Stubble Degradation

The conversion of polymeric lignin from plant biomass into renewable chemicals is an important unsolved problem in the biorefinery concept. This article summarises recent developments in the discovery of bacterial enzymes for lignin degradation, our current understanding of their molecular mechanism of action, and their use to convert lignin or lignocellulose into aromatic chemicals. The review also discusses the recent developments in screening of metagenomic libraries for new biocatalysts, and the use of protein engineering to enhance lignin degradation activity.

Lignin oxidation by bacterial dye-decolorizing peroxidase enzymes requires hydrogen peroxide as a cosubstrate, an unstable and corrosive oxidant. We have identified a glycolate oxidase enzyme from Rhodococcus jostii RHA1 that can couple effectively at pH 6.5 with DyP peroxidase enzymes from Agrobacterium sp. or Comamonas testosteroni to oxidise lignin substrates without addition of hydrogen peroxide. Rhodococcus jostii RHA1 glycolate oxidase (RjGlOx) has activity for oxidation of a range of α-ketoaldehyde and α-hydroxyacid substrates, and is also active for oxidation of hydroxymethylfurfural (HMF) to furandicarboxylic acid. The combination of RjGlOx with Agrobacterium sp. DyP or C. testosteroni DyP generated new and enhanced amounts of low molecular weight aromatic products from organosolv lignin substrates, and was able to generate high-value products from treatment of lignin residue from cellulosic biofuel production, and from a polymeric humin substrate.

Pathways by which the biopolymer lignin is broken down by soil microbes could be used to engineer new biocatalytic routes from lignin to renewable chemicals, but are currently not fully understood. In order to probe these pathways, we have prepared synthetic lignins containing 13 C at the sidechain b-carbon. Feeding of [b-13 C]-labelled DHP lignin to Rhodococcus jostii RHA1 has led to the incorporation of 13 C label into metabolites oxalic acid, 4-hydroxyphenylacetic acid, and 4-hydroxy-3methoxyphenylacetic acid, confirming that they are derived from lignin breakdown. We have identified a glycolate oxidase enzyme in Rhodococcus jostii RHA1 which is able to oxidise glycolaldehyde via glycolic acid to oxalic acid, thereby identifying a pathway for the formation of oxalic acid. R. jostii glycolate oxidase also catalyses the conversion of 4-hydroxyphenylacetic acid to 4-hydroxybenzoylformic acid, identifying another possible pathway to 4-hydroxybenzoylformic acid. Formation of labelled oxalic acid was also observed from [b-13 C]-polyferulic acid, which provides experimental evidence in favour of a radical mechanism for a,b-bond cleavage of b-aryl ether units.

The relative ability of the small laccase (sLac) and dye-decoloring peroxidase (DyP2) from Amycolatopsis sp. 75iv2 to transform a variety of lignins was investigated using time-of-flight secondary ion mass spectrometry (ToF-SIMS). The enzymes modified organosolv hardwood lignin to different extents even in the absence of an added mediator. More particularly, sLac decreased the lignin modification metric S (S-lignin)/Ar (total aromatics) by 58% over 16h, while DyP2 lowered this ratio by 31% in the absence of exogenous H2O2. When used on their own, both sLac and DyP2 also modified native lignin present in aspen wood powder, albeit to lesser extents than in the organosolv lignin. The addition of ABTS for sLac and Mn2+ as well as H2O2 for DyP2 led to increased lignin modification in aspen wood powder as reflected by a decrease in the G/Ar metric by up to a further 13%. This highlights the importance of exogenous mediators for transforming lignin within its native matrix. Furthermore, th...

The aim of the study was to determine the properties of pulp and paper produced from wheat stubble (Triticum aestivum L.), using soda-oxygen-sodium borohydride (NaBH 4) cooking method. Whole wheat straw (Triticum aestivum L.) was also used for comparison with stubbles. The morphological and chemical properties of the raw materials were investigated. The results showed that stubble had high holocellulose, cellulose, and alpha-cellulose contents and low lignin content. Fiber length, fi ber width, lumen diameter, and cell wall thickness were measured in order to determine suitability of the fi bers for pulp and paper production. The values were used to calculate fi ber parameters. The effects of these parameters on paper strength were also discussed. The effect of NaBH 4 on the yield and chemical, physical, and optical properties of pulp and paper were investigated. The addition of NaBH 4 increased pulp yield by 4.1 %, and improved the physical and optical properties of the pulp. The physical and optical properties of the stubble pulp were better than those of whole wheat straw pulp. It was concluded that these characteristics demonstrate the suitability of wheat stubbles for pulp and paper production.

