Enrichment of Aerobic Sludge for Microbial Protein Production from Palm Oil Mill Effluent (POME)

Sathishkumar Nalatambi*, Li Wan Yoon1*, Yoke Kin Wan1, Adeline Seak May Chua2

1School of Engineering, Taylor’s University, Malaysia

2Department of Chemical Engineering, Faculty of Engineering, University of Malaya, Malaysia

*sathishkumar.nalatambi@sd.taylors.edu.my

*Corresponding author: LiWan.Yoon@taylors.edu.my

Abstract. Production of microbial protein (MP) as alternative protein maintains the balance for highly nutritious protein as food and feed source, where enrichment process is necessary to yield high protein content. This research work investigates the effect of different types and dosage of nutrients for microbial protein production through aerobically enriched sludge. Mixed culture of aerobic sludge enriched with Palm Oil Mill Effluent (POME) as carbon (C) source and ammonium chloride and potassium dihydrogen phosphate added as nitrogen (N) and phosphorus (P) source respectively. Enrichment process operated under aerobic condition and pH, temperature, dissolved oxygen (DO), string rate and sludge retention time (SRT) and hydraulic retention time (HRT) fix at 7,, 1 vvm, 450 rpm and 2 days respectively. The operation run with different nutrients dosage, C: N: P ratio: a) 92.3: 8.4: 1 (N-50% excess), b) 92.3: 11.2: 1 (N-100% excess) and c) 92.3: 5.6: 1 (stoichiometric ratio). The process operated continuously until stable mixed liquor volatile suspended solid (MLVSS) (APHA, 1995) and chemical oxygen demand (COD) (COD Test kit) observed and the protein content (Kjeldhal Method, AOCA 981) analysed for the first and last day of operation. All three nutrients ratio reduced the COD concentration with range of 90-92%. N-50% excess nutrient ratio produced highest protein content, 55% and followed by N-100% excess at 40%. However, both conditions have similar specific protein production rate, which is 0.0031 mg/day. N-100% excess has the highest specific substrate consumption, which is 0.08 mg/day and followed by N-50% excess and stoichiometric ratio at 0.04 mg/day and 0.03 mg/day respectively. With that being said, the finding in this report demonstrates a new effective strategy for MP production through enrichment of aerobic sludge with POME and additional nutrients sources. Apart from promotion to a more sustainable POME management, it proved to function as value added waste feedstock.

Keywords: microbial protein, enrichment, mixed culture, palm oil mill effluent (POME), activated sludge

1.Introduction

Over decades, the need for alternative technologies were studied and explored in order to fulfil the need for sustainable food and feed source. Extensive researches since 60s are explored to practice the production of high quality protein from microorganisms. In current modernisation era, alternative protein or commonly referred as microbial protein (MP) has been receiving tremendous attention that led to a promising option as replacement of natural protein such as animal and plant based protein. There are several chains of scenario of MP as favourable protein option where global population increments sit at the top of the list. United Nation (UN) recently produced a report stating that the global population expected to increase from 7 billion to 9.6 billion by 2050 whereby the current global population as per 2017 is 7.6 billion [1]. Henchio et al [2] reported that with 7.6 billion global populations, the protein demand is approximately 202 million tonnes globally and expected incline to 206 million tonnes per year. In conjugation with protein demand fulfilment with reliance on animal and plant-based protein, 12% of greenhouse gas (GHG) emission can be expected alongside with negative implication on biodiversity of ecosystem due to vast deforestation and conversion of wetland and natural grassland into agriculture land [3]. In addition, plant-based proteins require acres of land for agriculture and the production is limited due to seasonal based production. These chains of scenario have been driving factors for MP technology to a feasible and cost-effective technology and mushrooming scholarly researches have been done especially MP derivation from waste.

