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    vironmental Potion 265 (2020) 15202 aseaeoas Contents lists available at ScionceDirect ¥ ag ROLRSN ron] Environmental Pollution ELSEVIER Journal homepage: www.clsevier.com/locate/envpol Synergistic effects of compost, cow bile and bacterial culture on bioremediation of hydrocarbon-contaminated drill mud waste* Daniel Osei-Twumasi *:", Bernard Fei-Baffoe *, Alexander Kofi Anning °, Kwabena Owusu Danquah ” Deen of het an Ape ot yo ens Cal See, um ama Une Sem nt ea Kaas * Deporomen of Medic! Digests acl of Aled Heath Sciences, Kwame Nkrumah Unversity of cence and Technolo. umes Chara ARTICLE INFO ABSTRACT ‘re Rony Recie 26 March 2020 30 une 2020, Accepted 5 fly 2020 ‘esate eatin 4 Aug 2020 Bioremediation has gained global prominence as an effective method for treating hydrocarbow- contaminated dil mod waste (HCDW). However, the problem of low nutrient content, bioavailability land microbial presence remain largely unresolved. In this study, the synergistic effects of compost, cow bile and bacterial culture om the degradation rate of HCDW was investigated. A homogenized HCDW sample (80 kz) obtained from 25 dillerent dil md tanks Was divided into 20 portions (4k each) and cach adjusted to LA% nitrogen content +20 ml cow bile (xe, basic treatment) Pure cultures of Brev- lngeteriu casei (Be) and Boils zhangzhouensi (Be) and their mixture (BeB2) were subsequently added t9 a Tear the amengedHebw (bs) o undergo 6meekncsaton A potion a he amen HED ‘as tn caton (2 he) ma sed conta al pr ekral conductty and sre tension ses he EDM oo sete 883.234 hon an 355 ny espe Correspning sles a al pecleam teen Son tf tol moyen and tt pe cunt acesa were 6S hg 08S an 44 ctu. The treatments led vo. substan eduction nT (p= 003) whe the conto ado sigan elect (p> 003) TMH feduton ater the experimental peor inthe rte fas Bae (Ena) base + Br (005%) > bse Be (G0) > bk (0528) > conta (O06) Mule eresin analysis revealed significant effect of total plate count, pH, CN ratio and electrical conductivity (R* — 0.87, 3a) onthe deraaton of inthe HCOW. The ly Semonsates org mace eects of Sompot om ie anaes ctr on te emedaon o CDNE wc one appl obs he cihceny ate Brenan techie © 2020 sever lights sere 1. Introduction DMs, although attention has manly been focused on ‘The generation of hydrocarbon-contaminated drill mud waste (HCDW) associated with crude oi production remains a major environmental concern throughout the world (Perryman, 2014; Romanus et al, 2015; Ramirez et al, 2017; Adetitun et al, 2018), despite the numerous economic benefits ofthe sector. HCDW is 3 well-established source of igh total petroleum hydrocarbon, and is commonly generated from Drill Mud Holding Tanks (DMHT) that stores drilling fluids for oil well drilling purposes. Large volumes of| this waste type is generated after periodic cleaning activities of the * This paper hasbeen recommended fo acceptance by Klaus Kammer, mal res ane wunasis70yna com (D Ose Tuma) ups:oto101015),envpt. 2020115202 (0268-74910 2020 sever i Al gs reserved hhydrocarbon-contaminated drill cuttings (Fink and Waltham, 2011; Fadairo et al, 2012). More importantly, indiscriminate disposal of HCDW can Tead to loss of soil quality, biodiversity, and water quality, among other environmental disturbances, underscoring the need for efficient, cost-effective and more environmentally- ftiendly remediation (Okparanma and Ayotamuno, 2008; Ghadebo et al, 2010; Noomen et al, 2012; Sharif et al, 2017) ‘A common goal of remediation is to reduce the level of total petroleum hydrocarbons (TPHs) in the HCDW. However, there is no direct guidelines regarding the permissible limit of TPH in HCDW. Yet, some individual studies and institutions have established TPH limits for ready-to-be-discharged _hydrocarbon-contaminated waste in general (Osuji, 2002; Australian Department of Environment and Conservation, 2010), For instance, Osuji, 2002 and the Australian Department of Environment and Conservation 2 Ons wumas et a / ironenta Pellation 265 (2020) 15202 (2010) reported 30 gikg and 1.0 g/kg respectively as the maximum ‘TPH permissible limits before discharge. The Oslo and Paris Commission (2015) also indicated 1% by weight for residual oil TPH asthe maximum allowable limit for