Int J Recycl Org Waste Agricult (2015) 4:105–119

DOI 10.1007/s40093-015-0090-6

ORIGINAL RESEARCH

Recycling of sewage sludge as production medium for cellulase

by a Bacillus megaterium strain
Adel Ali Saeed Al-Gheethi1

Received: 20 May 2014 / Accepted: 3 March 2015 / Published online: 19 March 2015
Ó The Author(s) 2015. This article is published with open access at Springerlink.com

Abstract animal feed, formulation of detergents, juice clarification,

Background Cellulase is one of the enzymes commonly paper industry and wine production.
used in several agricultural, industrial and sewage sludge
treatment processes. The present study aimed to investigate Keywords Cellulase
Bacillus megaterium
Sludge

the potential use sludge generated from sewage treatment Heavy metals
Recycle
plants as a production medium for cellulase by B. mega-
terium strain that was isolated from a sewage treatment
plant. The production of cellulase in the sludge medium
was compared to different cellulosic materials: cotton, fil- Introduction
ter paper, bagasse and sawdust as well as to galactose,
fructose, lactose, maltose, mannitol, mannose, ribose, su- Cellulose is the most abundant renewable natural biologi-
crose and xylose. The production of cellulase was con- cal resource produced in the biosphere (about 100 billion
ducted at optimum conditions (0.4 mL of the bacterial dry tons per years) (Zhang et al. 2006). Municipal solid
inoculum, 45 °C, 72 h, pH 6.5 and citrate phosphate wastes contain 40–50 % cellulose, 12 % hemi-cellulose
buffer) that were determined in this study. and 10–15 % lignin by dry weight (Wang et al. 1994).
Results The sludge medium has induced the cellulase Several anaerobic bacteria have the ability to degrade
production by B. megaterium strain compared to cotton, cellulose in anaerobic digestion (Chynoweth and Pratap
filter paper, bagasse and sawdust. However, B. megaterium 1996). The degradation of cellulose by cellulase(s) en-
produced high cellulase in the presence of carbohydrate zymes produced by numerous microorganisms is very
compounds as carbon source. More cellulase was produced important in several agricultural and waste treatment pro-
in the sludge medium containing low concentrations of cesses (Hamer 2003; Angenent et al. 2004; Schloss et al.
Ni2?, Zn2? and Cu2? ions. 2005). The aerobic microorganisms usually secrete copious
Discussion The ability of B. megaterium strain to pro- amounts of free cellulase, which acts synergistically to
duce cellulase in the sewage sludge medium was due to degrade cellulose (Blouzard et al. 2007; Mingardon et al.
that the strain has acclimatized to resist heavy metals and 2007).
produce the enzyme genetically. Moreover, B. megaterium The widely accepted mechanism for enzymatic cellulose
has an important environmental role for reuse of sewage hydrolysis involves synergistic actions by endo-glucanase
sludge as production medium for cellulase that could be or CMCase (EC 3.2.1.4), exoglucanase or cellobiohyrolase
used in many of applications, including production of (EC 3.1.1.91) and b-glucosidase (EC 3.2.1.21) (Lynd et al.
2002; Zhang and Lynd 2004; Sadhu et al. 2013). En-
doglucanase hydrolyzes accessible intra-molecular b-1-4
& Adel Ali Saeed Al-Gheethi glucosidic bonds of cellulose chain ends. Exoglucanases
[email protected]; [email protected]
processively cleave cellulose soluble cellobiose or glucose
1
Faculty of Medical Science, Yemeni Jordanian University, and b-glucosidase hydrolyzes cellobiose to glucose (Kr-
Sana’a, Yemen ishna 1999; Zhang et al. 2006).

123
106 Int J Recycl Org Waste Agricult (2015) 4:105–119

Applications of cellulase include production of animal respectively. The sewage flows to these plants comprise
feed, formulation of detergents, juice clarification, paper residential, commercial and industrial. TSTP and ASTP are
industry and wine production. Cellulase contributes to 8 % oxidation ponds. The treatment of sewage in the ISTP and
of the worldwide industrial enzyme demands and the de- SSTP is based on primary and secondary processes. The
mand is expected to increase by 100 % in future (Costa sludge samples were collected from each STP in sterile
et al. 2008). However, production of cellulase(s) by dif- paper bags. The samples were transported to the laboratory
ferent microorganisms was found to be affected by many in a cooler box and the microbiological analysis was carried
factors, e.g. inocula size, incubation temperature, incuba- out within 2 h of collection.
tion period, pH value, buffers, carbon and nitrogen sources
(Mawadza and Zvauya 1996; Camassola et al. 2004; Silva Determination of heavy metals (Cu21, Ni21
et al. 2005; Immanuel et al. 2006). and Zn21) concentrations
The type and concentration of carbohydrate are critical
for maximal cellulase production in the production medi- Heavy metals were extracted from dewatered sludge sam-
um. Krishna (1999) has suggested that the glucose has ples by nitric acid digestion method (APHA 1998). The
enhanced cellulase synthesis to a significant level. How- heavy metal concentrations (Zn2?, Cu2? and Ni2?) in the
ever, addition of cellulose, lactose or glucose at concen- digested samples were analysed and determined by atomic
tration above 1 % level led to a significant reduction in absorption. An atomic absorption spectrophotometer
enzyme synthesis. Alam et al. (2004) have studied the (AAS) was used for this purpose.
production of extracellular cellulase by S. omiyaensis under
different carbon source availability. Four carbon sources Screening of bacterial isolates for resistance
have been investigated: carboxyl methyl cellulose (CMC), to nickel ions and production of cellulase
avicel, rice bran and sawdust at the rate of 1.2 %. They
have revealed that the highest CMCase enzyme production Hundred and twenty-seven (127) bacterial isolates were
was recorded when CMC was used as a carbon source and isolated from sludge samples collected from four sewage
the lowest CMCase production when sawdust and rice bran treatment plants in Yemen. These bacterial isolates were
were used as carbon sources. purified according to APHA, 9225B (1999). The screening
Emtiazi et al. (2007) have revealed that Paenibacillus for the bacterial isolates resistant to nickel ions was con-
strain produced high CMCase when CMC was used as only ducted according to Hernández et al. (1998) and Abdel-
carbon sources. Karim et al. (2014) have revealed that B. Monem et al. (2010). The bacterial isolates were sub-cul-
licheniformis produced a significant amount of cellulase tured in BHI agar medium containing 15 and 10 mM Ni2?
when wheat bran and orange peel were used as a sole carbon for 24–48 h at 35 °C. The plates were exposed to sulph-
source. However, the production of CMCase in the sludge hydryl gas (resulted from the reaction of 2 g of sodium
medium has not been studied before. Therefore, the current sulphide with 10 mL of concentrated HCl in a sealed con-
work aimed to investigate the recycling of sludge as pro- tainer, the reagent was prepared before each experiment) in
duction medium of CMCase by Bacillus megaterium strain a sealed container for the formation of the metal sulphide.
in comparison to cellulosic materials such as cotton, filter Plates were carefully screened to detect any change in the
paper, bagasse and sawdust as well as to different carbon region surrounding or inside the bacterial colonies.
sources (galactose, fructose, lactose, maltose, mannitol, The bacterial isolates were screened for the growth on
mannose, ribose, sucrose and xylose). The effect of different CMC–Yeast Extract (CYE) agar medium containing
concentrations of nickel ions was also tested to investigate (g L-1): NaNO3, 2; KCl, 0.5; MgSO4, 0.5; K2HPO4
the potential of bacterial strain to produce the enzyme in (buffer), 1.0, yeast extract (growth factor), 1 and CMC as
different types of sludge contaminated with heavy metals. carbon source, 10; pH 7.0 ± 0.2. After incubation at 37 °C
for 48 h, the isolates that exhibited good growth were
recorded. To detect production of cellulase, the surface of
Materials and methods the plates was floated with iodine ZnCl2 solution (3 %
ZnCl2 was added to gram’s iodine solution). Diameters of
Collection of sewage sludge samples blue zones were measured. The bacterial isolates that ex-
hibited positive results for the production of cellulase were
Twelve sewage sludge samples were collected weekly from grown on CMC–Yeast Extract (CYE) broth medium for
four STPs (referred to as ISTP, TSTP, ASTP and SSTP) in 5 days at 37 °C. Cellulase production was determined by
Yemen. TSTP was located in Taiz and treat Industrial waste CMC cup-plate clearing zone (CCZ) assay.
generated from Industrial and Commercial Company. ISTP, In this assay, 2 % (w/v) CMC was dissolved in citrate
ASTP and SSTP were located in Ibb, Aden and Sana’a, buffers (pH 7) and supplemented with 1.5 % agar for

