Consolidating microbial process for sustainable ethanol production from poplar pairings.

Abstract

Lignocellulosic bioethanol represents the conversion of largest renewable biomass into transport fuel. For a particular plant biomass biological conversion economy is essentially dependent on the abundance and low/no cost abailability of the raw material / feedstock and development of efficient saccharification and fermentation processes. Simultaneous saccharification and fermentation (SSF) has been considered an appealing strategy in this regard. Recently natural feedstocks’ bioprocessing without the involvement of pre-treatment/detoxification processes is being investigated by workers for developing consolidated processes. The present study reports a simple consolidated bioprocess for conversion of poplar parings into ethanol. Poplar tree is cultivated in Pakistan to feed the needs of matchsticks, toothpics, ice creams stick and wooden crates. Resultantly its leaves and twigs are wasted, while the tree’s parings are rich in cellulosic content and thus represent renewable low/negative cost fermentable feedstock. A bacterium isolated from fish gut and preserved in the conservatory of Microbiology laboratory, Department of Zoology, University of the Punjab, Lahore was found cellulolytic as well as ethanologenic while saccharifying and fermenting the poplar parings. The bacterium had been identified as Bacillus cereus following its 16 S rDNA sequencing. In course of the present study its genome was sequenced commercially. An ethanologenic yeast was isolated from surface soil that had long been impregnated with fresh sugarcane juice. The yeast was found compatible for co culturing with the Bacillus cereus. The yeast grew best at 37oC; the optimum growth temperature of the bacterium (B. cereus) too. The yeast was identified as Candida tropicalis following its 18S rDNA sequencing. The present research reports pretreatment of poplar pairings by dilute acid, alkali and steam under pressure. For acid pretreatment three variables employed for Box-Bhenken Design (BBD) of response surface methodology (RSM) were sulphuric acid concentration, substrate loading and residence time. It was found that maximum amount of total sugars and total phenolic compounds with respective values of 163.056 and 57.386 (mg/ml) were liberated at 15% (w/v) substrate loading and 4 hrs of retention time in 0.8% (v/v) sulphuric acid at room temperature. Whereas highest level of reducing sugars (6.59 mg/ml) was obtained at 15% substrate loading and 6 hrs of soaking time in 0.6% sulphuric acid. The proposed model was found statistically significant (P<0.001) for removal of total phenolic compounds. Total sugars, reducing sugars and total phenolic compounds had the Fisher’s F-test values of 2.85, 48.39 and 8.08, respectively. Coefficient of determination values of total sugars, total phenolic compounds and reducing sugsrs (83.68%, 98.85% and 93.57%, respectively) predicted the goodness of fit of the model. Mass balance analysis of the acid pretreated substrate revealed that maximum degradation (80%) was obtained when 0.6% sulphuric acid was applied for 6 hrs at room temperature. When the acid treatment was coupled with autoclaving, it was found that maximum amounts of total sugars and phenolic compounds with respective values of 303.064 and 38.801 (mg/ml) were obtained when 15% substrate was treated with 0.6% sulphuric acid for 6 hrs. Whereas highest amount of reducing sugars upto 17.053 mg/ml was released when 10% substrate was soaked in 1.0% sulphuric acid for 8 hrs before autoclaving at 121oC for 20 minutes. The sugars production was vividly higher following the thermochemical pretreatment as compared to the acid alone. Whilst lower amount of total phenolic compounds was released following the thermochemical treatment. Mass balance analysis of the acid plus steam treated substrate showed 66.5% maximum degradation at 15% substrate loading treated with 0.8% sulphuric acid for 8 hrs before autoclaving. The total sugars, reducing sugars and total phenolic compounds released following acid plus steam pretreatment had Fisher’s F-test values of 17.18, 3.17 and 5.84, respectively. The substrate (poplar leaves and twigs 1:1) was