The activity of extracellular phenol oxidases is believed to play a critical role in decomposition processes in peatlands. The water logged, acidic conditions, and recalcitrant litter from the peatland vegetation, lead to exceptionally high phenolics in the peat. In order to quantify the activity of oxidative enzymes involved in the modification and break down of phenolic compounds two types

As Earth's climate warms, the massive stores of carbon found in soil are predicted to become depleted, and leave behind a smaller carbon pool that is less accessible to microbes. At a long-term forest soil-warming experiment in central Massachusetts, soil respiration and bacterial diversity have increased, while fungal biomass and microbially-accessible soil carbon have decreased. Here, we evaluate how warming has affected the microbial community's capability to degrade chemically-complex soil carbon using lignin-amended BioSep beads. We profiled the bacterial and fungal communities using PCR-based methods and completed extracellular enzyme assays as a proxy for potential community function. We found that lignin-amended beads selected for a distinct community containing bacterial taxa closely related to known lignin degraders, as well as members of many genera not previously noted as capable of degrading lignin. Warming tended to drive bacterial community structure more strongly in the lignin beads, while the effect on the fungal community was limited to unamended beads. Of those bacterial operational taxonomic units (OTUs) enriched by the warming treatment, many were enriched uniquely on lignin-amended beads. These taxa may be contributing to enhanced soil respiration under warming despite reduced readily available C availability. In aggregate, these results suggest that there is genetic potential for chemically complex soil carbon degradation that may lead to extended elevated soil respiration with long-term warming.

Humid tropical forest soils are characterized by low and fluctuating redox, conditions which are thought to inhibit organic matter degradation by microbes. However, evidence suggests that soil microbial communities are adapted to the redox conditions in these ecosystems. In this study we tested the hypothesis that soil oxygen (O 2) availability as an index of redox conditions structures patterns in litter decomposition and associated microbial community dynamics over space and time in humid tropical forests. We conducted a two year decomposition experiment on a common litter substrate in four sites along an elevational gradient with well described climate and redox dynamics. Microbial community sequencing, potential enzyme activities, and litter chemistry measurements were made on litter and soil to determine the relationship between soil and litter communities and biogeochemistry. Decomposition was slowest in the upper elevation site, which was the wettest and had the lowest average soil O 2 availability. However, soil hydrolytic and litter phenol oxidase activities were greatest at this site. Small subunit ribosomal RNA genes were sequenced with universal primers for bacteria, archaea and eukaryotes, yielding 40,850 unique taxa after quality filtering and clustering. Across all sites, microbial succession was observed as increasing litter richness, converging bacterial community profiles, and diverging fungal community profiles. Initial decomposers (1e4 weeks) included many r-selected bacteria, including Alpha-, Beta-and Gammaproteobacteria, Clostridia, Bacteroidetes. We also found evidence of anaerobic fungi such as Cryptococcus, as well as the plant-associated Phialocephala and Phyllachora species, suggesting that anaerobic and plant-associated fungi are prevalent later in decomposition in soils with low and fluctuating redox conditions. Because of the striking similarities between sites in functional potential despite differences in wet tropical soil decomposing communities and litter chemistry, we suggest that future climate-driven disruptions to redox fluctuations could significantly alter the terrestrial carbon (C) cycle in tropical forests.