123
Int J Recycl Org Waste Agricult (2015) 4:105–119 107

solidification. After sterilization, equal amounts of assay medium were dispensed in flasks of 250 mL capacity. The
medium (20 mL) were poured in sterilized petri dishes pH was adjusted at 6.5 and autoclaved at 121 °C for
(12 cm in diameter). Cups (10 mm in diameter) were made 15 min.
in each plate using a sterile cork borer. Equal amounts of
the enzyme solution (cultural supernatant) were put into Biomass yield
each cup. Plates with cups containing enzyme solutions
were incubated at 37 °C for 24 h, then the surface of the The bacterial growth was determined by dry weight
plates was floated with iodine ZnCl2 solution. Diameters of method. After collection of supernatant, the biomass resi-
blue zones were measured. The mean values of three due was dried at 80 °C for 24 h and the yield was ex-
readings were calculated. pressed as mg g-1 of substrate.

Identification of the most potent bacterial isolates b-1,4 Endoglucanase (CMCase activity)
determination
The bacterial isolates that exhibited the ability in the ac-
cumulation of Ni2? ions were identified based on mor- b-1,4 endoglucanase (CMCase activity) (EC 3.2.1.4) was
phological, culture and biochemical tests according to Noel determined by measuring the amount of reducing sugars
(1984), Sneath et al. (1986), Garrity et al. (2002) and released in the reaction mixtures containing 1.7 mL of
Brenner et al. (2004). 0.1 M acetate buffer (pH 5.5), 0.8 mL of 2 % (w/v) CMC
(sigma) solution and 0.5 mL of the culture supernatant. The
Cellulase production under catabolite repression mixture was maintained at 50 °C for 50 min. The reducing
sugars in the supernatant were assayed by 3,5-dinitros-
The purpose of this experiment was to investigate the alicylic acid (DNSA) methods (Miller 1959). Glucose was
ability of bacterial strains to produce cellulase enzyme as used as standard. Measurements were made in a spec-
inducible or genetically. The medium used was as men- trophotometer (Win. Aspect T 20, 031-2004, Germany) at
tioned previously with glucose (1 % w/v) equivalent to 540 nm wavelength in the presence of the blank. The re-
CMC as the only carbon source. After 48 h incubation at action mixtures containing heat-inactivated post-culture
37 °C, the cellulase enzyme assay was done by C.C.Z as- liquids (boiled for 5 min) were used as blanks. The cellu-
say as mentioned previously. lolytic enzyme activities were expressed in units defined as
the quantity of enzyme required to produce 1 lmol/h of
Factors affecting CMCase production by bacterial glucose, under the conditions of the assay.
isolate No. 1295S
Effect of different inocula sizes
The bacterial isolate No. 1295S, which secretes the highest
yield of cellulase under catabolic repression, was selected The effect of different inocula sizes on the production of
for studying different factors affecting CMCase produc- cellulase enzyme was investigated at 0.1, 0.2, 0.3, 0.4, 0.6,
tion: inocula size, temperatures, incubation periods, pH 0.8, 1, 1.5, 2 and 2.5 mL of bacterial inoculum (*2.7 9
values, buffers and nitrogen sources. 109 CFU mL-1). After the inoculation, the production
medium was incubated at 37 °C and pH 6.5 for 4 days.
Inoculum preparation Detection of CMCase production was performed by the
determination of reducing sugar using colorimetric tech-
The Bacterial isolate No. 1295S was maintained as stock nique as previously mentioned.
culture on Brain Heart Infusion agar (BHIA). The bacterial
strain was grown at 37 °C for 24 h and then stored at 4 °C Effect of temperature
for regular sub-culturing. The bacterium inoculum was
prepared using BHI broth in 250 mL conical flask. The For this purpose, the production medium was dispensed
inoculum was kept in shaker (200 rpm) at 37 °C for 14 h into flasks of 250 mL capacity; each flask containing
before it was used for the CMCase production. 50 mL of liquid medium was adjusted at pH 6.5. The flasks
were autoclaved at 121 °C for 15 min. The sterile medium
Production medium was inoculated with 0.4 mL (*2.7 9 109 CFU mL-1) of a
standardized bacterial inoculum, and incubated at the fol-
Carboxyl methyl cellulose yeast extract (CYE) liquid lowing temperatures 20, 30, 37, 45 and 60 °C. At the end
medium was used for studying factors affecting the of the incubation period of 4 days, the CMCase produced
CMCase production. The constituents of 50 mL of this was performed as described previously.

123
108 Int J Recycl Org Waste Agricult (2015) 4:105–119

Effect of different incubation periods Effect of different carbon sources applied

at different concentrations
The production medium was prepared as mentioned above
where bacterial isolate no. 1295S was incubated for 1, 2, 3, For this purpose, galactose, fructose, lactose, maltose,
4, 5, 6 and 7 days at 45 °C and pH 6.5. At the end of each mannitol, mannose, ribose, sucrose and xylose were used at
incubation period, the production of CMCase was deter- different concentrations (2, 4, 6, 8 and 10 mg mL-1) (w/v).
mined using the colorimetric technique. They were supplied singly to the sterilized carbon-free
production medium. The carbon sources used were steril-
Effect of pH values ized by membrane filter. The production medium with
CMC was used as control; other steps were carried out as
The pH values of the production medium were adjusted mentioned previously.
at 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 and 8 by the careful
addition of drop of (0.1 N) HCl and (0.2 N) NaOH Effect of different sludge media applied at different
using pH meter (IA 31-1114 WTW Germany). The concentrations of sludge
media were dispensed in flasks of 250 mL capacity each
containing 50 mL and then autoclaved at 121 °C for The ability of B. megaterium strain to produce enzyme in
15 min, then inoculated with 0.4 mL of a standardized the presence of sludge as only carbon and nitrogen source
bacterial inoculation. The inoculated media were incu- at different concentrations (2, 4, 6, 8 and 10 mg mL-1)
bated at 45 °C for 72 h. (w/v) was studied. The production medium was inoculated
with 0.4 mL of a standardized bacterial inoculum. At the
Effect of different buffers applied at various pH end of the incubation period 72 h, the CMCase produced
ranges was detected using colorimetric technique.

CYE liquid medium was used for detecting the suitable Effect of Ni21 ion concentrations
buffer for CMCase production. The following buffers
with their pH ranges were used for such a purpose, citrate To study the effect of Ni2? ion concentrations on CMCase
buffer with pH range from 5.6 to 6.2, citrate phosphate production, the (CYE) liquid medium was prepared with
buffer with pH range from 6 to 7 and phosphate buffer concentrations 117.2, 234.4, 351.6, 468.8, 586, 703.2 and
with pH range from 6 to 8. All these buffers with their 820.4 lg Ni2? mL-1. The medium was inoculated with
different pH were prepared according to Collee et al. bacterial strain and then incubated at 45 °C for 72 h. At the
(1989). The constituents of the production medium were end of the incubation period, CMCase concentration was
dissolved in the buffer solution and poured into the flask determined as described previously.
contained 50 mL of the buffered medium at the particular
pH. The media were inoculated with 0.4 mL of a stan- Data analysis
dardized bacterial inoculation. Then, the medium was
incubated at 45 °C for 72 h. Extraction of the crude en- All analyses were conducted in triplicate and values were
zyme was carried out as mentioned above at the end of reported as means with standard deviations. Data were
the incubation period and determined using colorimetric subjected to one-way analysis of variance (ANOVA) in the
technique. general linear model using the SPSS 11.5 statistical pack-
age. The statistical package (EASE, M-STAT) was used to
Effect of different cellulosic materials supplied perform the analyses of least significance difference (LSD).
at different concentration ANOVA was used to determine the significance (p \ 0.05)
of the differences between results.
This experiment was designed to test the effect of different
cellulosic materials on the CMCase production. DCY liq-
uid production medium was used without CMC. Substrates Results and discussion
were singly supplied to production medium. These sub-
strates were CMC, cellulose powder, cotton, filter paper, Selection of the most potent bacterial strains
sawdust, bagasse and tobacco leaves. All substrates were
supplied at concentrations 2, 4, 6, 8 and 10 mg mL-1 One-hundred and twenty-seven bacterial isolates were ob-
(w/v), respectively. pH value was adjusted to 6.5. The tained from sewage sludge samples collected from ISTP,
production medium was inoculated with bacterial inoculum TSTP, ASTP and SSTP at republic of Yemen. These iso-
and then incubated at 45 °C for 72 h. lates were purified and screened for nickel resistance at