also treated with dilute NaOH. Using BBD, it was found that maximum total sugars upto 184.18 mg/ml, reducing sugars upto 6.50 mg/ml and total phenolic compounds upto 47.73 mg/ml were released when 15% of the substrate was treated with 3% NaOH for 4 hrs at room temperature. The total sugars, reducing sugars and total phenolic compounds rebased following the alkaline pretreatment of the substrate had Fisher’s F-test values of 1.66, 0.95 and 3.69, respectively. Mass balance analysis of the alkaline treated substrate showed a maximum degradation of 76% at 5% substrate loading treated with 5% NaOH for 6 hrs. Following the alkaline plus steam pretreatement of the substrate it appeared that highest amount of total sugars upto 305.64 mg/ml was liberated at 15% substrate loading treated with 5% NaOH for 6 hrs before autoclaving. Maximum reducing sugars (16.65 mg/ml) were obtained at 10% substrate treated with 1% NaOH for 8 hrs. Whereas maximum total phenolic compounds measuring upto 166.91 mg/ml were obtained when 15% substrate was soaked in 3% NaOH for 8 hrs at room temperature before autoclaving. The Fisher’s F-values of 43.03, 10.68 and 139.12 were observed for total sugars reducing sugars, and total phenolic compounds, respectively. The mass balance analysis of the base plus steam pretreated substrate showed maximum degradation of 90% when 5% substrate was treated with 1% NaOH for 6 hrs before autoclaving. The Bacillus cereus yielded 0.698 IU/ml/min of exoglucanase and was thus selected for this study amongst the five Bacillus species screened initially. The cellulase optimization experiments revealed 0.5% yeast extract, 0.09% MgSO4 and 0.03% peptone as optimum concentrations for maximum cellulase production while using poplar substrate as carbon source. Initial pH 9.0, 37oC incubation temperature and 2% inoculum size were found optimum for maximum productions of exo as well as endogluconases ranging from 2.36 to 2.00 and 2.55 to 3.49 IU/ml/min, respectively, by the B. cereus. The saccharification experiments employing the crude enzymes were conducted at 50oC. The bacterial exoglucanase and the commercial cellulase released total sugars upto 31.42 mg/ml and 41.18 mg/ml, respectively after 6 hrs of incubation at 50oC using raw poplar biomass. Steam under pressure treated poplar biomass gave better results as compared to both the categories of acid and alkali pretreated substrates. Therefore the steam under pressure treatment was selected as the most simple and efficient pretreatment for the substrate for subsequent saccharification by cellulases from the B. cereus. Potential of the cellulolytic B. cereus for saccharification of lignocellulosic substrate and bioethanol fermentation was unveiled by employing the separate hydrolysis and fermentation (SHF) as well as simultaneous saccharification and fermentation (SSF) processes. The bacterium B. cereus grew successfully in a medium comprised of 2% substrate (poplar), 0.5% yeast extract, 0.03% peptone and 0.09% MgSO4 with 2% inoculum. The culture was incubated at 37oC with agitation of 120 rpm for 24 hrs. The crude enzyme was used to saccharify the poplar substrate and the sugars stream was then fermented with the help of bacterium, yeast and their co culture. The bacterium and the yeast were also employed for processing the non saccharified substrate to develop consolidated saccharification and fermentation processes. The bacterium grew well in the substrate hydrolyzate and a cell count of 2965 x 107/ml was recorded at first sampling which increased upto 19701x107 at 96 hr post fermentation. The yeast grew upto 513 x 106 cells/ml at 24 hrs which increased upto 852x106 cell/ml at the last sampling period. In case of co-culture the bacterium showed initially tremendous growth upto 10186 x 107 cell/ml at 24 hr which was 243.54% higher than the corresponding value of the cell count when its was monocultured. Thereafter the bacterial counts reduced but stablized over 8000 x 107 cell/ml at remaining sampling times. So that the co-cultured cell counts appeared 45.32% higher but 85% and 56.90% lesser than the corresponding monocultured bacterial counts at 48, 72 and 96 hrs, respectively. Whereas the co-cultured yeast grew moderately with cell