Innumerable studies have been reported for waste derived MP production, where Khan et al [4] and Steven et al [5] produced 58.62% of protein from waste banana skin with pure yeast strain of Saccharomyces Cerevisiae and 40% of crude protein from potato processing waste with pure culture of Cephalosporium Eichhorniae. Comparative to fruit waste, agriculture waste typically high in carbon, which is necessary food source for the microorganism where Asmamaw et al [6] utilized apple pomace to produce MP. Palm oil is one of Malaysia’s sustainable agriculture industries. Palm oil produced from fresh fruit bunch (FFB), whereby it undergoes a series of sterilization, threshing, digestion, pressing and oil purification process. Alongside palm oil production, high strength wastewater namely palm oil mill effluent (POME) produced. Typically, 2.5 tonnes of POME produced for every one-ton of palm oil production [7]. POME is a brownish acidic wastewater with extremely high chemical oxygen demand (COD) level, 22 500 – 74 000 ppm [8 and 9]. This is a clear indication of proper POME management is required. In general, POME undergoes several treatment processes including anaerobic and aerobic process with surface aerators prior to discharge. Countless studies have been done for treatment-oriented management of POME in accordance to discharge limit but most of the studies neglecting the facts of POME as an extremely reliable candidate for many waste derived products. Transformation of POME from waste to a value-added product has been continuously proven with polyhydroxyalkanoates (PHA) production from microorganism [10,11]. Nonetheless, very limited research has been done for MP production from POME.

In regard to direct MP production from POME, Onyekachi [12] reported one out seven-isolates yeast from POME yield 4.42 g/L of microbial protein. Most research methodology of MP production focused on pure culture. However, mixed culture has the tendency to yield even higher protein content and it is coupled with several advantages. Productions of MP through mixed culture will not only yield higher protein content but associated with cost reduction. Pure culture requires a specific operating condition and a single strain of microbe is far expensive than mixed culture and this concludes the reason of mixed culture being a cost-effective MP production methodology. Yadav et al [13] did a comparative study for MP production between pure and mixed culture of yeast and mixed culture yielded additional 8% higher protein content than pure culture and this finding tally with Yunus et al [14] and Dhanasekaran et al [15] findings whereby mixed culture has the tendency to yield higher protein content. Although, mixed culture of yeast has been widely explored for MP production, there are very limited studies have been done on mixed culture of bacteria. The most common source of bacterial mixed culture is activated sludge where Honda et al [16] reported 72.7% of MP production through mixed culture of bacteria. Despite higher protein yield through mixed culture condition, the result is much robust with enrichment process adaptation.

In the context of bio-operation, enrichment is imperative to consider once the process tailored to mixed culture condition. The reason is due to multiple species exists in the medium of mixed culture and there are possibilities of certain species does not has the ability to produce the desired product. Through enrichment, the species that able to produce the desired produced will continuously enriched and survive while the remaining will be eliminated from the system depending on the sludge retention time (SRT) and hydraulic retention time (HRT). In the available literature, enrichment process has repeatedly ameliorate the bioprocess efficiency and it is projected widely in PHA production where mixed culture aerobic sludge enriched with C: N: P ratio of 100:5:1. Chen et al [17], Mengmeng et al [18] and Valentino et al [19] enriched activated sludge with ammonium chloride as the nitrogen source and potassium dihyrogen phosphate as the phosphorus source and led greater yield of PHA. Recent novel discovery of Matassa et al [20] through production of MP with hydrogen-oxidizing bacteria showed that enrichment process resulted in 71% of protein content.

Although MP production has been receiving unveiling attention, there are several gaps that can be closed through this research. MP has been predominantly produced through either alternating anaerobic-aerobic or aerobic condition. In addition, most research on MP production was done on comparative basis of pure and mixed culture of yeasts. While each of these conditions has been extensively explored, enrichment of mixed culture sludge with nutrients dosage variation and POME as the sole carbon source is of particular interest in this research. POME lacks with nitrogen and phosphate source, which is necessary for protein production. As such, additional of nutrients (nitrogen (N) and phosphate (P)) source has the potential to be the decisive factor to yield higher protein content. In order to evaluate the feasibility of combined component of POME and activated mixed culture sludge for MP production, the activated sludge enriched with excess nutrients dosage under aerobic condition without pH variation. Henceforth, this study aims to investigate the effect of different types and dosage of nutrients on MP production, whereby the nutrients dosage set at a) 92.3: 5.6: 1 (stoichiometric ratio), b) 92.3: 8.4: 1 (N-50% excess) and c) 92.3: 11.2: 1 (N-100% excess).