During the year 1983, there was a breakthrough in the field of lignin biodegradation when fungal ligninases and their hydrogen peroxide requirement were described. A comparable progression has not yet occurred with ligninolytic bacteria, although it is expected to take place in the near future once depolymerizing enzymes are isolated. Several bacterial strains have been found to mineralize aerobically [14C-lignin]lignocellulose as well as 14C-labelled synthetic lignins, even though the most efficient are still far from reaching the rates exhibited by ligninolytic fungi. Actinomycetes follow a characteristic pattern of lignocellulose decomposition, with the release of lignin-rich, water-soluble fragments that are slowly metabolized thereafter. Research is being carried out to find the key enzymes involved in both lignin solubilization and mineralization by bacteria and uncover their mechanism of action. The ability of bacteria to grow on low-molecular-weight lignin oligomers as the sole source of carbon and energy indicates that bacteria produce enzymes catalysing cleavage of intermonomeric linkages. Various strains metabolize cyclic lignans, biphenyl structures, and other "dimeric" compounds, including those that possess the arylglycerol-fl-aryl ether (fl-O-4) linkage. Cleavage of the latter apparently is reductive, fl-O-4 dimers being metabolized by some bacteria through Cc~-Cfl cleavage. In contrast, no one has isolated a bacterium capable of decomposing a dimeric structure of the 1,2-diarylpropane-1,3-diol (fl-1) type, although strains metabolizing 1,2-diarylethane compounds have been found. In the absence of oxygen, only low-molecular-weight oligomers or chemically modified lignins are significantly degraded. The contribution of bacteria to the complete biodegradation of lignin in natural environments where fungi are also present is not known. However, bacteria seem to play a leading role in decomposing lignin in aquatic ecosystems.

Monocropping of rice is practiced in Assam (situated at north east part of India) throughout the year in different agro-ecosystems (upland and lowland) primarily under rainfed conditions. The estimation of methane emission has been realized investigating high yielding rice varieties viz. Ranjit and Mahsuri, grown under two different agro-ecosystems at Lower Brahmaputra Valley Zone of Assam with sandy to sandy loam type of soil. Variety 'Ranjit' grown at monsoon (Sali) rice ecosystem at lowland rainfed condition showed higher seasonal integrated methane flux value compared to variety 'Mahsuri' grown at pre monsoon (Ahu) rice ecosystem. Both varieties showed two methane peaks, one at the active tillering stage and the second at the reproductive stage of the crop. The observed variation in methane emission peaks are contributed mainly by physiological characteristics of the rice plant such as leaf numbers, tiller numbers, plant height, root shoot biomass and leaf area index. Statistical analysis of these parameters showed a positive correlation with methane emission. These physiological parameters are in turn governed by plant genotypes and environment i.e., the field water availability and climatological factors (rainfall and temperature) during the growing season. While comparing the two ecosystems it was found that methane emission is significantly less from upland rainfed rice ecosystem and this ecosystem can be considered a suitable option for biological mitigation of methane from rice paddies.

Pathways by which the biopolymer lignin is broken down by soil microbes could be used to engineer new biocatalytic routes from lignin to renewable chemicals, but are currently not fully understood. In order to probe these pathways, we have prepared synthetic lignins containing 13 C at the sidechain b-carbon. Feeding of [b-13 C]-labelled DHP lignin to Rhodococcus jostii RHA1 has led to the incorporation of 13 C label into metabolites oxalic acid, 4-hydroxyphenylacetic acid, and 4-hydroxy-3methoxyphenylacetic acid, confirming that they are derived from lignin breakdown. We have identified a glycolate oxidase enzyme in Rhodococcus jostii RHA1 which is able to oxidise glycolaldehyde via glycolic acid to oxalic acid, thereby identifying a pathway for the formation of oxalic acid. R. jostii glycolate oxidase also catalyses the conversion of 4-hydroxyphenylacetic acid to 4-hydroxybenzoylformic acid, identifying another possible pathway to 4-hydroxybenzoylformic acid. Formation of labelled oxalic acid was also observed from [b-13 C]-polyferulic acid, which provides experimental evidence in favour of a radical mechanism for a,b-bond cleavage of b-aryl ether units.

DESCRIPTION Recent demands for bio-based products from plant tissues led to an increased interest in the enzymatic activity of soil microbial communities that are potentially involved into the lignocellulose deconstruction. Lignocellulolytic microbes have developed a unique strategy to handle lignin degradation through the concerted action of lignocellulose-degrading enzymes, whose activity is regulated by soil properties, land use, biomass characteristics and the identity of their microbial decomposer. Recent studies have shown that the transgenic expression of microbial lignocellulolytic enzymes in plants is able to increase the degradability of resulting plant tissues. In order to unravel the microbial environment of plant litter and its associated enzymatic activity, we collected soil samples from sites characterized by the presence of different decomposing plants, Arundo donax and Phragmites australis. DNA extracted from soil was used for meta-genomic analyses using Roche 454 p...

