Abstract

The aim of this present study is to select high performance microbial strains for organic municipal solid waste biological pretreatment. Waste samples were collected at three municipal waste pre-collection centers in the city of Ouagadougou. Standard isolation and characterization methods have been used for strains selection in different biotopes. Waste biodegradation tests were carried out in bottle (300 mL) with 120 mL of useful volume composed of the buffer (K₂HPO₄ and NH₄Cl) and 2% of waste. Optimization tests of waste pretreatment were carried out in function of temperature and inoculum proportion (10 % and 25 %). The evolution of pH and total solid loss was monitored during fermentation. Sixteen (16) microbial strains were isolated from different matrices, including three (03) cellulolytic bacteria (CA1, CA2, CA3), three (03) Streptomyces sp (SS1, SS2, SS3), four (04) Bacillus sp (BS1, BS2, BDP, BAF), three (03) Yeasts (YBB, YDP, YEU) and three (03) molds (MS, MBB, MDP). The pH drops from 7 to 5.4 and persists until the 6^th day was followed by a gradual increase in pH to 9. Temperature rise at 37 °C allowed a sudden acidification from the 3^rd day (pH 5.6 at 30°C and pH 4.72 at 37°C) and increases to pH 10. The CA3-SS3-BDP-YBB consortium has been identified as the best combination for a pre-fermentation of municipal waste. The TS reduction on day 25 ranged from 9.9 g/L or 49.5% of TS removal for TNS, 6.7 g/L or 33.5 % for MDP, 9.3 g/L or 46.5% for SS3, 6.3 g/L or 31.5% for YDP, 8.7 g/L or 43.5% for CA3, 7 g/L or 35% for MBB and 4.8 g/L or 24% for YBB. The optimization allowed a reduction of the pretreatment time to 4 days, obtaining a biomass adapted to anaerobic digestion.

1. Introduction

The world population will know a surprising increase from 6.7 to 9.2 billion ¹. At the same time, urban population is projected to increase from 3.3 billion in 2007 to 6.4 billion in 2050. This strong growth of urban population will be mainly due to the strong urbanization of the African and Asian countries. The corollary of this is an increasing production of municipal solid waste. According to Amoo and Fagbenle ², from 2011, the world generates 2 billion tons of municipal solid waste (MSW) annually. These forms of municipal solid waste management are inadequate, the direct result being environmental pollution and degradation of population's living environment (microbial contamination, atmospheric air pollution by harmful odors, etc.). The organic fraction represents more than two-thirds ^{3, 4} and could be treated under controlled conditions to reduce environmental impact and recover energy ^{5, 6, 7}. Among the systems for managing the organic fraction, treatment with aerobic digestion or anaerobic digestion is gaining renewed interest. It could be seen as an economic, decentralized and ecological solution to these problems through energy autonomy and sustainable agricultural development in rural areas ⁸. Methanization becomes very delicate, in particular because of the complexity of the waste produced. According to Eleazer et al. ⁹, Lacour ¹⁰ and Bayard et al. ¹¹, the fermentable fraction of municipal waste consists mainly of leaves, branches, office paper, newspapers and food residues. It is made up of chemical compounds of different masses, sizes and chemical properties: hydrogen-carbon compounds including saccharides (soluble non-cellulosic compounds), cellulose, hemicellulose and lignin, lipid, protein and humic compounds. Pretreatment operations are then necessary to increase the anaerobic digestibility of the substrates and to improve process performance ^{10, 12}. Indeed, mechanical, thermal, chemical and biological pretreatment forms have been developed by various authors ^{12, 13, 14}. These preparations upstream aim to accelerate the kinetics of methane production on the one hand, and to increase the rate of conversion of waste to methane on the other hand. Indeed, forms of pretreatment reported by Yadvika et al. ¹⁵ are more or less likely to be applied in developing countries. This is the case of aerobic pre-fermentation, thermochemical treatment, physico-chemical treatment. Thermo-chemical or physicochemical treatments of organic solid wastes are extremely expensive and can result in compounds such as hydroxfurfural and furfural which are highly toxic to microorganisms ^{12, 16}. The use of microbial strains is one of the effective pretreatment techniques ^{16, 17, 18}. Many microorganisms, mainly bacteria, actinomycetes and fungi have the capacity to degrade organic matter ^{19, 20, 21, 22, 23}. Organic matter degradation is an extracellular process in which complex organic substances are decomposed into simple and soluble compounds. Several enzymes are involved in this process, hydrolytic enzymes which include cellulase, cellobiase, xylanase and amylase for degradation of polysaccharides to sugars, protease for protein degradation into amino acids, and lipase for degradation of lipids to glycerol and long chain fatty acids ^{24, 25, 26, 28}. In this study, the ability of different microbial strains to degrade municipal organic waste was investigated to realize efficient microbial complex for biological pre-treatment the first time. Also, pretreated product was characterized.