2. Methodology

2.1 Materials

One batch (35 L) raw POME collected from local palm oil mill, Seri Ulu Langat Palm Mill. Activated sludge (5 L) collected from local sewage treatment plant, Indah Water Konsortium (IWK), Puchong. For nutrients solution preparation, ammonium chloride , potassium dihydrogen phosphate used as nitrogen (N) and phosphate (P) source respectively as well as N-allylthiourea . In addition, iron (III) chloride hexahydrate, ethylenediamine tetra acetic acid (EDTA), potassium iodide, boric acid, cobalt (II) chloride hexahydrate, manganese (II) chloride tetrahydrate, zinc sulphate heptahydrate, sodium molybdate dehydrate and copper (II) sulphate pentahydrate purchased from Merck for trace nutrients preparation.

2.2 Characterization of POME

Upon collection of POME, it was placed in chiller and preserved at for 24 h in order to prevent biodegradation of POME due to microorganism activities and promote conventional gravitational solid settling to reduce its solid content. Through settling phase, the supernatant separated from raw POME and remained preserved at for enrichment process. Samples from both raw POME and supernatant of settled POME characterized for pH, soluble chemical oxygen demand (sCOD), total chemical oxygen demand (TCOD), total suspended solids (TSS) and volatile suspended solids (VSS).

2.3 Enrichment of Aerobic Sludge with POME

2.3.1 Operation of Aerobic Sludge Enrichment

An aerobic operating sequencing batch reactor (SBR, working volume of 0.6 L) was used for enrichment of aerobic sludge for microbial protein production from POME at room temperature without pH control. The reactor was inoculated with re-suspended activated sludge with nutrients solution at 3000 mg/L and POME with 3000 mg (COD basis) /L. Air was supplied to the reactor at 1 vvm without any control and monitoring of dissolved oxygen (DO). The reactor operated at 24 h per cycle. Each cycle consisted of 2 phases: (a) 10-min of simultaneous feeding of 0.15 L of POME (carbon substrate) and 0.15 L of nutrient solution (N and P source) and purging of 0.3 L through peristaltic pump (Cole Parmer/ Masterflex/ USA), (b) 23.8-h of aerobic reaction phase. There was no settling phase involved and resulting in equal sludge retention time (SRT) and hydraulic retention time (HRT) of 2 days and the reactor continuously stirred at 450 rpm. The operation undergoes enrichment process through daily addition of nutrient solution at 0.3 L during the filling phase where the nutrients solution stock neutralized to pH 7. The enrichment process started with stoichiometric ratio with C: N: P ratio of 92.3: 5.6:1. Ammonium chloride and potassium dihydrogen phosphate used as nitrogen (N) and phosphate (P) source respectively. Magnesium sulphate was added at fixed concentration of 660 mg/L while N-Allylthiourea added at fixed concentration of 50 mg/L to prevent nitrification. Alongside macronutrients, trace element adapted from Ong et al [20] was added in to provide additional supplements for microbial growth. The trace element consisted of (concentration in mg/L): EDTA (14400), (2160), (260), (220), (220), (170), (170), (80), (50). The process was run continuously until stable COD and mixed liquor volatile suspended solids (MLVSS) were achieved where sample was withdrawn at intervals of 2 days. At the end of enrichment operation, the sample was also subjected to Kjeldahl nitrogen content to evaluate the protein content as well as ammoniacal-nitrogen content.