123
Int J Recycl Org Waste Agricult (2015) 4:105–119 109

concentrations of 15 and 10 mM of nickel ions, where the reported that Bacillus sp., Pseudomonas sp., Chryseomonas
highest value of nickel ions was 15 mM in sludge sample sp., and Burkholderia sp. isolated from sewage effluent
at AWTP (Table 1). have the ability to tolerate 6 mM of Ni2? ions.
Among the 127 bacterial isolates, three bacterial isolates The results of screening for cellulase production showed
(586S, 1295S and 222W) exhibited good growth in the that 69 (almost 54.33 %) from 127 isolates exhibited high
presence of nickel concentrations of 15 mM (876 lg Ni2? growth, 59 from 69 bacterial isolates produced clearing
mL-1) and two bacterial isolates (117S and 120S) showed zones at varying degree and considered as positive for
high growth at 10 mM (584 lg Ni 2? mL-1). cellulase(s) production. It has been reported that the clear
Bacterial isolates forming a dark colour around or inside zone methodology was developed to isolate polymer-de-
the colony (possible reduction and precipitation of the grading strains from mixtures of microorganisms typically
metal) were considered to be possible nickel bio-sorbents. found in environments such as compost or soil. Microor-
Five bacterial isolates producing dark colour colonies ganisms capable of degrading a polymeric material are
grown in the presence of nickel were selected for further determined by looking for zones of clearing around mi-
studies. Neither clear halos nor dark colours were observed crobial colonies on agar media that are opaque due to the
around the colony when the clinical strains (E. coli, con- presence of powdered polymer (Pettigrew and Johanson
trol) were grown in the presence of nickel ion concentra- 1996; Ten et al. 2004). This screening process was per-
tions. The bacterial isolate Nos. 586S, 1295S and 222W formed in this work to choose the bacterial isolates, which
were characterized by a higher nickel tolerance than the have the potential to grow in the CYM medium containing
bacterial isolate Nos. 117S and 120S. CMC as carbon source.
Survey for nickel resistance among the obtained bacte- The second screening was carried out by growing the
rial isolates was chosen because nickel is among the most bacterial isolates, which have positive results in the pre-
toxic heavy metals and could be found in different indus- vious screening in the liquid medium for confirmation of
tries (Kaewchai and Prasertsan 2002). Ni2? is one of the the cellulase production using C.C.Z method. In this
most frequently encountered heavy metals in sewage screening, the diameters of the clearing zones surrounding
streams (Padmavathy 2008). Ni2? is a trace element and the wells on the plate screening medium ranged from
plays a role as cofactors for some of the bacterial enzymes 11 ± 0.4 to 77.5 ± 7.4 mm. This screening step was found
(Nies 1999; Dosanjh and Michel 2006). to give reliable indication of exhibited cellulolytic ac-
Only few bacterial strains were described to grow in the tivities. However, the enhancement of cellulase production
presence of higher concentration than 10 mM of Ni2?. was not linked with the amount of bacterial growth. The
Hernández et al. (1998) found that from 52 bacterial strains size of clearing zone diameter of each isolate is depicted in
isolated from contaminated soils of an oil refinery, only Fig. 1. Results showed that among the 59 bacterial isolates
two strains of bacteria Escherichia hermannii and Enter- which exhibited clear zone around the colony in the last
obacter cloacae were capable of accumulating either screening, 42 isolates (71.2 %) exhibited potential to pro-
nickel, vanadium or both metals at 10 mM. Leung et al. duce cellulase enzyme as determined by C.C.Z technique.
(2000) isolated nineteen metal resistant and non-resistant Among the forty-two cellulolytic bacterial isolates, five
bacteria of Ni2?, Pb2?, Cu2? and Zn2? from the activated bacterial isolates (586S, 1295S, 117S, 120S and 222W)
sludge. In the present work, among the 127 bacterial iso- showed nickel tolerance and selected for further studies as
lates obtained from sludge, five bacterial isolates exhibited will be described below.
resistance to 10 mM of Ni2? ions. Al-Gheethi et al. (2014)
Identification of the most potent bacterial isolates

Table 1 Heavy metal contents (mg kg-1 dry wt.) in sewage sludge at The bacterial isolates Nos. 586S, 1295S, 222W, 117S and
four sewage treatment plants (STPs) in Yemen (N = 3) 120S which showed high nickel tolerance were identified
STPs Heavy metals concentrations (mg kg-1) as Sporosarcina pasteurii 586S, Bacillus megaterium
1295S, Staphylococcus xylosus 222W, Bacillus subtilis
Cu2? Ni2? Zn2?
117S and Pseudomonas cepacia 120S (Table 2).
ISTP 575.6 369.3 3920.0
TSTP 733.3 156.0 9213.3 Production of cellulase under catabolic repression
ASTP 366.3 391.0 2425.6
SSTP 10.6 26.0 77.2 The considerations in the enzymatic treatment of sludge are
ISTP Ibb sewage treatment plant, TSTP Taiz sewage treatment plant,
the presence of substrates such as glucose, which may in-
ASTP Aden sewage treatment plant, SSTP Sana’a sewage treatment hibit the production of enzymes by the added bacterial
plant, S sludge strains. The ability of bacterial strains to produce

123
110 Int J Recycl Org Waste Agricult (2015) 4:105–119

90

80
Diameter of the clearing zones (mm)

70

60

50

40

30

20

10

Bacterial isolate

Fig. 1 Cellulase(s) production by 42 bacterial isolates obtained from four sewage treatment plants in Yemen in the presence of 1 % CMC as a
sole carbon source. Each point is the average of three determinations

detectable amounts of CMCase under catabolic repression the ability to produce the enzyme in the absence of
(genetically) was screened in the presence of glucose (1 % cephalexin (as inducibly). B. subtilis has produced b-lac-
w/v) equivalent to CMC as sole carbon source. As shown tamase in antibiotic-free and induced medium and con-
in Fig. 2, it could be observed that B. megaterium and B. sidered as producing b-lactamase genetically. In the current
cepacia could still synthesize cellulase with varying de- study, the bacterial species, which produced highest cel-
grees. These results were in agreement with Allcock and lulase in the presence of glucose, were regarded as
Woods (1981) who stated that the CMCase in Clostridium catabolite repression resistant and have constitutively
acetobutylicum induced by molasses and it was not re- CMCase production.
pressed by glucose. S. pasteurii, B. subtilis and S. xylosus
appeared to lose the ability to produce the enzyme in the Factors affecting production of CMCase by B.
presence of glucose. Bakare et al. (2005) demonstrated that megaterium strain
cellulase activity by P. fluorescens increased when cellu-
lose material was added to the culture medium than when B. megaterium strain was selected for studying the factors
glucose was used as sole carbon source. In this work, B. affecting CMCase production. The selection of B. mega-
megaterium 1295S produced CMCase in CYE2 medium terium strain was based on that the strain has produced
(CMC–yeast extract agar medium containing glucose as cellulase under catabolite repression and has the ability to
carbon source) more than that in CYE1 (CMC–yeast ex- grow at 15 mM Ni2? ions. The factors investigated were
tract agar medium containing CMC as carbon source) inocula size, incubation temperatures, incubation periods,
medium whereas B. cepacia 120S produced highest pH values, different buffers applied at various pH ranges. In
amounts of enzyme on CYE1 medium. B. megaterium these experiments, the amount of CMCase was determined
1295S has produced cellulase on CYE2 twofold than on by measuring reducing sugars using the 3,5-dinitrosalicylic
CYE1 and indicating the ability to produce cellulase ge- acid (DNSA) methods as described in ‘‘Materials and
netically. Conversely, B. cepacia 120S and S. xylosus methods’’ (Miller 1959). The DNSA methods were used
222W have described as inducible enzyme production. because the determination of differences in enzyme activity
Al-Gheethi and Norli (2014) studied the production of levels less than twofold is difficult using C.C.Z techniques
b-lactamase by bacteria isolated from sewage effluents in (Zhang et al. 2006). The DNSA method is one of the most
the presence or absence of cephalexin antibiotic as in- common assays for measuring reducing sugars for cellulase
ducible substrate. They found that B. stearothermophilus activity assays because of their relatively high sugar de-
and Burkholderia cepacia could still synthesize b-lacta- tection range (Coward-Kelly et al. 2003; Kongruang, et al.
mase at varying conditions. Chryseomonas luteola has lost 2004; Zhang and Lynd 2005).