2. Materials and Methods

Sampling of municipal solid waste was carried out in three (3) pre-collects centers of Ouagadougou’s town. For each tank, three (3) trash repetitions were performed. The first center is located in the district 2 (North latitude: 12° 22'; West longitude: 1° 32' and altitude: 335 m), the 2nd in the district 12 (North latitude: 12° 19', West longitude: 1° 31' and altitude: 349 m), and the third center in the district 3 (North latitude: 12° 23'; west longitude: 1° 32' and altitude: 326 m). Waste samples were mixed, sorted and dried under the sun for 7 days, then crushed and sieved (size ≤ 1 mm). Different matrices were used for the isolation of microbial strains. These include bovine dung, waste water collected at the national slaughterhouse in Ouagadougou, Burkina Faso (12°25'5.87"N, 1°28'29.23"O). Soil samples were collected from different locations in cultivated fields.

The pH, salinity, dissolved sediment rate (TDS), electrical conductivity (CE) and resistivity (R) were measured by a multi-parameter analyzer of the 9420 WTW multi-parameter type. The dry matter (DM) and were determined according to the method described by Charnay ²⁹. Five grams (5 g) of sample were placed in the oven at 105 ± 2°C to a constant weight, about 24 hours. Total ash (Ct) was determined using TAPPI 211Om-02 ³⁰. One gram (1 g) of waste powder was introduced into a crucible and placed in an oven at 525°C for 3 hours. The organic matter content (MO) in the sample was obtained by the difference between dry matter and total ash. The method of Nicholson et al ³⁰ was used to determine the lignin content (Klason lignin). The lignin content was achieved with 0.1 g of OFMSW in 1.5 ml of 72 % H₂SO₄ in a 50 ml polypropylene centrifuge tube and allowed to act for 2 h at 20°C. The mixture was diluted with 56.2 ml of distilled water and filtered under vacuum using the crucible previously weighed, before washing with distilled water and drying in a ventilated oven. Organic acids content were determined using the method described by Dilallo and Abertson ³¹. An amount of 25 mL of the sample was titrated with H₂SO₄ (0.1 M) at pH 4.0. The sample was then boiled lightly for 3 minutes, cooled, titrated by assay with Na₂CO₃ (0.05 M), and the titration amount was noted from pH 4.0 to 7.0.

The cellulolytic bacteria were isolated on a modified Nfb (N-free broth) base medium containing an antifungal, cycloheximide. Cellulose refined at 5 % (m/w) was carbon source according to method described by Chastrusse ³². Casein agar starch medium (CAA) was used for the isolation of Streptomyces ³³. Trypticase Soja Agar (BioMérieux SA, France) was used for isolation of Bacillus. The chloramphenicol sabouraud agar (Biokar, France) was used for the isolation of yeasts and molds (ISO Standard, 2007).

Morphological characteristics such as colony morphology and cell morphology (shape, Gram reaction and arrangement) were studies for identification ³⁶. Also, bacterial strain was subjected to different biochemical tests including Gram ‘staining, sporulation, catalase, oxidase, starch hydrolysis, citrate utilization, manitol mobility and nitrate reduction test ^{34, 35, 36, 37}. The use of sugars by strains was performed. Medium composition was: KH₂PO₄ 2.38 g/L, (NH₄)₂SO₄ 2.64 g/L, MgSO₄ 1 g/L, K₂HPO₄ 5.65 g/L, 1 ml of saline solution (CuSO₄ (5H₂O) 6.4 g/L, FeSO₄ (7H₂O) 1.1 g/L, MnCl₂ (4H₂O) 7.9 g/L, ZnSO₄ (7H₂O) 1.5 g/L), Agar 15 g/L pH: 7.0. The carbohydrates tested were added: glucose, mannose, maltose, arabinose, saccharose and lactose ³⁶.