2.3.2 MP production at different nutrients dosage

In order to yield higher protein content under similar condition, the effect of different nutrients dosage was investigated. Comparative to other parameters, the manipulation of nutrients dosage has a greater impact of the production of microbial protein. Henceforth, under similar condition as section 2.3.1 without remaining parameters alterations the nutrients dosage (C: N: P) was varied at 2 high levels: (a) 92.3:8.4:1 (N-50% excess), (b) 92.3: 11.2: 1 (N-100% excess). The enrichment process was started with nutrient dosage of 92.3:8.4:1 (N-50% excess) and new batch of re-suspended activated sludge. Upon stable condition, the enrichment process was continued with nutrient dosage of 92.3: 11.2: 1 (N-100% excess). Both conditions were operated one after another continuously until stable COD and MLVSS were achieved where samples were withdrawn at intervals of 2 days. At the end of enrichment operation, the sample was also subjected to Kjeldahl nitrogen content to evaluate the protein content as well as ammoniacal-nitrogen content.

2.4 Analytical Methods

Mixed liquor suspended solid (MLSS) and mixed liquor volatile suspended solid (MLVSS) were determined based on American Public Health Association (APHA, 1992) standard method [21]. For MLSS analysis, samples were filtered and heated with oven (Jouan/ Strarts Scientific/ Malaysia) at for 1 h. On the other hand, MLVSS analysis was heated at with furnace (Thermconcept/ Fisher Scientific/ Malaysia) for 15 min. COD was determined with COD test kit (Merck/ Rance: 25-1000 mg/L) where 3ml of filtered samples mixed with the test kit and heated for 2 h with thermo reactor. Once that, the COD measured with photometer (Merck/ Spectroquant Prove 300/ Germany). For protein analysis, Kjeldhal nitrogen content was adapted, where sample mixed with sulphuric acid and heated to . Upon digestion, samples was cooled down to room temperature and diluted with ultrapure water and transferred to distillation unit in which, ammonium ion converted to ammonia through addition of alkaline solution. Once that, the captured ammonium ion undergoes titration and the amount of ammonia was evaluated. Ion chromatography (861 Advanced Compact IC, Metrohm, Switzerland) with Metrosep C4-150/4 column was used to determine ammonium concentration.

2.5 Performance Evaluation

The MP production through enrichment process was evaluated based on Equation (1):

On the other hand, specific substrate consumption rate , specific protein production rate , specific and percentage COD reduction were computed based on Equation (2)-(4) respectively. The cell concentration was taken as the difference in concentration between MLVSS and protein [22].

(2)

(3)

(4)

The protein and biomass yields on the substrate consumed were computed based on Equation (5)-(6).

(5)

(6)

3. Results and Discussion

3.1 Characterization of POME

The characteristics of raw and supernatant of settled POME are presented in Table 1. Based on Table 1, it can be said that POME is an acidic wastewater rich in organic matter that contains lots of suspended solids. The reduction of suspended solids was achieved with 24 h of gravitational solid settling. This result was reflected with TSS value of raw and supernatant of settled POME. It was found that 91% of TSS was removed with simple yet conventional settling technique and resulting in final TSS concentration of 1450 mg/L.

Table 1. Characterization of POME and supernatant of settled POME.

Parameters

Raw POME

Supernatant of Settled POME

pH

TSS (mg/L)

VSS (mg/L)

TCOD (mg/L)

sCOD (Eg/L)

sCOD/TCOD

Ammoniacal-Nitrogen (mg/L)

95.43

The resultant effect of TSS reduction was observed alongside with TCOD reduction, where it was reduced from 70950 mg/L to 40250 mg/L. Regardless of such reduction, the value is still high enough to consider as suitable food source for MP production as it remained rich with organic matter. In addition, most organic matter is soluble as indicated by supernatant of settled POME sCOD/TCOD ratio of 0.92. This ratio is almost double as it sCOD/TCOD ratio of POME that is 0.48. The high fraction of soluble organic matters in the supernatant of settled POME is highly privileged to microorganism as they could easily attain and consume the rich organic matters for MP production.

3.2 Feasibility of direct application of activated sludge and POME for MP production

In this study, activated sludge from local sewage treatment plant was cultivated for MP production with POME. Prior for MP production, activated sludge was subjected to initial protein content and POME for nitrogen content. The results showed that raw POME and supernatant of settled POME has ammoniacal nitrogen content of 109.31 mg/L and 95.43 mg/L respectively as presented in Table 1. This is an indication that sole reliance of POME, as nitrogen source for MP production is not feasible. In addition, activated sludge showed an initial protein content of 10.4%. Henceforth, additional nitrogen source under excess condition will help to boast the MP production with high protein yield.