123
Int J Recycl Org Waste Agricult (2015) 4:105–119 111

Table 2 Morphological, physiological and biochemical tests of five bacterial strains isolated from four sewage treatment plants at Yemen
Test Bacterial isolates
Sporosarcina pasteurii Bacillus megaterium B. subtilis Pseudomonas cepacia Staphylococcus xylosus
586S 1295S 117S 120S 222 W

Gram stain ?(ve) ?(ve) ?(ve) -(ve) ?(ve)

Cell shape Bacilli Bacilli Bacilli Rod Cocci
Spore formation ? ? ? - -
Motility ? ? ? ? -
Anaerobic growth ? ? ? ? ?
Growth at
30 °C ? ? ? ? ?
37 °C ? ? ? ? ?
45 °C ? ? ? ? ?
60 °C ? ? - - -
Catalase production ? ? ? - ?
Indole production - - ? - -
Citrate utilization - ? ? ? ?
MR ? - - - -
VP - ? ? - -
TSIA R/Y R/Y R/Y Y/Y Y/Y
Urease production ? ? - - ?
Hydrolysis of
Gelatin ? ? ? ? ?
Starch ? ? ? ? -
Casein ? ? ? ? -
Acid from
Glucose ? ? ? ? ?
Lactose - ? ? - -
Mannose - ? ? ? -
B-galactose - ? ? - ?
Sucrose - - - - -
Fructose - ? ? ? -
Xylose - - ? ? ?
Maltose - ? ? - ?
Ribose - ? ? ? ?
Mannitol - ? ? ? -
Gas from glucose - - - - -
H2S production - - - - -
Esculin hydrolysis - - - - -
L-alanine - - - - -
L-cysteine ? ? ? ? ?
L-methionine - - - - -
L-arginine - - - - -
?ve Gram-positive stain, -ve Gram-negative stain, ? positive, - negative, R red, Y yellow, MR methyl red, VP Voges-Proskaure, TSIA triple
sugars iron agar

The maximum amount of CMCase production (16.8 U endoglucanase by B. subtilis and revealed that 15 % (v/w)
mL-1) was noted with 0.4 mL of the bacterial suspension of inoculum size was optimal for the production of both
(Table 3). The optimum inocula size for heavy growth was enzymes. Immanuel et al. (2006) reported that one millilitre
0.1 mL. Krishna (1999) studied the effect of inoculum size inoculum of Bacillus spp., Cellulomonas spp. and Micro-
(5–40 %) on production of exoglucanase and coccus spp. was optimal to produce cellulase enzyme.

123
112 Int J Recycl Org Waste Agricult (2015) 4:105–119

growth at 7 days. Krishna (1999) found similar trend in

cellulase production using B. subtilis and indicated that the
decrease in activity after 72 h might be due to denaturation
of the enzyme, resulting from variations in pH during in-
cubation. Maximal production in Bacillus strains has
achieved after 2–3 days (Kawai et al. 1988; Ito et al. 1989).
The production of CMCase by B. megaterium in a con-
tinuous-operating reactor may keep the bacterial growth at
the highest stage of enzyme production. However, the
current study focused on the batch scale of bacteria and
continuous-operating reactor will be conducted in the
Fig. 2 Production of cellulase(s) enzymes under catabolite repression possible future work.
by five bacterial strains. Each point is the average of three The maximum CMCase production was noted at pH
determinations. CYE1 CMC–Yeast Extract agar medium containing value of 6.5 (Table 6). Further increase in pH level to 8
carboxyl methyl cellulose (CMC) as carbon source. CYE2 CMC–
resulted in considerable decrease in enzyme production. At
Yeast Extract agar medium containing glucose as carbon source
pH 6, the B. megaterium showed heavy growth, but at pH 4
showed little growth and at pH 8 showed moderate growth,
The high production of CMCase (35.1 U mL-1) and respectively. The optimum pH value of cellulolytic or-
biomass yield (710 mg g-1) by B. megaterium strain was ganisms varied from acidic condition such as Trichoderma
recorded at temperature 45 °C (Table 4). Alam et al. reesei pH 2.8–pH 3.5 (Sternberg and Mandels 1979) to
(2004) observed similar results; the maximum production alkaline conditions such as Bacillus sp. pH 9–12 (Haka-
of CMCase by Streptomyces omiyaensis occurred at 45 °C, mada et al. 1997). Alam et al. (2004) revealed that S.
while the maximum growth was recorded at the range from omiyaensis showed heavy growth and high cellulase ac-
35 to 40 °C. The range between 35 and 60 °C was reported tivity at pH 6.5. The optimal condition of cellulase pro-
as optimal temperature of growth and production of cel- duction by Bacillus sp. was recorded at pH range from 5 to
lulase by Bacillus sp. (Chan and Au 1987; Krishna and 7.5 (Dhillon et al. 1985; Araujo and Ward 1990; Mawadza
Varma 1990; Mawadza and Zvauya 1996; Ito 1997; Kr- and Zvauya 1996; Krishna 1999; Immanuel et al. 2006;
ishna 1999; Immanuel et al. 2006; Sadhu et al. 2013; Sethi Karim et al. 2014).
et al. 2013). The effect of buffers on production of CMCase is il-
The overproduction of CMCase (32.5 U mL-1) was lustrated in Table 7. Highest CMCase production was at-
obtained after 72 h of incubation period (Table 5). The tained when the initial pH of the medium was adjusted to
amount of growth increased simultaneously with time; the pH 6.2 and 6.4 with phosphate buffers, where 38.5 U mL-1
maximum yield of cell growth of B. megaterium was noted was produced. The production of CMCase in the medium
after 4 days. The enzyme production was unstable and with citrate phosphate buffers was 38.6 and 38.4 U mL-1
decreased rapidly after 72 h. Reduction in CMCase yield at pH 6.4 and 6.6. The minimum production of CMCase
was accompanied by an accumulation of reducing sugars in was noted with the citrate buffer 33.7 U mL-1 at pH 6.2
the medium and compatible with increasing amount of and non-buffered production medium 34.2 U mL-1 at pH

Table 3 Effect of inocula sizes Inoculum size (mL)* CMCase concentrations Saccharification Biomass yield (g g-1)
on saccharification (%), biomass (U mL-1 ± SD) (%) cellulose
yield (mg g-1) and the CMCase
production (U mL-1) by B. 0.1 10.9 ± 1.0 1.1 0.63
megaterium
0.2 13.2 ± 1.8 2.3 0.28
0.3 12.7 ± 0.5 2.2 0.47
0.4 16.9 – 2.3 2.4 0.53
0.6 16.2 ± 1.8 2.4 0.24
0.8 15.5 ± 2.4 2.4 0.36
1 14.3 ± 0.7 2.2 0.24
1.5 14.6 ± 0.7 2.2 0.31
2 14.1 ± 1.9 2.4 0.26
2.5 14.2 ± 0.1 2.3 0.43
Bold values indicate the maximum CMCase production by B. megaterium strain
* mL contains *2.7 9 109 CFU mL-1 saline solution