hydrocarbon- contaminated soil resources that can be discharged into the environment. ‘To meet these recommended limits, oil producers have long relied on conventional methods such as incineration, landfilling and stabilization for treatment. Nonetheless, these methods are now considered expensive and unaeceptable from environmental standpoint (Das and Chandran, 2011; Rayu et al, 2012). Conse- quently, bioremediation has gained global prominence as an alternative treatment method for HCDW. This biotechnology, which {depends solely on micro-organisms or plants to treat hydrocarbons, has three widely reported limitations, namely; low nutrient con- tent, bioavailability, and low microbial presence (Yang, et al. 2009: ‘Acuia et al, 2010; Agamuthu et al, 2013; Maletie etal, 2013; Hua ‘and Wang, 2014), Despite several attempts, information regarding effective management of these limitations have yet to be scienif- ically resolved, spurring more research and leading to reports of significant hydrocarbon degradation (ie, TPH reduction) following ‘nutrient addition (Bira tal, 2014; Chen etal, 2015; Dadrasnia and Ismail, 2015; Paladino et al, 2016). Several studies into the ability of both chemical and biological surfactants to enhance bioavailability (Boroloi and Konvwar, 2009: Biria et al, 2014; Betancur-Corredor et al. 2015), have led to recommendation for use of the latter due to their lower toxicity, biodegradability and hhigher activity at extreme conditions (Silva et al, 2014). The potential use of cow bile as biosurfactant for bioremediation has come to the fore in recent times, although scanty information exists on its effects. Cow bile is readily available from commercial slaughterhouses (Jayathilakan et al, 2012) and contains, among other constituents, lecithin, which is an estab- lished surface tension-reducing bio-agent (Lock et al, 2008: Shikawa and Watanabe, 2011) This property of lecithin is derived ‘mainly from its amphipathic nature and the constituent phospho- lipid compounds such as the phosphatidyicholine, and phosphati- dylethanolamine, phosphatidylinositol, which often facilitate dispersion of oil in the aqueous layer for effective bioremediation ete, (Lock etal, 2006; Shikawa and Watanabe, 2011; Kapadia and Yagnik, 2013), In addition to high nutrient content and bioavailability, effective bioremediation requires optimum microbial population. High hy- drocarbon content tends to reduce the microbial population of the HCDW, effectively limiting its degradation (Brooijmans eta, 2008). This has led to suggestion of the application of environmental bacterial isolates to complement the work of the indigenous mi- robes in ensuring effective degradation (Gojgi-Cyijovie et al 2012; Mbadinga et al, 2012; Sagarkar et al, 2014; Szule et al. 2014 Nwankwegu and Onwosi 2017; Varjan! and Upasani, 2017). ‘These extraneous microbes may be directly obtained from the substrate, wild-type isolates or genetically modified and equipped with catabolic plasmids containing the relevant degradation genes (Fodelianakis et al, 2015). Application of wild-type isolates or ‘genetically-modified microbes during bioremediation is consid- ered useful, although their low survivability is still a concer (; Nikolopoulou et al, 2013; ABED et al, 2014; Hassanshahian et al, 2014; Fenu et al, 2015). For this reason, continuous search for tolerant microbes, preferably from the contaminated substrate, becomes crucial fr effective degradation. Since the discovery of oil in commercial quantities about a decade ago, Ghana has witnessed an increased crude ol production {peaking at 33 million barrels in 2017; Ghana Energy Commission, 2018), along with several other economic gains such as job creation ‘and revenue generation, To deal with the huge volumes of HCDW resulting from the increased oll production, many large companies operating in the country have often adopted bioremediation as a treatment option, but have yet to overcome the limitations of low nutrient content and bioavailability as well as low microbial pres- ence. The present study was designed fo evaluate the synergistic effects of compost, cow bile and bacterial isolates obtained from the HCDW to help find the most effective, environmentally friendly, and less costly treatment option of