counts of 166 x 106, 194 x 106, 394 x 106 and 506 x 106 at 24, 48, 72 and 96 hrs, respectively. These yeast cell counts were 67.64%, 60%, 23.35% and 40.61% lesser, respectively than their corresponding values when the yeast was mono-cultured in the hydrolyzate. In case of SSF, wherein the non-saccharified substrate was provided, the bacterial cell counts remained several folds less than the corresponding values for mono as well as co-cultures raised in the saccharified substrate. The yeast cell counts in case of SSF also remained less than all the values of its monoculture (SHF). Whereas the SSF yeast cell counts of the 72 and 96 hrs stages were 64.21% and 50.99% lesser, respectively than their corresponding values in the SHF co-culture. The batch fermentations of the saccharified substrate revealed that the amounts of HMF; one of the major inhibitors molecules generated during the lignocellulosic breakdown, in general, went down at end of the fermentation period. For the bacterially fermented saccharified substrate the HMF content reduced from 489 ±38.1 µg/L at 24 hrs sampling point to 232µg/L (52.56%) at the last (96 hr) sampling period. In case of co-culture this reduction in the HMF content decreased to 21.77%. However, in case of the yeast fermented poplar hydrolyzate the HMF content dropped down to 351.3 ±48.6 µg/L right at first sampling point and thereafter the inhibitor compound become un-detectable. In case of SSF, the HMF could appear only at 72 and 96 hrs of fermentation with respective values of 260.3 ±25.8 and 243 ±8.66 µg/L. Acetic acid content amongst the differently fermented substrates ranged from 460 ±230 to 5360 ± 503 mg/L. The highest acetic acid contents were encountered in case of SSF. Glucose and xylose monomers of 2% substrate saccharified by the bacterial cellulases measured upto 6.742 and 8.561 mg/ml, respectively. The bacterial inoculation caused 51.63% and 77.88% reductions in the glucose and xylose contents, respectively of the hydrolyzate at 24 hrs sampling point. Besides the bacterial cell mass formation, ethanol production at this level was 80.52 ± 24.2 mg/L. In case of yeast monoculture the glucose and xylose contents reduced down to 34.17% and 85.28%, respectively at 24 hrs post-inoculation with con-comittant ethanol production of 634 ±159 mg/L. Following 24 hrs of co-culturing of the microbes in the substrate hydrolyzate the glucose and xylose reduced down to 39.69% and 82%, respectively with accompanying ethanol fermentation level of 501.38 ±46.7 mg/L. Glucose content of 24 hrs incubated SSF fluids were 1568 ±226 mg/L, whereas the xylose remained non-detectable throughout the study period. Ethanol productions at 24, 48, 72 and 96 hrs of incubations for the SSF experiment were 140.43 ±44.8, 60.18 ±13.5, 177.78 ±23.9 and 83.48 ±10.3 mg/L, respectively. Excepting the SSF experiment the maximum ethanol productions were observed at first sampling period. In the present experiments no pre-treatment, except the autoclaving was applied. Whereas no attempt was made for detoxification of the inhibitors molecules. The bacterium as well as the yeast grew, in general, well in the media comprising of the substrate. Although the ethanol yields remained in general low. But owing to the less chemical and technological inputs these models can be upgraded by incorporating low-cost nutrient supplement, applying strict anaerobic conditions following initial optimum microbial growth to restrict further biomass formation and oxidative metabolism and introducing gas stripping procedure to the batch fermentor. It is likely that application of above mentioned strategies and other suitable processes will enhance the ethanol yield from lignocellulosic feedstocks in an environmentally sustainable way. Conclusively, the simple experiments reported here provide a workable model to assess the potential of suitable microbes for bioethanol production from plants’ biowastes by a simple consolidated bioprocess with incentive of animal feed without need of drastic pretreatment(s) and chemical detoxifications. However, upscaling of the process will require application of microorganisms exhibiting tolerance to high temperature and high resistance to ethanol and inhibitory substances.