123
Int J Recycl Org Waste Agricult (2015) 4:105–119 113

Table 4 Effect of different Temperature CMCase concentrations Saccharification Biomass yield (mg g-1)
incubation temperatures on (°C) (U mL-1 ± SD) (%) cellulose
saccharification (%), biomass
yield (mg g-1) and the CMCase 20 5.1 ± 1.9 0.8 640
production (U mL-1) by B.
30 22.8 ± 1.3 3.5 530
megaterium
37 30.7 ± 2.5 4.6 520
45 35.1 – 3.1 4.9 710
60 20.3 ± 0.5 3.1 530
Bold values indicate the maximum CMCase production by B. megaterium strain

Table 5 Effect of different Incubation period CMCase concentration Saccharification Biomass yield
incubation periods on (day) (U mL-1 ± SD) (%) (mg g-1) cellulose
saccharification (%), biomass
yield (mg g-1) and the CMCase 1 22.5 ± 3.5 1.8 360
production (U mL-1) by B.
2 27.4 ± 0.8 4.1 390
megaterium
3 32.5 – 0.0 4.9 400
4 16.1 ± 1.5 2.5 530
5 12.2 ± 3.0 1.9 320
6 11.7 ± 3.3 1.8 300
7 7.9 ± 0.7 1.2 520
Bold values indicate the maximum CMCase production by B. megaterium strain

Table 6 Effect of pH value on pH CMCase concentration Saccharification Biomass yield (mg g-1)
saccharification (%), biomass (U mL-1 ± SD) (%) cellulose
yield (mg g-1) and the CMCase
production (U mL-1) by B. 4 18.6 ± 4.4 4.1 120
megaterium
4.5 28.6 ± 1.6 4.2 150
5 29.7 ± 0.6 4.4 250
5.5 30.0 ± 2.0 4.5 180
6 32.0 ± 0.0 4.8 420
6.5 34.2 – 2.9 5.2 240
7 26.0 ± 1.2 4.6 310
7.5 25.6 ± 0.7 4.5 350
8 24.7 ± 2.0 4.2 280
Bold values indicate the maximum CMCase production by B. megaterium strain

6.5. The optimum buffers of heavy growth were observed Effect of different cellulosic material and carbon
as citrate phosphate 1260 mg g-1 at pH 6.2 and phosphate source on the production of CMCase
buffers 1400 mg g-1 at pH 7.4 were used in culture
medium, respectively. These results indicated that phos- The effect of different cellulosic material was studied to
phate buffer is an important buffer for cellulase production determine the optimum concentration of each substrate
by B. megaterium. Stutzenberger (1971) showed that supplied for the production of CMCase. Five concentra-
maximum cellulase production by Thermomonospora tions of each substrate were used: 2, 4, 6, 8 and
curvata was attained when the initial pH of the medium 10 mg mL-1 (w/v). The results (Table 8) show that the
was adjusted to pH 8.0 with phosphate buffer. However, maximum amount of CMCase production (52.7 U mL-1)
Duff et al. (1985) stated that sodium citrate buffer im- was recorded at 8 mg mL-1 of CMC compared to
proved the enzyme production in production medium by up 22.8 U mL-1 at 6 mg mL-1 of cellulose powder. The
to 40 %. Camassola et al. (2004) reported that production lowest cellulase production was noted at 2 mg mL-1 of
of cellulase enzyme by Penicillium echinulatum with ci- cotton and 6 mg mL-1 of sawdust. The highest saccha-
trate buffer was slightly higher than that in acetate buffer of rification (10 %) was observed when CMC was used as
the same pH. carbon source followed by cellulose powder.

123
114 Int J Recycl Org Waste Agricult (2015) 4:105–119

Table 7 Effect of different pH Buffers CMCase concentration Saccharification Biomass yield

buffers on saccharification (%), (U mL-1 ± SD) (%) (mg g-1) cellulose
biomass yield (mg g-1) and the
CMCase production (U mL-1) 5.6 Citrate buffer 30.8 ± 0.4 4.6 180
by B. megaterium
5.8 30.0 ± 0.9 5.0 420
6 31.6 ± 0.4 5.3 240
6.2 33.7 – 1.2 5.1 310
6 Citrate phosphate buffer 29.3 ± 0.0 4.5 990
6.2 36.6 ± 0.0 5.6 1260
6.4 38.6 – 4.3 5.8 710
6.6 38.4 ± 2.5 5.8 560
6.8 31.4 ± 1.5 4.7 890
7 24.1 ± 0.7 3.6 620
6 Phosphate buffer 31.8 ± 1.0 4.6 1130
6.2 38.5 ± 1.5 5.6 930
6.4 38.5 – 4.8 6.0 930
6.6 38.0 ± 1.7 5.6 880
6.8 32.9 ± 1.5 5.3 770
7 33.6 ± 0.9 5.0 650
7.2 31.6 ± 1.7 4.9 1030
7.4 31.8 ± 0.9 4.7 1400
7.6 31.6 ± 1.0 4.6 570
7.8 31.0 ± 1.4 4.5 1020
8 29.0 ± 3.2 4.2 750
Bold values indicate the maximum CMCase production by B. megaterium strain

The effect of fructose, galactose, lactose, maltose, Recycling of sludge as production medium
mannitol, mannose, ribose, sucrose and xylose on CMCase for CMCase
production is presented in Table 8. The results indicate that
the maximum production of CMCase (51.4 U mL-1) was The production of CMCase by B. megaterium inoculated to
recorded when 10 mg mL-1 of mannose was used as car- sludge samples was investigated in the present study. The
bon source followed by 8 mg mL-1 of ribose results are presented in Fig. 3. The maximum amount of
(47.4 U mL-1), 8 mg mL-1 of fructose (45.5 U mL-1), CMCase production (14.3 U mL-1) was recorded in
8 mg mL-1 of xylose (44.8 U mL-1), 2 mg mL-1 of lac- 6 mg mL-1 (w/v) of sludge from SSTP, followed by
tose (40.8 U mL-1) and 4 mg mL-1 of mannitol sludge collected from TWTP at concentration 4 mg mL-1,
(19.3 U mL-1). The lowest production of CMCase where 13.1 U mL-1 was produced. The lowest amount of
(11.7 U mL-1) was recorded with 8 mg mL-1 of galac- CMCase production (1.4 U mL-1) was observed in sludge
tose, maltose and 6 mg mL-1 of sucrose. The highest collected from ASTP and ISTP (1.6 U mL-1). The highest
saccharification (%) was observed with xylose and ribose, saccharification (7.5 %) was observed when 10 mg mL-1
9.7 and 9.5 %, respectively. The results obtained in the (w/v) of sludge from SSTP was used as production medi-
current study are similar to those reported by Alam et al. um, while the lowest saccharification (0.3 %) was noted
(2004) who found that the highest CMCase production was with 8 mg mL-1 (w/v) of sludge from ASTP. The pro-
recorded when CMC was used as carbon source and the duction of CMCase in sludge medium has not been re-
lowest CMCase production with sawdust as a carbon ported before. However, Barros et al. (2013) used cassava
source. Paenibacillus produced (4 U mL-1) CMCase when wastewater as production medium for amylase, protease
it was grown on CMC as the only source of carbon (Em- and lipase by B. subtilis strains and revealed that the bac-
tiazi et al. 2007). Krishna (1999) suggested that addition of teria produce detectable amounts of these enzymes in
cellulose powder, lactose or glucose at concentration above comparison to the synthetic liquid medium. Al-Gheethi and
1 % level led to a significant reduction in enzyme synthesis Norli (2014) investigated the production of b-lactamase in
by B. subtilis. sewage effluents medium by B. subtilis, C. luteola and