hydrocarbon-contaminated drill ‘mud waste. It was hypothesized that application of compost, cow bile and bacterial culture would interact to accelerate the degra dation rate ofthe contaminated drill mud, 2. Materials and methods 21. Study Area ‘The study was conducted atthe Kwame Nkrumah University of| Science and Technology (KNUST), located approximately 243.1 km. northwest from Accra, the capital of Ghana (Fig 1). The area falls within the wet semi-equatorial climatic zone. Rainfall pattern consists of annual double maxima; the main rainy season often ‘cers between March and July (peaks in May/june) and the minor season from September to November. Maximum temperatures ‘occur between January and April (range between 25 °C and 35°C) ‘whereas minimum values are recorded between May and December (range between 18°C and 24°C). Maximum, minimum and mean temperatures typically average 345 °C, 170 °C and 32.8 °C respectively. 22. Source of raw materials ‘The HCDW was obtained from twenty-five (25) drill mud tanks (MUTT) of Zeal Environmental Technology at Nyanekrom in the ‘Western Region of Ghana. A total of 1000 kg HCDW stock sample, consisting of 40 kg from each MHT, was collected forthe study. Cow bile samples were collected from the Kumasi Abattoir in the ‘Ashanti Region of Ghana, The cow bile samples were taken from 200 cows each day for three days and composited. The compost used for the study was obtained from the Accra Compost and Recycling Plant (ACARP), All samples were properly bagged, labeled and immediately transported to the laboratory for inital analysis and application in the field experiment. 23. Initial characterization of the HCDW, cow bile and compost 23.1, Physicochemical analysis Prior tothe field experiment, the HCDW, cow bile and compost samples were analyzed for vatious physicochemical parameters {pH electrical conductivity (EC), moisture content, surface tension, nickel, cadmium, lead, mercury, copper, cobalt, total petroleum hydrocarbon total phospholipid content, etc) in the Soil Science Laboratory of the Department of Crop and Soil Sciences, KNUST, Kumasi Total phospholipid concentration was analyzed at the Lipid ‘Analytical Laboratory Inc. In Canada. Additionally, the HCDW samples were tested for 16 selected PAH compounds (see Supple ‘mentary Material). Microbial analysis of the HCDW, cow bile and compost were carried out at the Department of Pharmaceutics Microbiology Laboratory, KNUST. Soil physical parameters such as pH, conductivity and moisture content of the HCDW were deter- ‘mined by the method described by Black (1965) and FAO (2008). ‘The Krusstensiometer (model k6) was used to measure the surface tension between the oil and moisture media. Surface tension ‘measurement was undertaken by immersed platinum-iridium ring of the instrument into the sample and slowly withdrawn by lowering the sample. The dial for controlling the dynesfmeter was uc tare nvoenta Paton 265 (2020) 15202 3 ig. toaton map ofthe sty rea adjusted until a defection of the ring from the sample surface is achieved. The dynes/meter on the instrument was read as the surface tension, ‘Total petroleum hydrocarbon (TPH) analysis was carried out to estimate the level of degradation of the contaminated oi! mud waste using the gravimetric method (EPA Method 16644, 1999). ‘The use of the gravimetric analysis was necessitated by the less volatile hydrocarbon composition of the oil-based mud (ie. either diesel or mineral oil. which lowers the risk of TPH underestima- tion, Ten grams (10 g) soil sample of each treatment was collected and dried at room temperature for 72 h. An aliquot of 150 ml of n- hexane was added to 5 g soil in 200 mi beaker. To ensure homo- -geneity the sample was stirred continuously for 30 min and left to stand in a fume cupboard for 2 h. The extract was then filtered through crystals of anhydrous NapSOs salt using Whatman No, 42 filter paper into a 50 mi beaker (EPA Method 3630 B 1996). The filtrate was subsequently placed on a water bath at 70 °C to evap- orate the n-hexane. The oil and grease extractants were dissolved into 100 mi of n-hexane. Silica gel (3 g) was added to the solution to selectively absorb the polar fatty acs. The sample was thoroughly ‘mixed on a magnetic stirrer for 5 min after which it was filtered