Lignin is an abundant cell wall component, and it has been used mainly for generating steam and electricity. Nevertheless, lignin valorization, i.e. the conversion of lignin into high value-added fuels, chemicals, or materials, is crucial for the full implementation of cost-effective lignocellulosic biorefineries. From this perspective, rapid screening methods are crucial for time-and resource-efficient development of novel microbial strains and enzymes with applications in the lignin biorefinery. The present review gives an overview of recent developments and applications of a vast arsenal of activity and sequence-based methodologies for uncovering novel microbial strains with ligninolytic potential, novel enzymes for lignin depolymerization and for unraveling the main metabolic routes during growth on lignin. Finally, perspectives on the use of each of the presented methods and their respective advantages and disadvantages are discussed.

The lignocellulosic biomass is comprised of three major components: cellulose, hemicellulose, and lignin. Among these three, cellulose and hemicellulose were already used for the generation of simple sugars and subsequent value-added products. However, lignin is the least applied material in this regard because of its complex and highly variable nature. Regardless, lignin is the most abundant material, and it can be used to produce value-added products such as lignin-modifying enzymes (LMEs), polyhydroxyalkanoates (PHAs), microbial lipids, vanillin, muconic acid, and many others. This review explores the potential of lignin as the microbial substrate to produce such products. A special focus was given to the different types of lignin and how each one can be used in different microbial and biochemical pathways to produce intermediate products, which can then be used as the value-added products or base to make other products. This review paper will summarize the effectiveness of ligni...

The relative ability of the small laccase (sLac) and dye-decoloring peroxidase (DyP2) from Amycolatopsis sp. 75iv2 to transform a variety of lignins was investigated using Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS). The enzymes modified organosolv hardwood lignin to different extents even in the absence of an added mediator. More particularly, sLac decreased the lignin modification metric S (S-lignin)/Ar (total aromatics) by 58% over 16 hours while DyP2 lowered this ratio by 31% in the absence of exogenous H2O2. When used on their own, both sLac and DyP2 also modified native lignin present in aspen wood powder, albeit to lesser extents than in the organosolv lignin. The addition of ABTS for sLac, and Mn2+ as well as H2O2 for DyP2 led to increased lignin modification in aspen wood powder as reflected by a decrease in the G/Ar metric by up to a further 13%. This highlights the importance of exogenous mediators for transforming lignin within its native matrix. Furthermore, the addition of ABTS reduced the selectivity of sLac for S-lignin over G-lignin, indicating that the mediator also altered the product profiles. Finally, when sLac was included in reactions containing DyP2, in part to generate H2O2 in situ, the relative abundance of lignin products differed from individual enzymatic treatments. Overall, these results identify possible routes to tuning lignin modification or delignification through choice of enzyme and mediator. Moreover, the current study expands the application of ToF-SIMS to evaluating enzyme action on technical lignins, which can accelerate the discovery and engineering of industrially relevant enzymes for lignin valorization.

The current demands for novel and sustainable biotechnological
processes, including new microbial enzymes with industrial
potential are constantly required. As a form to address the growing
need for industrially relevant enzymes, functional screenings of
microorganisms and/or (meta)genome mining techniques have
emerged as powerful strategies for the identification of promising
enzymes to novel or improved industrial processes [1]. Once
enzymes have been well characterized, they can be produced,
studied, and engineered about their biocatalytic, including possible
synergistic activities in multiple protein cocktails. Currently, there
is an increased interest in exploring and exploiting microbial
enzymes for selective degradation of plant biomass. The efforts
are concentrated on the bioconversion of lingo-cellulosic material.
While ample enzymes are nowadays available for the degradation
and modification of the polysaccharide content of plant biomass,
there is a need for effective lignin degrading enzymes.