123
Int J Recycl Org Waste Agricult (2015) 4:105–119 115

Table 8 Effect of different Carbon Conc. CMCase activity Saccharification

cellulosic material and carbon source (mg mL-1) (U mL-1 ± SD) (%)
source applied at different
concentrations on CMC 2 11 ± 0.2 10
saccharification and CMCase 4 25 ± 0.5 10.1
production by B. megaterium
6 40 ± 1.3 9.8
8 52.7 – 2.5 10
10 15 ± 0.9 2
Cellulose powder 2 10 ± 0.3 8.9
4 13 ± 1.2 5.6
6 22.8 – 0.5 5.8
8 15 ± 1.3 5
10 12 ± 0.7 2
Cotton 2 4 – 1.3 6
4 1.5 ± 1.2 0.7
6 1.5 ± 0.6 0.4
8 1.4 ± 0.3 0.3
10 1.3 ± 0.1 0.2
Filter paper 2 8.5 – 1.4 5.5
4 2.8 ± 0.1 1
6 1.8 ± 0.5 0.2
8 1.5 ± 0.2 1
10 1.5 ± 0.0 0.1
Tobacco leaves 2 5 ± 0.1 4.1
4 23 – 1.4 3.5
6 13.5 ± 0.4 3.2
8 8.3 ± 0.1 1.2
10 4.2 ± 0.3 0.4
Sawdust 2 2.8 ± 0.2 2
4 2.7 ± 0.2 1.3
6 5.6 – 0.6 1
8 4.2 ± 0.1 0.8
10 3 ± 0.1 8.5
Bagasse 2 1.8 ± 0.2 1
4 5.2 ± 0.5 2
6 6.7 ± 0.2 1.8
8 9.8 – 0.4 1
10 5 ± 0.3 1
Fructose 2 8 ± 0.2 8.5
4 20 ± 0.3 8
6 33.2 ± 0.5 8.5
8 45.5 – 1.1 8.9
10 44.2 ± 0.4 6.8
Galactose 2 1.6 ± 0.1 1.3
4 2.3 ± 0.5 1
6 8.1 ± 1.2 2
8 11.7 – 0.4 2.1
10 1.9 ± 0.2 0.1
Lactose 2 \0.1 0
4 10.3 ± 2.4 3.7
6 15.2 ± 0.5 4.1
8 40.8 – 3.2 7.5
10 34.5 ± 0.3 4.9

123
116 Int J Recycl Org Waste Agricult (2015) 4:105–119

Table 8 continued Carbon Conc. CMCase activity Saccharification

source (mg mL-1) (U mL-1 ± SD) (%)

Maltose 2 11.6 – 0.6 8.3

4 8.1 ± 0.3 3.1
6 [0.1 \0.1
8 [0.1 \0.1
10 [0.1 \0.1
Mannitol 2 4.8 ± 0.2 7.1
4 19.3 – 1.2 7.5
6 10 ± 1.5 2
8 2.3 ± 0.1 0.2
10 [0.1 \0.1
Mannose 2 1.2 ± 0.0 1.1
4 2.4 ± 0.1 2.7
6 32 ± 0.8 9.4
8 35 ± 1.5 7
10 51.4 – 1.4 8
Ribose 2 11.2 ± 0.3 9.5
4 24.5 ± 0.4 9.2
6 29.7 ± 0.6 7.5
8 47.4 – 0.8 9
10 30 ± 0.4 4.8
Sucrose 2 5.2 ± 1.3 4.3
4 6.2 ± 1.5 2.3
6 11.7 – 1.5 3
8 10 ± 0.8 1.7
10 7.3 ± 0.3 1
Xylose 2 13.7 ± 0.4 9.7
4 18.8 ± 1.2 7
6 30.5 ± 1.1 8
8 44.8 – 1.3 8.5
10 40.2 ± 0.6 5.9

Bold values indicate the maximum CMCase production by B. megaterium strain

B. cepacia. They revealed that the bacterial strains pro- ability of bacteria to resist Cu2? was reported to be more
duced b-lactamase at levels above 0.333 U mL-1. than Ni2? at the same concentrations (Al-Gheethi et al.
In comparison to the concentrations of heavy metals in 2014). Lankinen et al. (2011) reported that the production of
sludge collected from the STPs, it can be noted that the b-glucosidase and b-cellobiosidase by decomposing fungi
concentration of Cu2? and Zn2? ions in the sludge from in heavy nickel-contaminated soil (more than 20 mg kg-1)
TSTP was more than that in the sludge from ASTP and was less than that in the non-contaminated soil. In this
ISTP. However, the production of CMCase and percentage study, however, the ability of B. megaterium to produce
of saccharification in sludge from TSTP were more than detectable amount of CMCase in sludge contaminated with
those in the sludge from ASTP and ISTP. These results Ni2? ions is because B. megaterium was tolerant to 15 mM
could be explained based on the toxicity of Ni2? ions in of nickel. Therefore, this bacterium would be used to pro-
comparison to Cu2? and Zn2? at the high concentration. It duce cellulase from the sludge.
has reported that Ni2?, Cu2? and Zn2? improve the enzy-
matic reaction at low concentration (Nies 1999). However, Effect of different nickel ion concentrations
at high concentrations, Ni2? becomes more toxic than Cu2?
and Zn2?. This is because the transport of Zn2? and Cu2? The effect of different nickel ion concentrations was in-
through bacterial cells membrane is by specific transport vestigated to know the minimal Ni2? ion concentrations at
system (Fath and Kolter 1993; Fagan and Saier 1994), while which B. megaterium strain produces maximum amount of
Ni2? is accumulating by the fast and unspecific system CMCase. Ni 2? ion concentrations of 117.2–468.8 lg Ni2?
(Smith and Maguire 1995; Tao et al. 1995). Besides, the mL-1 have provided a broad range for the growth of

123
Int J Recycl Org Waste Agricult (2015) 4:105–119 117

CMCase Saccharification CMCase Saccharification

10 10 20 10
9
A 9 18
B 9
CMCase activity (U mL-1)

Saccharification (%)
8 8 16 8
7 7 14 7
6 6 12 6
5 5 10 5
4 4 8 4
3 3 6 3
2 2 4 2
1 1 2 1
0 0 0 0
0 2 4 6 8 10 0 2 4 6 8 10
Sludge concentrations (mg mL-1) Sludge concentrations (mg mL-1)

CMCase Saccharification
10 CMCase Saccharification 10 20 10
D
9 C 9 18 9
CMCase activity (U mL

Saccharification (%)
-1)

8 8 16 8
7 7 14 7
6 6 12 6
5 5 10 5
4 4 8 4
3 3 6 3
2 2 4 2
1 1 2 1
0 0 0 0
0 2 4 6 8 10 0 2 4 6 8 10
Sludge concentrations (mg mL-1) Sludge concentrations (mg mL-1)

Fig. 3 Effect of different sludge media applied at different concen- Taiz sewage treatment plant, c sludge from Aden sewage treatment
trations of sludge on saccharification and CMCase production by B. plant, d sludge from Sana’a sewage treatment plant
megaterium. a Sludge from Ibb sewage treatment plant, b sludge from

20
CMCase Biomass yield
500
was obtained at 117.2 and 468.8 lg Ni2? mL-1 of the
nickel ions, respectively (Fig. 4).
CMCase activity (U mL-1)

18 450
Biomass yield (mg g-1)

16 400 Nickel has been demonstrated for many bacteria and

14 350
12 300 plant species, where eight nickel-containing enzymes pre-
10 250 sent in one or more of these species have been identified
8 200
(Ragsdale 1998; Wattt and Ludden 1999). Nickel is iden-
6 150
4 100 tified as micronutrients at trace concentrations. On the
2 50 other hand, it has been noted that the addition of Ni2? ions
0 0
117.2 234.4 351.6 468.8 586 703.2 820.4 at low concentrations enhanced biomass yield (Sujarit-
Ni2+ ion concentraions (µg L-1) tanonta and Sherrard 1981). Husain et al. (2013) revealed
that the biomass of P. fluorescens increased with increasing
Fig. 4 Effect of different Ni2? ion concentrations in carboxyl methyl Ni2? ions in the broth medium from 250 to 1000 mg L-1;
cellulose yeast extract (CYE) broth medium on biomass yield and
CMCase production by B. megaterium. Each point is the average of
the maximum growth was recorded at 1000 mg L-1.
three determinations However, the ability of Ni2? ions to induce the CMCase
production by B. megaterium strain has not been reported
bacteria and stimulated CMCase production with optimal before.
production at 468.8 lg Ni2? mL-1. There was little de- In comparison to the production of CMCase in the
tectable growth above 468.8 lg Ni2? mL-1 or cellulase sludge medium, the concentrations of nickel ions in the
production below 351.6 lg Ni2? mL-1 or above 586 lg sludge from TSTP and SSTP were 156 and 26 mg kg-1,
Ni2? mL-1. The maximum biomass yield and CMCase while the maximum concentrations of nickel added to the
production (450 mg g-1 and 9.1 U mL-1 respectively) production medium were 820.4 lg L-1. However, the