through a Whatman No. 42 filter pre-moistened with n-hexane solvent. To ensure complete removal ofthe hydrocarbons, the silica ‘gel was further washed with 10 mi of n-hexane into the receiving, beaker. The final weight of the beaker was taken and the TPH content in the HCDW was calculated using the following formula: rr nga, ey weigh As tt ow where:® » final weight of beaker and residue, corrected for blank (g)-A= initial weight of beaker, corrected for blank (g)- M = weight ‘of sample taken (x). ~ moisture factor (MF ~ 100/(100:Xmoisture) In determining the PAH, samples were extracted with n-hexane using the FEX-IKA Fluidized Bed Extraction system (USEPA. 1995: USEPA, 1998)-About 2-pL aliquot of the filtrate was injected into the ‘gas chromatograph (GC) mass spectrophotometer (GC/MS) via a narrow-bore fused-silica capillary column, The GC column was femperature-programmed to separate the analytes, which were then detected with a mass spectrometer (MS) connected tothe gas chromatograph. In identifying the PAM compounds, their mass spectra were compared with the electron impact (or electron impact-like) spectra of authentic standards. Quantitation was ‘accomplished by comparing the response of a major (quantitation) jon to an internal standard using a five-point calibration curve. ‘Total phospholipid concentration was analyzed by LAL-SOP-2 (Total Phospholipid and Fatty Acid Methyl Ester Composition) ‘method (Bligh and Dyer, 1959; Morrison and Smith, 1964; Holub ct al, 2011). Lipid content of pre-weighed cow bile was extracted with chloroform and methanol followed by filtration through a through Whatman No. 1 filter paper. Separation of extracts was done via solid-phase microextraction by spotting of lipid extracts fn silica gel 60 Thin layer chromatography (TLC) plate (Merck 5721-7), After this, standard solution was spotted in the adjacent lane to sample lanes. The thin layer ciromatography plate was subsequently developed in mobile phase heptane: isopropyl ether: acetic acid 60:40:3 vjvjv. After the solvent had moved 80% up the plate, the plate from the TLC tank was removed to allow drying in the fume hood for 5 min. Further, the plates were sprayed with 2.7- dichlorofloresin. or $-Anilino-1-Naphthalene Sulfonic Acid in ‘methanol in order to identify bands of interest based on RF com- parisons to appropriate standards by using long wave UV. Using a Single edged razor blade, bands of interest were scraped off into a Screw top 16 mm x 125 mm glass test tubes. Fatty acid methyl esters were prepared using boron trichloride in methanol, and the ‘methylation tubes were heated at 95°C on hotplate for 30 min. The resulting fatty acid methyl esters were then analyzed on Agilent 7890 B gas-liquid chromatograph with 60 m DB-23 capillary col- ‘umn (0.32 mm internal diameter). ‘Total nitrogen (TN) and moisture contents were determined using the methods described by Abeka (2014). Heavy metals (ie. Hg, Ni Ba, Co, Cu, Cd, Al) were determined by the Atomic by the ‘method prescribed by the FAQ (2008) 4 Ons wumas et a / ironenta Pellation 265 (2020) 15202 22. Estimation of total microbial population ‘The samples were analyzed for microbial populations at the Microbiology Laboratory of the Department of Pharmaceutics, KNUST. One gram (1 g) subsample of the HCDW was weighed from the homogenized stock and placed in a test tube that contained 10m of sterile deionized water, and thoroughly mixed manually to homogenize it (Ayotamuno et al, 2006), Total heterotrophic bac terial (THB) and fungal populations were quantified by the pour plate method to screen hydrocarbon-utilizing microorganisms. One imiliitre (1 mil) aliquot each of the serially diluted samples (10"*, 10> and 10 *) was aseptically added into different petri dishes that contained mutrient and Sabouraud dextrose agar media for the determination of bacterial and fungal load, respectively Bacterial and fungal population samples were plated at a temper- ature of 37 °C for 24 and 72 h, respectively. Subsequent to that, colonies of the microbes were enumerated, Heterotrophic bacteria and fungi (hydrocarbon degraders)col- tonies were isolated and plated using the Vapour Phase Transfer {(VPF) technique (Chikere and Chijoke-Osuji, 2006). It involved the tse of mineral sat agar (10 g NaCl; 0.42 