There is increasing interest in research on lignin biodegradation compounds as potential building blocks in applications related to renewable products. More attention is necessary to evaluate the effects of the initial pH conditions during the bacterial degradation of lignin. In this study we performed experiments on lignin biodegradation under acidic and mild alkaline conditions. For acidic biodegradation, lignin was chemically pretreated with hydrogen peroxide. Alkaline biodegradation was achieved by developing the bacterial growth on Luria and Bertani medium with alkali lignin as the sole carbon source. The mutant strain Escherichia coli BL21(Lacc) was used to carry out lignin biodegradation over 10 days of incubation. Results demonstrated that under acidic conditions there was a predominance of aliphatic compounds of the C3–C4 type. Alkaline biodegradation was produced in the context of oxidative stress, with a greater abundance of aryl compounds. The final pH values of acidic and alkaline biodegradation of lignin were 2.53 and 7.90, respectively. The results of the gas chromatography mass spectrometry analysis detected compounds such as crotonic acid, lactic acid and 3-hydroxybutanoic acid for acidic conditions, with potential applications for adhesives and polymer precursors. Under alkaline conditions, detected compounds included 2-phenylethanol and dehydroabietic acid, with potential applications for perfumery and anti tumor/anti-inflammatory medications. Size-exclusion chromatography analysis showed that the weight-average molecular weight of the alkaline biodegraded lignin increased by 6.75-fold compared to the acidic method, resulting in a repolymerization of its molecular structure. Lignin repolymerization coincided with an increase in the relative abundance of dehydroabietic acid and isovanillyl alcohol, from 2.70 and 3.96% on day zero to 13.43 and 10.26% on 10th day. The results of the Fourier-transformed Infrared spectroscopy detected the presence of C = O bond and OH functional group associated with carboxylic acids in the acidic method. In the alkaline method there was a greater preponderance of signals related to skeletal aromatic structures, the amine functional group and the C – O – bond. Lignin biodegradation products from E. coli BL21(Lacc), under different initial pH conditions, demonstrated a promising potential to enlarge the spectrum of renewable products for biorefinery activities.

Biological lignin valorization to fuels and value-added chemicals enables sustainable and economic biorefineries. While significant progress has been made, several major challenges arose due to high recalcitrance and het-erogeneity of lignin, which needs to be addressed to improve lignin processing. This work provides an overview of biological lignin conversion and its regulation from a metabolic engineering and systems biology viewpoint. Biological lignin valorization includes three stages: lignin depolymerization, aromatics degradation, and target product biosynthesis. Ligninolytic microorganisms have an extensive enzymatic toolbox to break down the lignin and convert heterogeneous lignin derivatives to central intermediates, such as protocatechuate or catechol, through a peripheral pathway. These intermediates undergo ring cleavage via the β-ketoadipate pathway and are ultimately transformed into metabolites by yielding acetyl-CoA for internal product biosynthesis, such as tria-cylglycerols, polyhydroxyalkanoates, etc. Bioprospecting will expand the knowledge base of ligninolytic microbial communities, strains, and enzymes to facilitate the understanding of aromatics metabolism. Systems biology analyses achieve an understanding of molecular and systems-level degradation mechanisms and metabolic pathways of lignin and aromatics. By identifying these mechanisms, synthetic biology provides promising approaches to create the lignin conversion pathways and engineer ligninolytic strains suitable as potential hosts for lignin conversion. Techno-economic analysis of biological lignin upgrading to coproducts in biorefineries will guide the implementation of lignin valorization by mitigating technical risk for scale-up and improving the profitability of biorefinery. By improving the understanding of biological lignin valorization, it should be possible to create biological lignin valorization route to effectively produce value-added products from lignin.

The degradation of dimeric phenylpropanoid lignin model compounds using mixed bacterial cultures was studied. The six model compounds contained the most common linkages of lignin: fl-O-4, fl-fl, fl-5, and fl-1. The results indicate that it is possible to enrich bacteria which are able to degrade all these compounds. Bacteria were also able to use these dimers as the sole source of carbon for growth. In view of these results it seems probable that bacterial inability to degrade polymeric lignin is due to the physical properties such as the molecular size of lignin.

Stubbles of rice varieties Mahsuri (Taichung 65/ Mayang Ebos 6080/2) and Ranjit (Pankaj x Mahsuri) were treated in situ after harvest of the crop by spraying microbial inoculums – laboratory culture of cellulose degrading microorganism (CDM) or commercial yogurt or both with glyphosate, and with or without sugar for aerobic decomposition. Microbial inoculums were also sprayed with glyphosate and urea and the treatments were compared with control plots, viz. untreated, water spray and glyphosate spray. At the end of the fourth month, Mahsuri showed faster decomposition than Ranjit with corresponding decreases in dry weight and organic carbon content of the stubbles due to spraying of CDM culture or commercial yogurt with glyphosate. The effect of addition of sugar to the spray mixture was not significant. The reduction in stubble dry weight and organic carbon content was up to 61.1 and 45.3 per cent in Mahsuri and up to 47.1 and 46.4 per cent in Ranjit, respectively after four months of the treatments. Significant increase in diversity of weeds in terms of number of species was observed in plots with higher stubble decomposition.