The demonstration of cellulolytic activity of bacteria was carried out by the technique described by Chastrusse ³². The bacterial colonies were subcultured on a 3% TSA rich medium (3 g of Trypcase Soja Broth and 15 g of Agar per 1 L of deionized water) and incubated for 5 days at 30°C. The strains were again transferred onto the refined cellulose Nfb medium, and placed at 30 °C for two weeks. Cellulolytic activity of strains were visualized with a solution of Red Congo 2.5 % (m/w) for 20 min. The excess of unbound dye was removed and replaced with a 5M NaCl solution which remained in contact with the medium for 30 min. Finally, the NaCl was removed and the dishes were rinsed gently with water. The lysis ranges then appear discolored, that is to say yellow-orange with respect to the rest of the medium which is red.

The protocol described by Dutta et al. ³⁷ was modified to test the biodegradation of organic waste with isolated strains. The waste medium was prepared by mixing K₂HPO₄ (2g) and NH₄Cl (2g) in 1000 ml of distilled water. The media were prepared in 300 mL flasks with 120 mL of useful volume. In each flask, 2.4 g of waste powder was introduced for a 2% charge. After 24 hours of culture, the strains were transplanted into the 250 mL Erlenmeyer flakes containing nutrient broth. The cultures were incubated for 24 hours with stirring at 65 rpm at 30°C. The waste after sterilization at 121°C for 15 min was inoculated with cultures (10%) (v/v).

A combination of the efficient strains was carried out; and strains were coded CA3, SS3, BDP and YB. The protocols described by Dutta et al. ³⁷ and Poszytek et al. ³⁸ were adapted for the optimization of the activity of these strains. The combination was cultured in broth (yeast extract: 2.5 g/L, peptone: 5 g/L, glucose: 1 g/L, NaCl: 5 g/L) with stirring at 75 rpm for 72 hours. The culture was supplemented in 24 hours with 25 mL of cellulose 5% (v/w). The degradation tests were carried out at different temperatures (30 and 37°C).

XLSTAT software was used for statistical analysis of data. A one-way ANOVA and Fisher’s test LSD was performed to compare the mean values of the different variables. P value of less than 0.05 was considered to indicate statistical significance.

3. Results and Discussion

The organic fraction of the municipal waste sampled was mainly composed of green waste (grass, dead leaves) and food waste (kitchen waste, condiment residues from markets). The major components of this fraction are shown in Table 1. High organic matter contents (85.14%) indicate a substrate of choice for microorganisms. The high lignin composition (29.21%) could be explained by the richness of the organic fraction of municipal waste in lignocellulosic biomass such as tree leaves, branches and grasses ⁹. Indeed, the secondary cell walls of the wood and herb tissues are composed mainly of cellulose, hemicelluloses and lignin ³⁹. Lignin is extremely resistant to biodegradation ^{40, 41}. The C/N ratio of the sample in the order of 31.65 is favorable for biodegradation because it is very close to the optimum between 25 and 30 ^{42, 43}. The maximum tolerated value is 35 for urban waste. Beyond this value, several cycles of oxidation are necessary to reach the optimum value ^{29, 44}.

Table 2 shows the various isolated microorganisms and the matrices used. Five different microorganisms were isolated. Three types of cellulolytic bacteria and Streptomyces sp, two types of Bacillus sp were isolated. Cellulolytic bacteria and Streptomyces sp were isolated only from soil. Two strains of Bacillus were isolated from all matrices excepted from the cow dung. The maximum of microorganism was isolated from soil and putrefaction ‘waste. Three strain of yeast were isolated from waste putrefaction, Bovine dung and wastewater. And three strains of mold were isolated from soil, waste putrefaction and cow dung.

Based on the pH-monitoring results for 20 days showed on Figure 1, initial pH of various assays is around 7, which is due to phosphate buffer used as reaction medium. The pH drops from 7 to 5.4 and persists until the 6^th day. The pH gradually increases from the 6^th day to 7-7.5 on the 15^th day, which corresponds to the maturity of the tests. The decrease of pH is due to the fermentative activity of microorganisms ^{45, 46}. Indeed, the decrease of pH could be due to the degradation of certain molecules, such as simple carbohydrates, starch, hemicelluloses, pectins and amino acids contained in waste releasing organic acids ^{28, 45, 46}. Cellulose, a more bulky polymer, is more resistant. Lignin and other highly aromatic aromatic polymers will be degraded later, slower and incompletely. According to Aissam ⁴⁷, pH decrease could be attributed to release of H⁺ ions by cells in reaction medium. This release occurs at the time of transporting antiport organic compounds from the culture medium to cells. pH increase from the 6^th day could be explained by consumption of organic acids produced and hydrolytic action of bacteria on organic nitrogen forms such as proteins, urea ^{48, 49, 50} but also as a result of reduced microorganisms activity ^{47, 51}. Equally, reaction of denitrification during which the consumption of protons necessary to reduce nitrates or nitrites in molecular nitrogen and / or gaseous nitrogen oxide would also explains pH increase according to Berthe ⁵². The results obtained show that all strains present capacity to degrade organic fraction of municipal solid waste. The strains CA3, SS3, BDP, YBB and MBB showed a high production of acids, that reflects their performance in degrading organic matter. The production of acids with TNS control was more increased; this could be explained by diversity of microorganisms in the waste. Flora action could be complementary and allow a better degradation of organic matter. Indeed, in waste there is a great diversity of microorganisms, mainly bacteria, Actinomyces and fungi, which are responsible for mineralization of organic matter ^{47, 57}.

