Use of the newly isolated bacteria E. coli (SAY) in bioethanol production from organic wastes

This work was carried out to find a new and cheap material as source for alternative energy. In the present study a newly recorded bacterial sub-strain (E. coli SAY) was used for the bio-fermentation of latus leaves. The results show that the bacteria were able to fermenting the sugar presented in the latus leaves and produce ethanol. It was found that the pretreatment increases the amount of sugar available for fermentation and the best results were found with HCL pretreatment. The resulted ethanol concentrations were increased with distillation as well as with dehydration process.


Introduction
During the human live history, energy play a crucial role in human development.Especially through the last few decades, humans are building their future depending on the various energy sources in different life aspects such as agriculture, industrial and economic [1].The increasing consumption of fossil fuel such as coal, oil will lead to an increasing amount of greenhouse gases (GHG) such as methane and CO2 which are main causes of global warming as well as climate change [2].At the present time the whole world, is on search for clean, renewable, environmental friendly, reliable and economically feasible energy resources [3], and one of these alternative sources is biofuels which is sustainable, accessible and dependable fuels that are gained from sustainable sources [4], it gives several advantages including an increased number of rural manufacturing jobs, reduced GHG emissions, and reduce the dependence on fossil fuels as well as reduce fluctuations in oil price [5], and from theses biofuels, bioethanol is considered as an attractive biofuel that can be defined as a renewable and alternative energy source that produced from the fermentation of biomass [6].Moreover, it's possible to produce ethanol by fermentation from any organic materials that rich in carbohydrate [7].Since that there is a lack of researches that being applied on the use of endogenous isolated bacteria in the production of bioethanol through fermentation process., this study was carried out to investigate the possibility of producing bioethanol from organic waste (latus leaves) by using the newly recorded isolated bacteria (sub strain) E. coli (SAY) that have been recorded in Iraq for the first time with different pretreatment methods that improve bio-digestibility of cellulose for ethanol production.

Isolation and Identification of Bacterial species
The bacteria samples were isolated from organic waste (Latus leaves) collected from AL-Rasheed grand vegetables market in Baghdad/Iraq, a serial of dilution was prepared and 1 ml of each dilution was activated in nutrient broth for 24 hours and then cultured on EMB selective media.The purified colonies were sent for 16 RNA tests [8], at Wahaj AL-DNA Laboratory in Baghdad.The identified bacteria were recorded in Iraq for the first time as (E. coli SAY) in NCBI at the link below: https://www.ncbi.nlm.nih.gov/nuccore/OR186522.1 and then the identified bacteria were used in the following experiments.

Bacterial Optimization
The optimal growth condition for the newly recoded E. coli (SAY) was tested and the optimal condition were measured according to [9] shown in Table 1 Table 1. the optimal growth conditions for E. coli SAY Parameter Optimal condition Temperature 35°C pH 8 Time (life cycle) 7 days

Samples Collection and Processing
The vegetables samples (latus leaves) were collected from AL-Rasheed grand vegetables market in Baghdad/Iraq.The collected leaves were washed and sun dried then the dried leaves were grinded with an electric grinder to fine powder, kept at 4ºC in refrigerator in packed in polyethylene bags for consecutive experiments [10].The experiments were performed in two ways: 2.3.1.Without pretreatment: fifty gram of dried latus leaves were weighted and soaked in 1 liter of distil water and prepared for fermentation process.
After that it was filtrated and the resulted liquid was complete to 1 liter with distilled water.Then the pH value was adjusted to 8, and it was ready for fermentation.

2.3.2.B. Pretreatment with Hydrochloric acid (HCL):
fifty grams of dried latus leaves were pretreating with 500 ml of diluted HCL (5%) then kept in conical flask (1000 ml volume) that placed on hot plate stirrer for two hours at 130 °C and 500 rpm [12].
After that it was filtrated and the resulted liquid was complete to 1 liter with distilled water.Then the pH value was adjusted to 8, and it was ready for fermentation.

2.3.2.C. Pretreatment with Sodium hydroxide (NaOH): fifty grams of dried latus leaves
were pretreating with 500 ml of diluted NaOH (1%) then kept in conical flask (1000 ml volume) that placed on hot plate stirrer at room temperature for three hours.then it was filtrated and the resulted liquid was complete to 1 liter with distilled water distilled water.Then the pH value was adjusted to 8, and it was ready for fermentation [13].

Fermentation process
Each prepared sample (with pretreatment and without pretreatment) were put in glass container with two liters capacity and then autoclave in 121ºC for 15 min.The activated bacteria were added to sample for each experiment in sterilized circumstances in order to prevent any contamination.then left in incubator for fermentation for seven days with anaerobic condition [14].During that the ethanol concentration and sugar content were determined every two days.After 7 days, the resulted fermented solution was distilled through distillator.The ethanol was collected depending on the differences in the boiling points of water and ethanol [15].fermented raw materials (one liter) was kept in conical flask that set in a digital heating mantle at 80 ºC for 3 hours.The vapor was condensed and collected in a flask for each sample.To increase ethanol concentration, a second distillation was used in the present work.