The residues of crop and weeds in rainfed upland rice ecosystem need rapid, viable utilization before establishment of the succeeding crop. Enhanced decomposition reducing the volume and C:N ratio of the biomass may be a good proposition towards efficient residue management facilitating tillage and decomposition. Cellulose degrading microbe (CDM) or commercial yogurt was evaluated with glyphosate for in situ decomposition of rice stubbles after harvest. A field experiment was conducted by spraying glyphosate, and CDM or yogurt with or without sugar on rice stubbles after harvest of the crop. Glyphosate and, CDM or yogurt significantly reduced the dry weight, carbon and nitrogen content of stubbles at 25, 50 and 75 days after spray compared to untreated plot or spraying glyphosate and bacterial cultures separately, and the corresponding organic carbon content and C:N ratio of the stubble reduced significantly. The effect of adding sugar with the spray solution was not significant. The species diversity of the weeds for grass and sedge was unaffected by the treatments, but number of broadleaved weed species significantly increased due to spraying of glyphosate and, CDM or yogurt.

Lignocellulosic biofuels are promising as sustainable alternative fuels, but lignin inhibits access of enzymes to cellulose, and by-products of lignin degradation can be toxic to cells. The fast growth, high efficiency and specificity of enzymes employed in the anaerobic litter deconstruction carried out by tropical soil bacteria make these organisms useful templates for improving biofuel production. The facultative anaerobe Enterobacter lignolyticus SCF1 was initially cultivated from Cloud Forest soils in the Luquillo Experimental Forest in Puerto Rico, based on anaerobic growth on lignin as sole carbon source. The source of the isolate was tropical forest soils that decompose litter rapidly with low and fluctuating redox potentials, where bacteria using oxygen-independent enzymes likely play an important role in decomposition. We have used transcriptomics and proteomics to examine the observed increased growth of SCF1 grown on media amended with lignin compared to unamended growth. Proteomics suggested accelerated xylose uptake and metabolism under lignin-amended growth, with up-regulation of proteins involved in lignin degradation via the 4-hydroxyphenylacetate degradation pathway, catalase/peroxidase enzymes, and the glutathione biosynthesis and glutathione S-transferase (GST) proteins. We also observed increased production of NADH-quinone oxidoreductase, other electron transport chain proteins, and ATP synthase and ATP-binding cassette (ABC) transporters. This suggested the use of lignin as terminal electron acceptor. We detected significant lignin degradation over time by absorbance, and also used metabolomics to demonstrate moderately significant decreased xylose concentrations as well as increased metabolic products acetate and formate in stationary phase in lignin-amended compared to unamended growth conditions. Our data show the advantages of a multi-omics approach toward providing insights as to how lignin may be used in nature by microorganisms coping with poor carbon availability.

El cultivo de piña en Costa Rica ha permitido exportar hasta dos millones de toneladas por año, sin embargo genera un importante residuo, el rastrojo. Este residuo representa un problema ambiental debido a que permite la proliferación de la mosca de establo. Los productores recurren al uso de herbicidas quemantes para evitar esta situación.
Un proyecto de investigación de la Universidad Nacional de Costa Rica busca generar una metodología de tratamiento alterna de este residuo con un consorcio de microrganismos. Como primera fase de este proyecto, se determinó que el rastrojo de piña puede ser degradado por un consorcio de microorganismos compuestos por cepas del hongo Pleurotus ostreatus, las bacterias Lactobacillus sp, Bacillus subtilis, y la levadura Saccharomyces cerevisiae. Este poster refleja el trabajo de evaluar si el crecimiento de este consorcio es posible en suelos
Se ha comprobado cualitativamente con el trabajo objeto de este poster que el crecimiento del consorcio de hongos y bacterias objeto de la investigación es posible en suelos tanto de la Universidad Nacional en Heredia, como de Pital de San Carlos. El crecimiento óptimo se da cuando se tienen 6 gramos de suelo, 3 gramos de rastrojo y 1 mL de microorganismos y la humedad es del 75%. Se observa mayor crecimiento del consorcio microbiano cuando el suelo está sin esterilizar.

Since most renewable carbon is either in lignin or in compounds protected by lignin, there is wider scope for degradation of lignin in a sustainable way.
In this article, efforts are made to understand the gravity of the situation and various concepts to achieve these goals are narrated.