Figure 2 shows evolution of total solid (TS) during waste fermentation. Total solid control reveals amount of material converted to volatile matter following the biotransformation process ^{46, 54}. The results obtained show capacity of strains to degrade organic matter contained in waste. The Figure 3 and Table 3 show respectively effect of different strain on TS % removal and reduction of TS after 25 days. The TS reduction on day 25 ranged from 9.9 g/L or 49.5 % of TS removal for TNS, 6.7 g/L or 33.5 % for MDP, 9.3 g/L or 46.5% for SS3, 6.3 g/L or 31.5% for YDP, 8.7 g/L or 43.5% for CA3, 7 g/L or 35 % for MBB and 4.8 g/L 24 % for YBB. The biodegradation experiments carried out in this study have assessed the efficiency of five different strain based exclusively on the percentage of TS removal and organic acids production. Strains CA3, SS3, BDP and YBB were retained. The TNS control showed TS reduction 9.9 g/L, which could be explained by a very wide diversity of pre-existing microorganisms in waste (Bacteria, Actinomyces and fungi) which consumes oxygen gas to mineralize organic matter in CO₂ and H₂O ^{55, 56}. Few microorganisms are capable of completely mineralizing organic matter, organic by-products may be consumed directly by other microorganisms or they may subsist for a longer or shorter time in waste ⁵⁷. Bacteria and fungi had a broad spectrum of activity over a wide pH range, especially in high humidity ^{27, 51}. They are responsible for start of degradation of organic matter. They degrade easily oxidizable products such as proteins, carbohydrates (sugars) and lipids. Streptomyces belong to the group of Actinomyces and intervene according to Crawford and Pometto ⁵⁸ very late in degradation of organic matter. They attack organic substances not degraded by bacteria and fungi (chitin or tannins). Neutrophilic, they tolerate slightly basic pH and are not very competitive with other groups.

The characteristics of different strains are given in Table 4. CA3 was stick-shaped, filamentous, Gram positive, oxidase positive and produced spore. This strain utilized various sugars including glucose, maltose, lactose, arabinose and saccharose. It showed negative growth on acetate, starch, urea and nitrates. CA3 showed cellulolytic activity visualized using Congo red, which is in accord with Chastrusse ³⁴. The SS3 strain was of filamentous shell shape, Gram positive, oxidase positive and produced spore. It was able to use glucose, maltose, lactose, arabinose and saccharose. SS3 utilized starch, urea and nitrates. SS3 registered negative growth acetate. The strain BDP was found to be Gram positive, oxidase positive and produced spore. It was able to use glucose, maltose, arabinose, lactose, saccharose except mannose. BDP was capable to use other substrates such as nitrates and acetate except starch. YBB yeast strain used glucose, maltose, lactose, arabinose, mannose, manitol and saccharose. It utilized nitrates and acetate except starch. The MDP strain gave white colonies, the texture was cottony. MDP had very rapid and extensive growth. Microscopic observation showed an aspergillum head. CA3 and BDP could belong to Bacillus genus, SS3 to Streptomyces genus, YBB could belong to Candida genus and MDP strain could belong to Aspergillus genus.

