranged from 8 to 24 g/L, according to sugarcane > switchgrass > paper = stover > poplar. Although the workshop was performed in a laboratory with specialized instruments dedicated to bioenergy research, the workshop could also be conducted in a typical chemistry laboratory with access to an autoclave and distillation column. A workbook was created to document the experimental procedures and to record data and perform calculations related to feedstock supply and ethanol yields. The workshop built on this technical foundation to include analysis of social, ethical, and ecological issues including environmental impacts, food security, and rural development.

Combining Compost Application and Soil Solarization for Control of Soilborne Plant Pathogens

In soil solarization, moist soil is covered with transparent plastic tarps, resulting in passive solar heating of the soil and a pasteurization effect, which reduces plant pathogen populations without the use of chemical pesticides. By combining compost application and soil solarization, there is the added benefit of utilizing organic wastes and the potential for decreasing the duration of solarization treatments. Compost stimulates microbial activity in the soil, which may lead to heat generation by aerobic microorganisms, higher soil temperatures, and thus more rapid thermal inactivation of pathogen populations. The objective of these initial laboratory studies was to assess the potential for compost incorporation to produce higher soil temperatures during solarization. These studies were carried out using insulated 1-L bioreactors, which were placed in an incubator to approximate diurnal soil temperature fluctuations typically achieved during solarization. Bioreactors were filled with soil mixed with compost substrates at various loading rates (0, 2% (v/v), or 10% (v/v)). Oxygen was supplied to bioreactors through humidified airflow or by diffusion through a piece of solarization plastic. Temperatures in aerated bioreactors with compost substrates were up to 3-10 °C higher than bioreactors with soil alone. Increased respiration in aerated bioreactors with compost substrates suggested that increased temperatures were related to increased aerobic microbial activity.However, the temperature of bioreactors receiving oxygen by diffusion through solarization tarp was not greater when soil was amended with compost substrates. These results show the potential for compost addition to increase soil solarization temperatures by increasing aerobic microbial activity, if sufficient oxygen is available. Future experiments will focus on variables that influence soil oxygen content during solarization, such as soil moisture content, compost loading rate, and compost stability. New bioreactors are under construction that will provide oxygen to the soil only by diffusion through solarization plastic, will measure temperature more accurately using thermistor probes, and will characterize the oxygen limitation by measuring redox potential in soil and oxygen concentration in headspace.