123
118 Int J Recycl Org Waste Agricult (2015) 4:105–119

amount of CMCase produced in the sludge medium was cephalexin from secondary effluents by tolerant bacteria. Clean
more than that in the CYE medium. The increasing Technol Environ Policy 16:137–148
Allcock ER, Woods DR (1981) Carboxymethyl cellulase and
CMCase production in the sludge may be related to the cellobiase production by Clostridium acetobutylicum in an
sludge which is rich in nutrients and trace elements that industrial fermentation medium. Appl Environ Microbiol
induced the enzyme production more than CYE medium 41(2):539–541
that is a synthetic medium. These findings are in consistent Angenent LT, Karim K, Al-Dahhan MH, Wrenn BA, Domı́guez-
Espinosa R (2004) Production of bioenergy and biochemicals
with those reported by Barros et al. (2013). They revealed from industrial and agricultural wastewater. Trends Biotechnol
that the production of amylase, protease and lipase by B. 22:477–485
subtilis strains in cassava wastewater was more than that in APHA (1998) Standard methods for the examination of water and
the synthetic liquid medium. Another explanation of high wastewater, 20th edn. American Public Health Association,
Washington, DC
production of CMCase in the sludge medium may be due to APHA (1999) Standard methods for the examination of water and
the nitrogen source. The sludge is rich with the amino acid wastewater, 9225B, 21st edn. American Public Health Asso-
and organic compounds, which represent as nitrogen ciation, Washington, DC
source of bacteria, while in CYE the nitrogen source used Araujo A, Ward OP (1990) Hemicellulases of Bacillus sp. preliminary
comparative studies of production and properties of mamma-
was inorganic (sodium nitrate). Al-Gheethi (2008) revealed mases and galactanases. J Appl Bacteriol 68:1253–1261
that the production of cellulase in the presence of peptone Bakare MK, Adewale IO, Ajayi AO, Shonukan OO (2005) Purification
as nitrogen source was more when sodium nitrate was used and characterization of cellulase from the wild-type and two
as nitrogen source. improved mutants of P. fluorescens. Afr J Biotechnol 4(9):898–904
Barros FFC, Simiqueli APR, de Andrade JC, Pastore GM (2013)
Production of enzymes from agroindustrial wastes by biosurfac-
tant-producing strains of Bacillus subtilis. Biotechnol Res Int
Conclusions 2013:1–9
Blouzard JC, Bourgeois C, Philip P, Valette O, Bélaı̈ch A, Tardif C,
Bélaı̈ch JP, Pagès S (2007) Enzyme diversity of the cellulolytic
It can be concluded that B. megaterium strain isolated from system produced by Clostridium cellulolyticum explored by two-
the sludge possesses an important potential to produce dimensional analysis: identification of seven genes encoding new
CMCase in sewage sludge medium. The potential of bac- dockerin-containing proteins. J Bacteriol 189(6):2300–2309
terial strain to use sewage sludge as production medium Brenner DJ, Krieg NT, Staley JT (2004) Bergeys manual of
systematic bacteriology, The Proteobacteria, 2nd edn, vol 2,
would lead to biodegradation of cellulose in the sludge and, part A, B and C. Williams and Wilkins Awaverly Co, USA
thus, can be used as a source of biofuel. Camassola M, De Bittencourt LR, Shenem NT, Andreaus J, Dillon
AJP (2004) Characterization of cellulase complex of Penicillium
Acknowledgments This work was conducted at Taiz University echinulatum. Biocat Biotrans 22(5–6):391–396
(TU)-Yemen. I gratefully thank TU for their technical assistance and Chan KY, Au KS (1987) Studies on cellulase production by Bacillus
academic staff. Many thanks for Mr. Jamil Ali Saeed Al-Gheethi for subtilis. Antonie Van Leeuwenhoek 53(2):125–136
his support during the study. Chynoweth DP, Pratap P (1996) Anaerobic digestion of municipal
solid wastes. In: Palmisano AC, Barlaz MA (eds) Microbiology
Open Access This article is distributed under the terms of the of solid waste. CRC Press Inc, Florida, pp 71–104
Creative Commons Attribution License which permits any use, dis- Collee JG, Duguid JP, Fraser AG, Marmion BP (1989) Practical
tribution, and reproduction in any medium, provided the original medical microbiology, 13th edn. Longman-FE. Ltd, London
author(s) and the source are credited. Costa RB, Silva MVA, Freitas FC, Leitao VSF, Lacerda PSB, Ferrara
MA, Bon EPS (2008) Mercado e Perspectivas de Uso de
Enzimas Industriais e Especiais no Brasil. In: Bon EPS, Ferrara
MA, Corvo ML, Vermelho AB, Paiva CLA, Alencastro RB,
References Coelho RRR (eds) Enzimas em Biotecnologia, Producao,
Aplicac oes e Mercados, 1st edn. Rio de Janeiro, Intercie^ncia,
Abdel-Monem MO, Al-Zubeiry AH, Al-Gheethi AAS (2010) p 463–488
Biosorption of nickel by Pseudomonas cepacia 120S and Coward-Kelly G, Alello-Mazzari C, Kim S, Granda C, Holtzapple M
Bacillus subtilis 117S. Water Sci Technol 61:2994–3007 (2003) Suggested improvement to the standard filter paper assay
Alam MZ, Manchur MA, Anwar MN (2004) Isolation, purification used to measure cellulase activity. Biotechnol Bioeng
and characterization of cellulolytic enzyme produced by the 82:745–749
isolate Streptomyces omiyaensis A2. Pak J Biotechnol Sci Dhillon N, Chhibber S, Saxena M, Pajni S, Vadehra DV (1985) A
7(10):1647–1653 constitutive endoglucanase (CMCase) from Bacillus licheni-
Al-Gheethi AAS (2008) Bacteriological studies on sludge from some formis. Biotechnol Lett 7(9):695–697
municipals wastewater treatment plants in Yemen. MSc Thesis, Dosanjh NS, Michel SL (2006) Microbial nickel metal-loregulation
Department of Applied Microbiology, Faculty of Applied NikRs for nickel ions. Curr Opinion Chem Biol 10(2):123–130
Science, Taiz University, Taiz, Yemen Duff SJB, Cooper DG, Fuller OM (1985) Effect of colloidal materials
Al-Gheethi AAS, Norli I (2014) Biodegradation of pharmaceutical on cellulase production by Trichoderma reesei Rut-C30. Appl
wastes in treated sewage effluents by Bacillus subtilis Environ Microbiol 49(4):934–938
1556WTNC. Environ Process 1:459–489 Emtiazi G, Pooyan M, Shamalnasab M (2007) Cellulase activities in
Al-Gheethi AAS, Norli I, Lalung J, Megat-Azlan A, Nur-Farehah ZA, nitrogen fixing Paenibacillus sp. isolated from soil in N-free
Ab. Kadir MO (2014) Biosorption of heavy metals and media. World J Agri Sci 3(5):602–608