3.3 Enrichment of activated sludge

In order to enrich activated sludge with POME and additional nutrient sources for MP production, an aerobic enrichment operating SBR was established. The process was operated at with SRT and HRT of equal 2 days as no settling phase involved. Besides that, air was supplied at 1 vvm without any DO control and no pH control was taken as well. POME and nutrients feed daily into the reactor at a combined volume of 0.3 L. The first enrichment process started with stoichiometric C: N: P ratio, which is 92.3: 5.6:1 and lasted for 23 days. This ratio is well within recommended ratio for active biomass growth [23].

Figure 1. Concentration profiles of COD and MLVSS based on stoichiometric nutrients ratio (92.3: 5.6: 1).

Fig 1 displays the concentration profiles of COD and MLVSS of the enrichment operation based on stoichiometric ratio. On the 0th day, the influent COD and MLVSS to the SBR was at 2095 mg (COD)/L and 2630 mg/L respectively. Drastic drop of COD at 615 mg/L was noticed on day 2 onwards based on the initial COD concentration and similar trend was noticed on MLVSS trend. From 2nd to 8th day onwards, the COD concentration profile remained at a considerably steady level within the range of 615 mg/L to 530 mg/L. The immense decline of COD within short period of time (2 day) explained with the facts that the bacteria consumed about 71% of the initial influent COD and continuously retained the COD consumption rate within the range until the 8th day. Similarly, the MLVSS reduced from 2630 mg/L to 1300 mg/L within 5 days. The tremendous reduction in MLVSS concentration is due short SRT of 2 days that would have washed out the entire slow grower from the system. Short SRT generally reduced the number of surviving species on the system. This implies the facts that on the first 5 days, the system was not dominated with MP producing species of bacteria. Similar findings were reported by Lee et al [24] under similar operating condition. However, 5th day onwards until 10th day a stable increment of MLVSS concentration was noticed and remained steady until day 15th. There was a notable COD increment from 8th to 10th day and the concentration progressively reduced to 203 mg/L. The continuous steady state consumption and reduction of COD concentration promoted the growth of microbes and new cells where it is reflected through the increment of MLVSS concentration. The increment of MLVSS concentration upon progressive drop of COD concentration from the 10th day onwards showed that MP producing organism had dominated the system. The enrichment operation was stopped upon stable COD concentration achieved on day 19th onwards. Toward end operation, total of 90% COD reduction was achieved with continuous progression of MLVSS concentration. Nevertheless, if the system was cultivated for even longer period of time, there are possibilities for the MLVSS to continuously increase and reach steady state alongside constant COD consumption rate.

3.4 Enrichment of activated sludge with excess nutrients dosage

A comparative study was done under similar condition, where nutrients dosage of nitrogen source was varied in excess of 50% and 100%. The enrichment process was started with nutrients dosage of 92.3: 8.4: 1 (N-50% excess) and upon stable condition, the process continued with nutrients dosage of 92.3: 11.2: 1 (N-100% excess).

Figure 2. Concentration profiles of COD and MLVSS at excess nutrients dosage: (b) N-50% excess, (c) N-100% excess.

Fig 2 (b) is the concentration profiles of COD and MLVSS at N-50% excess nutrients dosage and Fig 2 (c) is the concentration profiles of COD and MLVSS at N-100% excess nutrients dosage. In the available literature, limited nutrients dosage has the tendency to suppress microbial growth for MP production and favours towards PHA production. Thus, excess nutrients dosage expected to yield higher protein content. Comparative to enrichment based on stoichiometric nutrients dosage, the COD and MLVSS concentration profile trend of N-50% excess enrichment processes through excess nutrients dosage is more or less the same. The relationship between COD and MLVSS concentration profiles for both conditions are much apparent in contrast to stoichiometric nutrients dosage. Generally, with presence of biodegradable COD heterotrophic bacteria consume oxygen to oxidize the biodegradable COD in order to generate energy and build up new cells [25]. Production of new cells deduced based on the increased of MLVSS concentration.