Dehydration
The final step of process was the dehydration process in which 5 gm of calcium oxide (CaO) was used as a drying agent that added to the ethanol and mixed well for 10 minutes.The mixture was distillated again at 80°C [16] .

Measuring sugar content:
The sugar in the sample was measured by using phenol-sulfuric acid method, 10 ml of the sample was transfer to centrifuge, then 1 ml of supernatant was taken and mix well with 1 ml of phenol reagent (5%) and 5 ml of concentrated H2SO4, then the mixture was transfer to water bath with 25-30°C for 30 minute.Then the samples were measured by using spectrophotometer [17].

Ethanol measurement
Ethanol concentration of each sample was measured at different levels of the process: daily in the air above the aqueous sample by using ethanol sensor [18].After the first and second distillation of each sample by using HPLC techniques [19].After dehydration the ethanol concentrations was measured.

RESULTS
This study was carried out to find an alternative and cheap energy source.latus leaves were used as a raw material to produce bioethanol by fermentation process.Newly recorded sub strain E. coli (SAY) was isolated and classified from organic wastes and recorded for the first time in Iraq.At first the composition of the latus leaves was measured and the result show that the leaves is rich with the cellulose and hemicellulose as shown in Table 2.The latus leaves were characterized by their chemical structure that composed of cellulose, hemicellulose, lignin, proteins, crude fibers [20] .As shown in the table 2 the plant is rich with cellulose, hemicellulose and lignin which can offer a good contender for the production of bioethanol since that cellulose and hemi cellulose are polymers of glucose and can act as raw material for the production of bioethanol [21].The concentrations of sugar available for fermentation was highly affected by the different types of pretreatment as shown in table 3. The use of pretreatment was for defragmenting the cellulose and hemicellulose to a simpler sugar that can be used by the microorganism [22].On the other hand the process of acid pretreatment is very important for increasing the efficiency of the ethanol production through slow breakdown of the cellulosic component into simpler sugar [23] the result show that the highest sugar concentration was found when the raw materials was pretreated with HCL followed by H2SO4, NaOH and the lowest was without pretreatment, respectively, as shown in Table 3.The results of this study were much higher than the findings of Al-Rubaie [17] who found that the total sugar results from the olive oil were 2.05 and 1.5 mg/ml when pretreated with H2SO4 and HCl, respectively.The concentration of the sugar was declined with time as is it being consumed by the bacteria as shown in figure 1, and that can be related to the fact that the sugar being consumed by the bacteria to produce bioethanol through a process called saccharification [24].The results are similar to the findings of Salim T, et al. [25] who found that as the bacteria grow, they will consume sugar in the medium and that there was a reverse correlation between the bacterial growth and sugar content.

Figure 1. The sugar consumption through the fermentation process
The ethanol concentration was measured during the experiment period by using the ethanol sensor.The results show that the highest ethanol concentration was resulted from the pretreatment with HCL followed by H2SO4 with ethanol concentrations up to 45283 and 6228 ppm, respectively, while there was no much difference between NaOH and without pretreatment with ethanol concentrations up to 2323 and 2319 ppm, respectively as shown in Table 4.That indicate that HCl was more effective in de-crystalizing the cellulose to mono-sugars that is available for fermentation [26].The results of this study are similar to the findings of Yahaya U, et al. [24] who found that using diluted HCl can increase the release of sugar and subsequently increase the resulted ethanol.While Aznury M, et al. [27] found that the H2SO4 increase the ethanol production from the empty fruit bunch.After seven days of the experiment the resulted solution from fermentation process was distillated for two times in order to increase the purity of the ethanol, the resulted solution from the first and second distillation was measured by using the HPLC technique as shown in figure 2, the ethanol concentration after first and second distillation is shown in the Table 5.The results show that the second distillation increase the purity of the ethanol as shown in Table 5.The results of this study agree with the findings Salim et al. [28] how found that there was an increase in the purity of the ethanol resulted from fermentation process as it being distillated for the second time.The final step was the dehydration in which the second distillation for the samples was mixed together with CaO was used as a drying agent, which increase the ethanol concentration to 47.17 ppm, the results of this study agree with the findings of Al-Rubaie and Abdulhay [17] who found that the dehydration process increase the purity of the ethanol resulted from fermentation process.

Conclusions
The findings of this study indicated that the newly recorded bacteria have the ability to ferment sugar and produce ethanol and the lettuce leaves can be used as raw material for the production of ethanol, it also clear that using the pretreatment can increase the ethanol production.

Figure 2 .
Figure 2. ethanol concentrations measured by HPLC A) the 1 st distillation withoutpretreatment., B) the 2 nd distillation without pretreatment.,C) the 1 st distillation pretreated with NaOH., D) the 2 nd distillation pretreated with NaOH., E) the 1 st distillation pretreated with HCL., F) the 2 nd distillation pretreated with HCL., G)the 1 st distillation pretreated with H2SO4., H) the 2 nd distillation pretreated with H2SO4

Table 2 :
The chemical compositions of latus leaves.

Table 3 .
sugar concentrations resulted from different pretreatment of the raw material

Table 4 .
the ethanol concentration produced by E. coli SAY through seven days