123
Int J Recycl Org Waste Agricult (2015) 4:105–119 119

Fagan MJ, Saier MHJ (1994) P-type ATPases of eukaryotes and Mingardon F, Chanal A, López-Contreras AM, Dray C, Bayer EA,
bacteria: sequence comparisons and construction of phylogenetic Fierobe HP (2007) Incorporation of fungal cellulases in bacterial
trees. J Mol Evol 38(1):57–99 minicellulosomes yields viable, synergistically acting cellulolyt-
Fath MJ, Kolter R (1993) ABC-transporters: bacterial exporters. ic complexes. Appl Environ Microbiol 73(12):3822–3832
Microbiol Rev 57(4):995–1017 Nies DH (1999) Microbial heavy metals resistance. Appl Microbiol
Garrity GM, Johnson KL, Bell J, Searles D (2002) Bergey’s manual Biotechnol 51(6):730–750
of systematic bacteriology: taxonomic outline of the prokaryotes, Noel RK (1984) Bergey’s manual of systematic bacteriology, vol 1.
2nd edn. Eds. Release 3.0 July 2002. Springer-Verlag, New York Williams and Wilkins Awaverly Co, USA, p 409–602
Hakamada Y, Koike K, Yoshimatsu T, Mori H, Kobayashi T, Ito S Padmavathy V (2008) Biosorption of nickel (II) ions by baker’s yeast:
(1997) Thermostable alkaline cellulase from an alkaliphilic kinetic, thermodynamic and desorption studies. Bioresour
isolate Bacillus sp. KSM-S237. Extremophiles 1(3):151–156 Technol 99(8):3100–3109
Hamer G (2003) Solid waste treatment and disposal: effect on public Pettigrew CA, Johanson BN (1996) Testing the biodegradability of
health and environmental safety. Biotechnol Adv 22:71–79 synthetic polymeric materials in solid waste. In: Palmisano AC,
Hernández A, Mellado RP, Martı́nez JL (1998) Metal accumulation Barlaz MA (eds) Microbiology of solid waste. CRC Press Inc,
and vanadium-induced multidrug resistance by environmental Florida, pp 71–104
isolates of Escherichia hermannii and Enterobacter cloacae. Ragsdale SW (1998) Nickel biochemistry. Curr Opin Chem Biol
Appl Environ Microbiol 64(11):4317–4320 2:208–215
Husain RSA, Thatheyus AJ, Ramya D (2013) Bioremoval of nickel Sadhu S, Saha P, Sen SK, Mayilraj S, Maiti TK (2013) Production,
using Pseudomonas fluorescens. Am J Microbiol Res purification and characterization of a novel thermotolerant
1(3):48–52 endoglucanase (CMCase) from strain isolated from cow dung.
Immanuel G, Dhanusha R, Prema P, Palavesam A (2006) Effect of Springer plus 2:1–10
different growth parameters on endoglucanase enzyme activity Schloss PD, Hay AG, Wilson DB, Gossett JM, Walker LP (2005)
by bacteria isolated from coir retting effluents of estuarine Quantifying bacterial population dynamics in compost using 16S
environment. Int J Environ Sci Technol 3(1):25–34 rRNA gene probes. Appl Microbiol Biotechnol 66:457–463
Ito S (1997) Alkaline cellulases from alkaliphilic Bacillus: enzymatic Sethi S, Datta A, Gupta BL, Gupta S (2013) Optimization of cellulase
properties, genetics and application to detergents. Extremophiles production from bacteria isolated from soil. ISRN Biotechnol
1(2):61–66 2013:1–7
Ito S, Shikata S, Ozaki K, Kawai S, Okamoto K, Inoue S, Takei A, Silva R, Lago ES, Merheb CW, Macchione MM, Park YK, Gomes E
Ohta YI, Satoh T (1989) Alkaline cellulase for laundry (2005) Production of xylanase and CMCase on solid state
detergents production by Bacillus sp. KSM 635 and enzymatic fermentation in different residues by Thermoascus aurantiacus
properties. Agri Biol Chem 53:1275–1281 miehe. Brazilian J Microbiol 36:235–241
Kaewchai S, Prasertsan P (2002) Biosorption of heavy metal by Smith RL, Maguire ME (1995) Distribution of the CorA Mg2?
thermotolerant polymer-producing bacterial cells and the transport system in gram-negative bacteria. J Bacteriol
bioflocculant. Songklanakarin J Sci Technol 24(3):421–430 177(6):1638–1640
Karim A, Nawaz MA, Aman A, Qader SAU (2014) Hyper production Sneath P, Mair NS, Sharpe EM (1986) Bergey’s manual of systematic
of cellulose degrading endo (1,4) b-D-glucanase from Bacillus bacteriology, vol 2. Williams and Wilkins Awaverly Co, USA
licheniformis KIBGE-IB2. J Rad Res Appl Sci. doi:10.1016/j. Sternberg D, Mandels GR (1979) Induction of cellulolytic enzymes in
jrras.2014.06.004 T. reesei by sophorose. J Bacteriol 139:761–769
Kawai S, Okoshi H, Ozaki K, Shikata S, Ara K, Ito S (1988) Stutzenberger FJ (1971) Cellulase production by Thermomonospora
Neutrophilic Bacillus strain KSM.522 that products an alkaline curvata isolated from municipal solid waste compost. Appl
carboxy methyl cellulase. Agri Biotechnol Chem 52:1425–1431 Microbiol 22:147–152
Kongruang S, Han MJ, Breton CIG, Penner MH (2004) Quantitative Sujarittanonta S, Sherrard JH (1981) Activated sludge nickel toxicity
analysis of cellulase-reducing ends. Appl Biochem Biotechnol studies. J Water Poll Control Fed 53(8):1314–1322
113–116:213–231 Tao T, Snavely MD, Farr SG, Maguire ME (1995) Magnesium
Krishna C (1999) Production of bacterial cellulases by solid state transport in Salmonella typhimurium: mgtA encodes a P-type
bioprocessing of banana wastes. Biores Technol J 69:231–239 ATPase and is regulated by Mg2? in a manner similar of the mgt
Krishna KB, Varma A (1990) Endoglucanase production by en- B P-type ATPase. J Bacteriol 177(10):2654–2662
trapped Bacillus thermoalkalophilus. World J Microbiol Ten LN, Im WT, Kim MK, Kang MS, Lee ST (2004) Development of
Biotechnol 6(3):267–272 a plate technique for screening of polysaccharide-degrading
Lankinen P, Kähkönen MA, Rajasärkkä J, Virta M, Hatakka A (2011) microorganism by using a mixture of insoluble chromogenic
The effect of nickel concentration on growth of litter-decom- substances. J Microbiol Meth 56:375–382
posing fungi, extracellular enzyme activities and toxicity in soil. Wang YS, Byrd CS, Barlaz MA (1994) Anaerobic biodegradability of
Boreal Environ Res 16(3):229–239 cellulose and hemicellulose in excavated refuse samples. J Indus
Leung WC, Wong MF, Chua H, Lo W, Yu PH, Leung CK (2000) Microbiol 13:147–153
Removal and recovery of heavy metals by bacteria isolated from Wattt RK, Ludden PW (1999) Nickel binding proteins. Cell Mol Life
activated sludge treating industrial effluents and municipal Sci 56:604–625
wastewater. Water Sci Technol 41(12):233–240 Zhang YH, Lynd LR (2004) Toward an aggregated understanding of
Lynd LR, Weimer PJ, Van Zyl WH, Pretorius IS (2002) Microbial enzymatic hydrolysis of cellulose. Non complexed cellulose
cellulose utilization: fundamentals and biotechnology microbi- systems. Biotechnol Bioeng 88:797–824
ology. Microbiol Mol Biol Rev 66:506–577 Zhang YH, Lynd LR (2005) Determination of the number average
Mawadza C, Zvauya R (1996) Some factors affecting endo-b-1,4- degree of polymerization cellodextrans and cellulose. Biomacro-
glucanase production by two Bacillus strains isolated from molecules 6:1510–1515
Zimbabwean hot springs. J Basic Microbiol 36:177–185 Zhang YHP, Himmel ME, Mielenz JR (2006) Outlook for cellulase
Miller GL (1959) Use of dinitrosalicylic acid reagent for determina- improvement: screening and selection strategies. Biotechnol Adv
tion of reducing sugar. Anal Chem 31:426–428 24:452–481

123