On the 0th day, the influent COD and MLVSS to the SBR was at 2760 mg (COD)/L, 3325 mg/L and 1950 mg (COD)/L, 2151 mg/L respectively for N-50% excess and N-100% excess. For both conditions, the COD concentration dropped progressively until 3rd day, where 79% and 91% of COD was consumed respectively for N-50% and N-100% excess. For N-50% excess, the COD concentration slowly decreased from 975 mg (COD)/L to 218 mg (COD)/L. Alongside the decrement, the MLVSS concentration continuously fluctuate with COD concentration and remained stable on day 14th onwards. The operation was stopped on day 16th continues with N-100% excess condition. Disparity to N-50% excess condition, the COD and MLVSS profile is slightly different. The enriched sludge from N-50% excess condition was used for N-100% excess condition. Henceforth, this has reduced the acclimatization period for the system to reach fast steady state system. This is proven with constant COD consumption from 3rd day onwards and MLVSS concentrations augment linearly from 4th day onwards. Nevertheless, if the system was cultivated for even longer period of time, there are possibilities for the MLVSS to continuously increase and reach steady state alongside constant COD consumption rate. Towards the end, both condition reached total COD reduction of 92% and 90% respectively.

3.4 MP Production at different nutrients dosage

Towards the end of each enrichment process, samples were withdrawn and subjected to performance evaluation for MP production. Protein content, specific protein production rate, specific substrate consumption, biomass yield and protein yield were evaluated and tabulated in Table 2.

Table 2. Performance evaluation of MP production.

Nutrients Dosage

(mg COD/mg X/h)

(mg protein/ mg X/h)

Protein content (%)

92.3:8.4:1 (N-50% excess)

0.0477

0.0031

0.8595

0.0641

55

92.3: 11.2:1 (N-100% excess)

0.0818

0.0031

0.1876

0.0375

40

Based on Table 2, N-50% excess resulted a higher protein content compared to N-100% excess, which is 55% and 40% respectively. Comparatively, similar protein content of 54.5% was reported by Rajoka et al [26] through mixed culture of C.utilis and Brevibacterium lactofermentum. However, the specific protein production rate for condition is the same, which is 0.0031 mg protein/ mg X/h. The possibilities of such results are as explained in section 3.3, where N-50% excess condition was continuously run until stable COD was noticed alongside with stable MLVSS concentration. Stable MLVSS concentration is an indication of maximum protein content production. Hence, it can be concluded that N-50% excess has the ability to produce 55% of protein content. Comparative to N-50% excess, N-100% excess protein content is only 40%. However, the system only stabled with COD concentration but the MLVSS concentration still increases constantly. Thus, 40% protein content is only measured on the final day for N-100% excess and there are possibilities that the protein content to increase even higher than 50% if the system was enriched and cultivated for even longer period with constant MLVSS concentration observed as well. This protein content results tally alongside with protein yield where N-50% and N-100% excess is 0.0641 mg protein/ mg substrate and 0.0375 mg protein/ mg substrate.

In the available literature, it was proven that the initial COD concentration greatly affects the biomass yield [27]. High substrate COD concentration expected to lower the biomass yield. Although the initial COD set to 3000 mg/L, upon dilution the concentration dropped slightly. N-50% and N-100% excess nutrients dosage has initial COD concentration of 2760 mg/L and 1950 mg/L respectively. Based on observation, the biomass yield for N-50% excess is far greater than N-100% excess, where the yield is 0.8595 mg (VSS)/ mg (COD) and 0.1876 mg (VSS)/ mg (COD). At a stable MLVSS concentration, the biomass yield approximated at mg (VSS)/ mg (COD). Henceforth, the biomass yield for N-50% excess is acceptable. Comparatively, the biomass yield for N-100% excess is lower than theoretical value as the MLVSS concentration has not stabled. Similar findings were reported by Lim et al [27], where the biomass yield is higher for initial COD of 2310 mg/L than 1040 mg/L. Hitherto, the biomass yield calculation was done on assumption that the complete substrate consumption results on biomass growth. In reality, the substrate consumption by microbes is not only for biomass growth but also for non-growth related processes where portion of substrate consumed by microbe used for cellular maintenance [28]. Thus, the biomass yield based on biomass growth only is underestimated. Although lower yield obtained for N-100% excess, 90% COD reduction within a short period of time where in most biological wastewater treatment plant does not require high biomass yield.