g MgSO4.7H,0; 0.29 g KCI; (083 g KH, POs: 125 g KzHPOy, 0.42 g NaNOs: 15 g Agar Agar: TL Distilled water) for 7 and 10 days, respectively. Fungi growth was ‘ot observed at this stage. Using pure cultures ofthe hykrocarbon- degrading bacteria, further sub-culturing was carried out on min- eral salt medium using VPF with respective checks (ie. without filter paper soakeal in the pollutant) to ascertain their individual degradation potentials. Bacteria cultures were allowed to prolifer- ate in this medium for a week before the resulting colonies were Counted. The colonies were further cultured in nutrient broth, and the turbidity level was used as an indicator of bacteria presence. Pure bacteria cultures were inoculated in a stock culture and incubated at room temperature for 24 h. Molecular identification of the bacteria strains was cartied out in the CABI Laboratory, UK. Identification involved extraction of DNA, amplification by poly- ‘merase chain reaction (PCR) and sequencing using the Sanger sequencing approach (Sanger etal, 1977) 233, Feld application of hydrocarbon-uilizng bacteria “Twenty (20) ml of mineral salt was placed in conical flasks followed by 0.1 mi of pure bacteria cultures at room temperature to {get a stack solution for use on the field, The conical flasks were covered with cotton wool and incubated at three different periods {l.e.24,36, 48 h). Trypan de (20 iL) was added to the same volume Of the cultured solution before the living and dead bacterial cells ‘were separated using a haemocytometer. Following this, non- staining and staining cells were classified as living cells and dead cells, respectively, The above-mentioned procedure was repeated for 48 and 72 h, but cell density was determined after 72 h to be 16 x 10 ® cfujmL. The density of bacterial cells reported by (Okparanma etal (2009) was used as the reference, and the bacteria colonies were diluted by the mineral salt medium to 26 » 10" fu) ‘ml. Mixed bacteria cultures were obtained by mixing two different bacteria cultures in a ratio of 1:1 (Le, 0/50 ml). These, together with the pure culture (50 ml), were applied to the HCDW in the field experiment. 2A. Field experiment to determine the effects of compost, cow bile ‘and bacteria culture on HCDW degradation ‘A homogenized HCDW sample (80 kg) obtained from 25 diferent drill mud tanks was divided into 20 portions (4 kg each) and adjusted to 1.4% nitrogen content + 20 mi cow bile (as the basic treatment) and 4 portions without amendment (Le. control). Pure cultures of Brevibacerium case (Be) and Bacillus zhangzhouensi(Bz) and their mixture (BcBz) were subsequently added to 12 of the amended HCDW to begin a 6-week incubation period. The 14% nitrogen content +20 ml cow bile formulation was used in this study because a previous local study comparing the degradation of | HICDW with varying quantities of compost (as nitrogen source) and cow bile (as biosurfactant) proved that it was the most effective in reducing TPH (Osei-Twumasi etal, under review). Total petroleum hydrocarbon, surface tension, carbon-nitrogen ratio, pH, electrical conductivity and total plate count (TPC) were monitored weekly as indicators of hydrocarbon degradation. 25, Statistical analysis Repeated measure analysis of variance (ANOVA) was run to compare residual TPH, CN ratio, EC, pH, and surface tension of the samples among the various treatments. To determine the effects of DH, TPC, EC and surface tension on residual TPH, a multiple linear Fegression was run on these variables. Analyses were preceded by tests of normality and homogeneity of variance using Shapiro Wilk Normality test and Levene test. respectively The above mentioned analyses were conducted using the R software (Core Team, 2017). 