pH evolution during fermentation with consortium CA3-SS3-BDP-YBB proportions 10 % and 25 % is shown in Figure 4. pH evolution with 10 % and 25 % of inoculum did not show a significant difference at 30°C (p > 0.05). The pH drop from 7 to 5.75 until the 6^th day was followed by its gradual increase about 9. Temperature increase at 37°C resulted a high acidification after day 3 (pH 5.6 for 30°C and 4.08 for 37°C). Analysis of pH variance during pretreatment as function of inoculums proportion showed significant differences at 10 % and 25% inoculum at 37°C (p < 0.05). This difference is not perceptible at 30°C. This indicates that temperature has effect on organic matter biodegradation. Total solid reduction was 5.25 g/L (26.25 % of organic matter reduction) with 10 % of inoculums (Figure 5a), 6.5 g/L with 25 % at 30 °C (32.5 %). At 37 °C, TS reduction was 5.9 g/L (29 %) for 10 % inoculums and 8.8 g/L for 25 % (44 %). Evolution of organic acids in function of inoculums proportion (10% and 25%) is given in Figure 6. Inoculums quantity has significant effect on organic acids production the 4th days (P < 0.05). Initially organic acids concentrations increased rapidly to maximum value of 12400 acetic acid/L for 10% and 21500 mg acetic acid/L for 25%. According to Ugwuanyi et al. ⁶³ accumulation of organic acids in aerobic system is considered evidence of fermentative metabolism. Similar variations in total organic acids production patterns were observed by Parawira et al. ⁵⁹ and Suchowska-Kisielewicz et al. ⁶⁵ when they studied the production of organic acids from vegetable, tea, solid potato and municipal solid wastes. Mshandete et al. ⁶⁰ was found initially the concentrations increased rapidly to maximum value of 12 g/l during 84 h for untreated waste during anaerobic digestion, in case the high organic acids production affects the performance of the subsequent methane bioreactor. Pretreatment have effect on hydrolases and VFAs profiles during acidogenic phase of anaerobic digestion and is suitable both during start-up and subsequent process stability ⁶⁰.

After 25 days of fermentation of the organic solid waste, the pH profile and the loss in total solid made it possible to determine an optimal time of biodegradation which was four (04) days at 37 °C. The pH was 6.55 and 4.08 respectively for 10 % and 25 % of inoculums. The total solid reduction on day 4 was 1.825 g/L for 10 % of inoculums and 1.725 g/L for 25 % of inoculums.

The total solid (TS) of the product after fermentation was 1.82 % to 10 % inoculums and 1.72 % to 25 % of inoculums. Better biogas production has been found with total solid ratios of between 2 and 4 % on bovine dung, food waste and jatropha oil residues (68). The mean values of some physicochemical parameters of the final product in terms of salinity, total dissolved sediment (TDS), electrical conductivity (EC) and resistivity were respectively 3.91 g/L, 7.03 g/L, 6, 45 mS/cm, 105.5 Ω.cm to 10 % of inoculums and 3.55 g/L, 6.42 g/L, 6.45 mS/cm 116.2 Ω.cm to 25 %). Indeed, the authors have expressed influence of these parameters on anaerobic digestion ^{62, 63, 64, 65}. The results obtained was in agreement with those obtained by Manonmani et al. ⁶¹, which found 3.88 - 11.9 mS/cm of conductivity, 2.5 - 8.92 g/L of salinity, 2.89 - 6.98 of TDS and 6.3 -8.1 of pH. Also Zeng et al. ⁶⁶ found similar TDS values of 6.80 g/L but a high conductivity of 13.61 mS/cm in the animals dung, the preferred substrate for anaerobic digestion. In a study about industrial wastewater anaerobic digestion, Graterol ⁶⁷ found similar pH, conductivity and TDS values between 6.05 - 6.97, 2.58 - 4.28 and 1613 - 2675 mg/L at the stability point of the process.

4. Conclusion

This work allowed realizing a microbial consortium presenting a very wide spectrum of organic waste degradation that will be used in biological pre-treatment upstream of anaerobic digestion. This is complex consisting in a combination of cellulolytic bacteria (CA3) of Bacillus genus, Streptomyces sp (SS3), Bacillus sp (BDP) and Candida sp (YBB). MBB molds of the genus Aspergillus also exhibit a broad spectrum of biodegradation. The optimization of biodegradation by varying the temperature and the proportion of inoculums allowed a reduction of the pre-fermentation time at four (04) days and a partially digested stable product. The finished product is a preferred substrate for launch of anaerobic digestion.

Acknowledgements

We thank the West African Research Association (WARA) and UEMOA through the CRSBAN / University Ouaga I Prof. Joseph KI-ZERBO for their financial support.

Conflict of Interest Statement

We declare that we have no conflict of interest.

List of Abbreviations

MSW= Municipal Solid Waste

TS= Total Solid;

TSD= Total Solid Dissolved;

EC= Electrical Conductivity

R= Resistivity

VFAs = Volatile Fatty Acids

References