Conclusion

This work demonstrated an effective strategy for enrichment of aerobic sludge with POME as carbon source and additional nitrogen source for higher MP production. Throughout the enrichment process, all three nutrients ratio reduced the COD concentration to 90%, 92% and 90% for stoichiometric, N-50% excess and N-100% excess nutrients ratio respectively. However, the MLVSS concentration noticed to continuously increased for stoichiometric and N-50% excess nutrient ratio despite constant COD consumption. In contrast, N-50% excess conditions reached constant MLVSS and COD concentration and resulted in 55% protein content while N-100% excess resulted in 40% protein content. It was noticed that both conditions has similar specific protein production rate, which is 0.0031 mg/day and protein yield for N-50% excess is twice compared to N-100% excess. In future work, prolonged cultivation should be considered until stable MLVSS and COD concentration achieved, where stable MLVSS concentration proves the system dominated by MP producing bacteria. Henceforth, the protein content for N-100% excess will be higher than N-50% excess.

References

[1]United Nation, “World Population Prospects 2017 Revision”, Department of Economic and Social Affairs, New York, (2017)

[2]M. Henchion, M. Hayes, A. Mullen, M. Fenelon and B. Tiwari, “Future Protein Supply and Demand: Strategies and Factors Influencing a Sustainable Equilibrium”, Foods, 6, no. 7, p. 53, (2017)

[3]H. van Zanten, H. Mollenhorst, C. Klootwijk, C. van Middelaar and I. de Boer, "Global food supply: land use efficiency of livestock systems", The International Journal of Life Cycle Assessment, 21, no. 5, pp. 747-758, (2015).

[4]M. Khan, S. Khan, Z. Ahmed and A. Tanveer, “Production of Single Cell Protein from Saccharomyces Cerevisiae by utilizing Fruit Waste”, 1,no. 2, pp. 95-178, (2010)

[5]C. Stevens and K. Gregory, “ Production of Microbial Biomass Protein from Potato Processing Wastes by Cephalosporium Eichhorniae”, Applied and Environmental Microbiology, 2, no.53, pp. 284-291, (1986).

[6]T. Asmamaw and A. Fassil, "Co-culture: A great promising method in single cell protein production”, Biotechnology and Molecular Biology Reviews, 9, no. 2, pp. 12-20, (2014)

[7]A. Latif Ahmad, S. Ismail and S. Bhatia, "Water recycling from palm oil mill effluent (POME) using membrane technology", Desalination, 157, no. 1-3, pp. 87-95, (2003)

[8]M. Chin, P. Poh, B. Tey, E. Chan and K. Chin, "Biogas from palm oil mill effluent (POME): Opportunities and challenges from Malaysia's perspective", Renewable and Sustainable Energy Reviews, 26, pp. 717-726, (2013)

[9] Y. Chin, S. Loh and C. Fong, “ Palm Oil Potential”, (2017)

[10]W. Lee, A. Chua, H. Yeoh and G. Ngoh, "Influence of temperature on the bioconversion of palm oil mill effluent into volatile fatty acids as precursor to the production of polyhydroxyalkanoates", Journal of Chemical Technology & Biotechnology, 89, no. 7, pp. 1038-1043, (2013)

[11]M. Md. Din, P. Mohanadoss, Z. Ujang, M. van Loosdrecht, S. Yunus, S. Chelliapan, V. Zambare and G. Olsson, "Development of Bio-PORec® system for polyhydroxyalkanoates (PHA) production and its storage in mixed cultures of palm oil mill effluent (POME)", Bioresource Technology, 124, pp. 208-216, (2012)

[12]I. Onyekachi, "Treatment and Valorization Palm Oil Mill Effluent (POME) through Production of Microbial Biomass", University of Nigeria, (2015)