3. Results 31, tial physicochemical properties of HCDW, cow bile and ‘compost ‘TPH concentration in the untreated HCDW (16.50 g/kg) excee- ded the tolerable levels of 1.0 g/kg and 30 g/kg, respectively sug gested by the Australian Department of Environment and Conservation (2010) and. However, no detectable concentrations of polycyclic aromatic compounds (PAH) such as acenapthalene, anthracene, benzo (a) anthracene and benzo (a) pyrene were recorded, Concentration of heavy metals were also lower than their respective Australan Department of Environment and Conservation (2010) Standards (Table 1). Mean pH, EC, surface tension and moisture content of the HCDW used for the study were 8.83, 2.34 mSjem, 365 N/m and 76.4%, respectively, Mean values of TPH, TN and TPC were 16.5 g/kg, 0.04% and 40 x 10? cfu/ml respectively. These physicochemical properties did not vary sta- tistically (p> 0.05) among the 25 drill mud tanks. For the cow bile, values obtained for pH, EC, and TN were 76,48 mSjem and 0.04%, respectively. Corresponclingly. total phospholipid concentration, ‘TPH and TPC of 483 mg/100 g, < 100 jigimg and 400 cfu/ml were recorded, The values of pH, % moisture, EC, organic matter, TN, TPC and TPH for compost were 8.26, 20.0%, L8mSjcm, 48.31%, 140% 40, ‘fuji and <100.00 g/kg in that order. 3.2. Bffects of compost, cow bile and bacterial culture on HCDW degradation ‘The treatments generally reduced (p < 0.05) TPH concentration in the HCDW to levels below the tolerable limits suggested by the ‘Australian Department of Environment and Conservation (10 glk 2010) and Osuji et al. (30 g/kg; 2005), and were very effective relative to the control (Fig 1). Across the treatments, residual TPH during the first weck varied from 88.1 to 93.7 g/kg. The reduction continued rapidly between the second and fourth weeks and thereafter marginally. At the end of the experiment, residual TPH concentration ranged between 154 g/kg (control) and 0.145 g/kg (basic + BcB), Statistical analysis showed that the treatments as ‘well as their interaction with time had significant effect on changes in PH (p < 0.05). With respect to surface tension, ll te treatments, but the control recorded values below the threshold of 30 mN/m suggested by Nitschke et al (2004; Fig. 2). These values ranged from 22.8 mN/m for the basic treatment to 28:5 mN/m for the uc tare nvoenta Paton 265 (2020) 15202 5 ‘ble Ina plysochemicl properties of the hyocahon contaminated il mad comport and cow ile wed the ty. Paneer Mean Concetta tan dev) Sindaed tesa HeDW TPH (eis) 185.00 «600 rae oH ass +032 S tial conducvity (mem) 2342022 Tosa topes (2) 01001 . Moisture comtent (2) 7540 +920 Surface tension (Nm) 330-6450 ita) aos 128 came) 197+ us, Pomel) 4017 2644 He (mas) a8 «028 comes) 01 = 003 ‘cuvmaie) 2506 «1222 sre ab 103 = Compost WH (eig) <<1on00 oH 820 5585: Moisture content (2) 2o00 or lei conducty (sf) 10 = ‘Orzic mater 2). a1 230" Toa topes) 140) 205 Toa plate count (tue) aoa0 = Cow ble oH 760.2013 tial conductivity (nem) S04 = Toa trogen (2) 004 = 00 Total posphii (g/100 3) 18100 «040 Tighe) Sieae0 Toa plate count (tae sono © (hasan Department of ivrooment aa Conservation, 2010) * Coat 2002, * (anton, 2000, ig. 2. Changes in 1 of HCDW folowing the eeament with vanous amendment. basic + BeBe treatment. Furthermore, surface tension values for treatments increased slightly (345-35.7 mNJm) after the frst week, following which the basic, basic + Bc and basic + Bz treat- ments dropped gradually. However, no significant differences {(p > 0.05) in surface tension were found among the treatments. Changes in CN ratio (Fig 3) largely mirrored those of TPH, reducing steadily (p < 0.05) from the beginning ofthe experiment, Mean CN ratio for the first week ranged from 648 (For basic) to 67.0 (for basic + Bz). From the second tothe sixth week, CN ratio across the Fi 3 Changs in surface tension aos the eaten treatments dropped continuously but the control showed minimal changes. Residual CN ratio values of 42.4, 17.1, 146, 13:2 and 11.6 were recorded for the basic, basic + Bc, basic Bz and basic + BeB, respectively, with the controt recording 71.1. Results from statistical analysis indicated strong interactive effect of the treatments and time on the CN ratio of the HCDW. Unlike the TPH and CN ratio, the treatments induced minimal bur statistically significant (p < 0.05) changes in pH during incu- bation (Fg. 4) By the sixth Week, the pH had dropped to 74 across the treatment (fom a mean of 88 recorded at the start of the study), Both the treatments and their interactions with time significantly influenced the pH (p < 0.05). EC for the treatments basic + Bc, basic + 82 and basic + BcB2 peaked during the second week of the experiment (with a mean value of 77 mSlem) and

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