[13]J. Yadav, S. Yan, C. Ajila, J. Bezawada, R. Tyagi and R. Surampalli, "Food-grade single-cell protein production, characterization and ultrafiltration recovery of residual fermented whey proteins from whey", Food and Bioproducts Processing, 9, pp.156-165, (2016)

[14]F. Yunus, M. Nadeem and F. Rashid, "Single-cell protein production through microbial conversion of lignocellulosic residue (wheat bran) for animal feed", Journal of the Institute of Brewing, 121, no. 4, pp. 553-557, (2015)

[15]D. Dhanasekaran, S. Lawanya, S. Saha, N. Thajuddin and A. Pannerselvam,” Production of Single Cell Protein from Pineapple Waste using Yeast”, Innovative Romanian Food Biotechnology, 8, pp. 26-32, (2011)

[16]R. Honda, K. Fukushi and K. Yamamoto, "Optimization of wastewater feeding for single-cell protein production in an anaerobic wastewater treatment process utilizing purple non-sulfur bacteria in mixed culture condition", Journal of Biotechnology, 125, no. 4, pp. 565-573, (2006)

[17]Z. Chen, L. Huang, Q. Wen, H. Zhang and Z. Guo, "Effects of sludge retention time, carbon and initial biomass concentrations on selection process: From activated sludge to polyhydroxyalkanoate accumulating cultures", Journal of Environmental Sciences, 52, pp. 76-84, (2017)

[18]C. Mengmeng, C. Hong, Z. Qingliang, S. Shirley and R. Jie, "Optimal production of polyhydroxyalkanoates (PHA) in activated sludge fed by volatile fatty acids (VFAs) generated from alkaline excess sludge fermentation", Bioresource Technology, 100, no. 3, pp. 1399-1405, (2009)

[19]F. Valentino, L. Karabegovic, M. Majone, F. Morgan-Sagastume and A. Werker, "Polyhydroxyalkanoate (PHA) storage within a mixed-culture biomass with simultaneous growth as a function of accumulation substrate nitrogen and phosphorus levels", Water Research, 77, pp. 49-63, (2015)

[20]Y. Ong, A. Chua, B. Lee, G. Ngoh and M. Hashim, "An Observation on Sludge Granulation in an Enhanced Biological Phosphorus Removal Process", Water Environment Research, 84, no. 1, pp. 3-8, (2012)

[21]APHA, Standard Methods for the Examination of Water and Wastewater, 18th edn. APHA, USA (1992).

[22]M. Albuquerque, V. Martino, E. Pollet, L. Avérous and M. Reis, "Mixed culture polyhydroxyalkanoate (PHA) production from volatile fatty acid (VFA)-rich streams: Effect of substrate composition and feeding regime on PHA productivity, composition and properties", Journal of Biotechnology, 151, no. 1, pp. 66-76, (2011)

[23]J. Willey, L. Sherwood, C. Woolverton and L. Prescott, Prescott's principles of microbiology. New York: McGraw-Hill, (2012)

[24]W. Lee, A. Chua, H. Yeoh, T. Nittami and G. Ngoh, "Strategy for the biotransformation of fermented palm oil mill effluent into biodegradable polyhydroxyalkanoates by activated sludge", Chemical Engineering Journal, 269, pp. 288-297, (2015)

[25]Y. Hung, L. Wang and N. Shammas, Handbook of environment and waste management. Singapore: World Scientific Pub. Co., (2012)

[26]M. Rajoka, S. Ahmed, A. Hashmi and M. Athar, "Production of microbial biomass protein from mixed substrates by sequential culture fermentation of Candida utilis and Brevibacterium lactofermentum", Annals of Microbiology, 62, no. 3, pp. 1173-1179, (2011)

[27]J. Lim and V. Vadivelu, "Treatment of agro based industrial wastewater in sequencing batch reactor: Performance evaluation and growth kinetics of aerobic biomass", Journal of Environmental Management, 146, pp. 217-225, (2014)

[28]G. Bitton, Wastewater microbiology. New Delhi: John Wiley & Sons, (2013)

13